New method for the detection of acid-resistant micro-organisms in stool

A number of published documents are cited in the description of this invention. The subject matter of these documents is incorporated into the description by reference.

The invention relates to a method for detecting infection of a mammal with an acid-resistant microorganism, wherein (a) a stool sample of the mammal is incubated with at least two different monoclonal antibodies, fragments or derivatives thereof or aptamers under conditions which cause complexation of antigens allow the acid-resistant microorganism with the antibodies, fragments or derivatives thereof or the aptamers, and wherein (aa) the first monoclonal antibody or the fragment or derivative thereof or the first aptamer specifically binds an epitope of the first antigen which has a structure in at least part of the mammals after the intestine passage which has a structure which corresponds to the native structure or the structure, against which a mammal produces antibodies after infection or immunization with the acid-resistant microorganism or an extract or lysate thereof or a protein thereof or a fragment thereof or a synthetic peptide; (ab) the second monoclonal antibody or the fragment or derivative thereof or the second aptamer specifically binds an epitope of a second antigen which differs from the epitope of the first antigen and which, at least in part of the mammals after the intestine passage, has a structure which corresponds to the native structure or corresponds to the structure, against which a mammal produces antibodies after infection or immunization with the acid-resistant microorganism or an extract or lysate thereof or a protein thereof or a fragment thereof or a synthetic peptide, the parts of the mammals according to (aa) and (ab) overlapping can and in total make up essentially the total number of infected mammals; and (b) detects the formation of at least one antigen-antibody complex / antigen-aptamer complex according to (aa) or (ab).

The acid-resistant microorganism is preferably a bacterium, in particular <i>Helicobacter pylori, Helicobacter hepaticus</i> or <i>Mycobacterium tuberculosis</i> or <i>Campylobacter jejuni</i> or <i>Campylobacter pylori.</i> It is further preferred that the two epitopes epitopes of a urease and a heat shock protein, preferably of Hsp60, an alkyl hydroperoxide reductase, preferably the 26kDa protein, the 20kDa protein (3-dehydroquinase type II), the 16.9kDa protein (Neutrophil activating protein) or the 33.8 kDa protein (fructose bisphosphate aldolase). In a further preferred embodiment of the invention, the stool sample is examined with three different monoclonal antibodies, fragments or derivatives thereof or aptamers, the first antibody, the fragment or derivative thereof or the first aptamer being an epitope of a urease and the second antibody and the third antibody (or the derivative, fragment or aptamer) each an epitope of different proteins mentioned above, preferably Hsp60 (if one of the first two epitopes is an epitope of an alkyl hydroperoxide reductase) or specifically binds an alkyl hydroperoxide reductase if one of the first two epitopes is an epitope of Hsp60. The invention further relates to diagnostic and pharmaceutical compositions and test devices which contain the aforementioned components and packaging containing them.

The detection of infection of a mammalian organism with a microbial pathogen or parasite can today be carried out in various, invasive as well as non-invasive, direct and indirect ways. When using invasive techniques, the physical integrity of the person being examined is violated, for example when a biopsy or serum is taken. Non-invasive diagnostic techniques detect changes in parameters that can be measured without intervention in the organism. Body fluids and excretions such as breathing air, urine, saliva, sweat or stool are preferably sampled and analyzed. In the case of direct methods, the presence of the pathogen or parasite, its constituents or their breakdown products is detected, for example by staining and microscopic examination or specific enzymatic reactions. Indirect methods rely on the detection of reactions of the host organism to the pathogen or the parasite, e.g. the presence of antibodies to antigens of the pathogen in the serum, urine or saliva of the host. In a further indirect method, indicator substances must be supplied to the patient that are specifically modified by the microbial pathogen or parasite, the modified form being detected in samples (breath test).

An intervention in the organism, for example to obtain a tissue sample, is stressful for the organism in most cases and often also involves a lot of equipment and a health risk. The comparatively simple acquisition of samples of the body fluids and excretions described above therefore make non-invasive techniques the method of choice. Furthermore, since not every host responds in the same way to a particular pathogen or parasite, and the host's reaction may start with a delay and may persist even after the pathogen or parasite has been removed from the organism, direct methods are always preferable. Ideally, a diagnosis is made through the non-invasive, direct detection of the pathogen or parasite in body fluids or excretions.

In addition, a diagnostic procedure should also be optimized in terms of other aspects: high reproducibility, sensitivity and specificity, guaranteed availability of the materials to be used in constant quality, low costs for production and implementation, and simple use regardless of complex equipment are parameters to be considered here .

For the reasons mentioned above, methods based on the high selectivity and binding affinity of certain classes of substances (e.g. antibodies, receptors, lectins, aptamers) for molecular structures, which can be chosen in such a way that they are highly specific for the substance / microorganism to be determined in particular medical diagnostics an increasingly wide space. In particular, the possibility of immobilizing these substances on solid surfaces and coupling them with radioactive nuclides, with enzymes that trigger color reactions in combination with suitable substrates, or with colored particles with a highly specific binding affinity (e.g. ELISA = enzyme linked immunosorbent assay) led to the development at a lower cost , simple and less time-consuming detection procedure for the body's own and foreign substances.

In the early stages of the development of these detection methods, only polyclonal antibodies were used. However, these have some disadvantages which are well known to the person skilled in the art, above all limited availability and often also cross-reactivities. The development of methods for the production of monoclonal antibodies (Köhler &amp; Milstein (1975)), the progress in isolating receptors and their directed expression in cellular host systems, the development of lectins with high affinity for certain carbohydrates and the discovery that nucleic acids (aptamers ) specifically bind molecular structures, these disadvantages were largely eliminated. Today, the specificity and sensitivity of detection methods can be optimized using comparatively simple methods.

Due to the high specificity, such methods are particularly suitable for the detection of individual, defined substances such as haptens, peptides or proteins, provided that the recognized structural element is constant within the sample population to be examined and is specific for the substance to be detected. They are also well suited for measurement in body fluids and are therefore an obvious option for the direct detection of pathogens in this sample matrix. Accordingly, methods for diagnosing eg of <i>Entamoeba histolytica</i> (<nplcit id="ncit0001" npl-type="s"><text>Haque (1993), J. Infect. Dis. 167: 247-9</text></nplcit>), enterohemorrhagic <i>Escherichia coli</i> (EHEC; <nplcit id="ncit0002" npl-type="s"><text>Park (1996) J. Clin. Microbiol. 34: 988-990</text></nplcit>), <i>Vibrio cholerae</i> (<nplcit id="ncit0003" npl-type="s"><text>Hasan (1994), FEMS Microbiol. Lett. 120: 143-148</text></nplcit>), Torovirus-like particles (<nplcit id="ncit0004" npl-type="s"><text>Koopmans (1993) J. Clin. Microbiol. 31: 2738-2744</text></nplcit>) or <i>Taenia saginata</i> (<nplcit id="ncit0005" npl-type="s"><text>Machnicka (1996), Appl. Parasitol. 37: 106-110</text></nplcit>) described from the chair. A diagnostic procedure for adenovirus-related gastroenteritis (<nplcit id="ncit0006" npl-type="s"><text>Hermann (1987), J. Infect. Dis. 155: 1167-1171</text></nplcit>) uses the combination of two monoclonal antibodies directed against different enteric adenovirus serotypes in order to be able to detect these serotypes unaffected by the presence of other, non-enteric adenoviruses. With knowledge of the adenovirus serotype with which a patient is identified, the detection is also reliable with the respective monoclonal antibody alone.

The pathogens described above have in common that they are viable and multiply in the intestine, in the intestinal mucosa or in the intestinal lumen of their host, in all cases of humans. So you have mechanisms that allow you to survive and multiply in the presence of the digestive and digestive systems active in the intestine. It is therefore likely that a large number of intact or almost intact pathogens or parasites will go off when excreted with the stool. With detection reagents, for example antibodies, which recognize components of the pathogens or parasites, these can usually be easily detected in the stool or in prepared stool samples.

However, there are a number of pathogens and parasites that can occur in the stool on the one hand due to the relationships of the affected tissue (e.g. lungs, stomach, pancreas, duodenum, liver) to the gastrointestinal tract, but on the other hand they do not live in the intestine itself - and / or are capable of reproduction. These pathogens and parasites are referred to here as acid-resistant microorganisms. These pathogens and parasites include, for example<i>Helicobacter pylori (H. pylori)</i> and <i>Helicobacter hepaticus, Mycobacterium tuberculosis</i> and other mycobacteria, <i>Chlamydia pneumoniae, Legionella pneumophilae, Pneumocystis carinii, Campylobacter jejuni, Campylobacter pylori</i> and other. Some of these pathogens can be detected in sputum, for example, but the detection of<i>Mycobacterium tuberculosis</i> only possible in the sputum for a short period of time after a cavern containing the pathogen has opened. Evidence is also made more difficult by the fact that it is not always possible to obtain a sputum sample from a person to be examined. This applies, for example, to small children, confused patients or animals. Other pathogens such as<i>Legionella pneumophilae</i> can be specifically identified using antigens that enter the urine via the kidney. However, this only works if the amount in the urine is sufficient for the detection. Proof of this in the stool would be a welcome alternative. The passage of the intestine in these organisms is, however, with a strong attack by the digestive and degradation mechanisms of the mammal and especially the intestinal flora. connected. Molecular structures specific to the pathogen under consideration can be destroyed or their concentration can be greatly reduced.

The degradation of the pathogens in the intestine has proven to be a problem for reliable detection in stool samples in acid-resistant bacteria. The number of germs that enter an infected person's stomach is small compared to other bacteria that colonize the intestine. In addition, germs and germ fragments have to travel a long way through the protease-rich intestine after leaving the stomach. This means that only small amounts of intact proteins can be found in the stool. This also means that the combination of two epitopes on an antigen required for an ELISA test is no longer necessarily the same as in the native protein, and that epitopes located close to each other have the greatest probability in a detection that requires two epitopes on the same molecule, to show a positive result. The individually different distribution of antigens detected in the stool of infected people also indicates individual properties in the processing of the antigens during intestinal passage. A first approach to reducing this problem was through the disclosure content of the<patcit id="pcit0001" dnum="EP0806667A"><text>EP-A 0 806 667</text></patcit> provided. In this application it was shown that polyclonal antibodies with the lysate of a particular<i>H</i>. <i>pylori</i>Strain could be induced which recognize a broader variability of strains from different geographic regions. However, this application does not show which antigens are recognized by the serum. A corresponding diagnosis using monoclonal antibodies was also excluded. In view of the fact that immune sera can fluctuate in spite of all standardization efforts, this must be stated in the above Application developed methods can be considered as suboptimal for a wide application. In addition, new animals have to be immunized again and again to provide the polyclonal sera. The corresponding procedures are both time-consuming and costly.

The object of the present invention was therefore to improve the aforementioned disadvantageous methods from the prior art and to provide an easily standardized and inexpensive method for the reliable detection of acid-resistant microorganisms.

This object is achieved by the embodiments characterized in the claims.

The invention thus relates to a method for detecting an infection of a mammal with an acid-resistant microorganism, wherein<ul id="ul0001" list-style="none"><li>(a) incubating a mammalian stool sample with at least two different monoclonal antibodies, fragments or derivatives thereof or aptamers under conditions which allow antigen from the acid-resistant microorganism to complex with the antibodies, fragments or derivatives thereof or the aptamers, and wherein<ul id="ul0002" list-style="none"><li>(aa) the first monoclonal antibody or the fragment or derivative thereof or the first aptamer specifically binds an epitope of the first antigen which has a structure in at least some of the mammals after the intestine passage which has a structure which corresponds to the native structure or the structure, against which a mammal produces antibodies after infection or immunization with the acid-resistant microorganism or an extract or lysate thereof or a protein thereof or a fragment thereof or a synthetic peptide;</li><li>(ab) the second monoclonal antibody or the fragment or derivative thereof or the second aptamer specifically binds an epitope of a second antigen which differs from the epitope of the first antigen and which, at least in part of the mammals after the intestine passage, has a structure which corresponds to the native structure or corresponds to the structure, against which a mammal produces antibodies after infection or immunization with the acid-resistant microorganism or an extract or lysate thereof or a protein thereof or a fragment thereof or a synthetic peptide,</li></ul>wherein the parts of the mammals according to (aa) and according to (ab) can overlap and together make up essentially the total number of infected mammals; and</li><li>(b) Detects the formation of at least one antigen-antibody complex or antigen-aptamer complex according to (aa) or (ab).</li></ul>

The term "acid-resistant microorganism" in the sense of this invention encompasses any microorganism which, due to its properties / adaptation mechanisms to the host, resists the physical and chemical influences of the digestive tract, so that it can be detected by preferably immunological detection or using aptamers. Examples of such acid-resistant microorganisms are<i>Helicobacter pylori, Helicobacter hepaticum, Mycobacterium tuberculosis, Mycobacterium pseudotuberculosis, Mycobacterium cansassii, Camplyobacter jejuni</i> and <i>Campylobacter pylori.</i>

For the purposes of this invention, the term “stool sample of the mammal” means any stool sample that can be used for the detection method according to the invention. In particular, this includes stool samples that have been prepared for diagnostic tests using methods known per se. The preparation is carried out, for example, according to the RIDASCREEN® Entamoeba enzyme immunoassay (R-Biopharm GmbH, Darmstadt).

For the purposes of this invention, “fragments” or “derivatives” of monoclonal antibodies have the same binding specificity as the monoclonal antibodies. Such fragments or derivatives can be produced by conventional methods; see. e.g.<nplcit id="ncit0007" npl-type="b"><text>Harlow and Lane "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, USA, 1988</text></nplcit>. Examples of fragments are Fab-, F (ab ')₂ or Fv fragments. Examples of derivatives are scFv fragments. Derivatives can also be chemically produced substances that have the same or improved binding properties as the antibodies. Such substances can be produced, for example, by peptidomimetics or by various rounds of phage display and subsequent selection for improved binding properties. According to the invention, aptamers are understood to mean nucleic acids such as RNA, ssDNA (ss = single strand), modified RNA or modified ssDNA, which bind a large number of target sequences with high specificity and affinity. The term "aptamer" is known in the prior art and is defined, for example, in<nplcit id="ncit0008" npl-type="s"><text>Osborne et al., Curr. Opin. Chem. Biol. 1 (1997), 5-9</text></nplcit>, or in <nplcit id="ncit0009" npl-type="s"><text>Stull and Szoka, Pharm. Res. 12 (1995), 465-483</text></nplcit>.

The term "at least two" is not limited in terms of the number of antibodies etc. used and means, for example, three, four, five, six, seven, eight, nine or ten antibodies etc.

"Conditions that allow complex formation" can be easily set by the person skilled in the art; s. also Harlow and Lane, op. cit., and are, for example, physiological conditions.

The term "after the intestinal passage has a structure which corresponds to the native structure" in the sense of this invention means that the epitope of an antigen after the intestinal passage is recognized by a monoclonal antibody, derivative or fragment thereof or the aptamer which acts against the same Antigen / epitope was obtained or binds to it, which has not passed the intestinal passage. In other words, the epitope / antigen which is derived from the above-mentioned antibody or Fragments or derivatives thereof or aptamers is specifically bound, the intestinal passage survived in its structure without damage or essentially undamaged and has not been degraded. As a source for the native structure of the epitope / antigen, for example, a bacterial extract digested with a French press can be prepared using conventional methods (cf. e.g.<nplcit id="ncit0010" npl-type="b"><text>Sambrook et al., "Molecular Cloning, A Laboratory Manual", 2nd edition 1989, CSH Press, Cold Spring Harbor, USA</text></nplcit>) was further purified, or a bacterial lysate that was further purified by standard methods (eg Sambrook et al., loc. cit.)

The term "after passage through the intestine has a structure which corresponds to the structure against which a mammal produces antibodies after infection or immunization with the acid-resistant microorganism or an extract or lysate thereof or a protein thereof or a fragment thereof or a synthetic peptide" means according to the invention that the epitope recognized by the monoclonal antibody, fragment, derivative or aptamer corresponds to an epitope which is derived from the immune system of a mammal, preferably a human being. The mechanisms of antigen presentation as well as mechanisms that lead to the processing of antigens and the resulting variety of antibodies are known in the prior art and, for example, in<nplcit id="ncit0011" npl-type="b"><text>Janeway and Travers, Immunology, 2nd edition 1997, Spektrum Akademischer Verlag GmbH</text></nplcit>, Heidelberg described. These epitopes can differ from the native epitopes. The mammal may come into contact with the microorganisms or the proteins or fragments or the synthetic peptides by natural infection (except for the synthetic peptides) or by immunization. Extracts, lysates, synthetic peptides etc. of the microorganism / protein can also be used for the immunization. Suitable immunization schemes are known in the prior art and are described, for example, in Harlow and Lane, loc. Cit. Suitable antibodies can also be obtained, for example, by immunization and / or screening for surrogates such as synthetic peptides, recombinantly produced proteins, extracts, lysates or partially digested proteins.

According to the invention, the term “specifically binds” means that the monoclonal antibody, the fragment or derivative thereof or aptamer has no or essentially no reactivity with other epitopes in samples from uninfected mammals.

The term "where the parts of the mammals according to (aa) and according to (ab) can overlap and together make up essentially the total number of infected mammals" means that at least one, but possibly also two or more epitopes / antigens occur in the stool that are specifically bound by the monoclonal antibodies, fragments or derivatives or the aptamers and that the monoclonal antibodies, Detect fragments or derivatives or the aptamers of at least one epitope of an antigen in essentially any infected mammal. The term "essentially the total number" means that the epitopes and thus a corresponding infection with the microorganism are more than 70%, preferably at least 75%, more preferably more than 85%, particularly preferably more than 90% and most preferred more than 95% of those affected can be recorded. Ideally, infections are detected in 100% of those affected.

The term "antigen-antibody complex" in the sense of this invention includes not only complexes which the antigen enters into with the native antibody, but also complexes which it enters into with its fragments or derivatives.

Surprisingly, it was found according to the invention that a relatively certain diagnosis of the infection with these bacteria / pathogens can be carried out with a limited number of monoclonal antibodies, fragments or derivatives thereof or aptamers which specifically bind at least two different epitopes of different antigens of acid-resistant microorganisms . In this embodiment of the invention, for example, antigens of a prepared stool sample can be bound to a solid phase, for example, and the infecting agent can be detected with the limited number of monoclonal antibodies, fragments or derivatives thereof or aptamers present in labeled form. If the antigen present after the intestinal passage is (still) in dimeric or multimeric form, the same monoclonal antibodies, fragments or derivatives thereof or aptamers can be used both as a catcher and as a detector. This embodiment also includes the possibility that an epitope occurs several times on the same subunit. The invention includes embodiments in which further epitopes which have the abovementioned properties are recognized by further monoclonal antibodies or fragments or derivatives thereof or by further aptamers. An antigen can also be detected from different monoclonal antibodies or aptamers that recognize the same or different epitopes of the antigen. For example, three monoclonal antibodies or three aptamers can recognize three different epitopes on two protein fragments. The latter embodiments are suitable for further increasing the certainty when making the diagnosis. In the case of higher-order structures, for example of dimers or multimers, the same monoclonal antibody can also bind several epitopes in the same multimeric protein in the process according to the invention. Examples of such embodiments are described in more detail below.

The invention encompasses embodiments in which only monoclonal antibodies or fragments or derivatives thereof or only aptamers are used, as well as embodiments in which different types of detection reagents are used in a test. It is thus possible for a first monoclonal antibody with a second antibody derivative or a first aptamer with a second antibody fragment to be used, to name just two examples. In this respect, the terms "first" and "second" designate the first and second detection reagents. This does not mean that two antibodies, derivatives or fragments thereof or two aptamers are always used.

The results according to the invention are surprising, above all, because the prior art had taught them away. For example, at<i>H. pylori</i> found that major antigens in ELISA tests do not have the desired specificity and sensitivity; see.<nplcit id="ncit0012" npl-type="s"><text>Newell et al., Serodiag. Immunother. Infect. Dis. 3 (1989), 1-6</text></nplcit>. It also teaches<patcit id="pcit0002" dnum="EP0806667A"><text>EP-A 0 806 667</text></patcit>that a sure proof of <i>H. pylori</i>-Infections with monoclonal antibodies due to the genetic variability of the <i>H. pylori</i>Tribes is not possible.

The method according to the invention is particularly advantageous over the prior art mentioned because only two monoclonal antibodies or fragments or derivatives thereof or aptamers enable a relatively reliable diagnosis. For the detection, for example in the ELISA, pairs of antibodies, fragments, derivatives thereof or aptamers are preferably used, the two antibodies of the pair binding the same or different epitopes on the same antigen. For example, forms<i>H</i>. <i>pylori</i>-Urease multimeric structures from several identical as well as different subunits. The same antibodies, fragments or derivatives thereof or aptamers can thus be used as capture antibodies / aptamers as well as detection antibodies / aptamers in the ELISA or other assays. On the one hand, the use of monoclonal antibodies, fragments or derivatives thereof or of aptamers provides an easy-to-maintain standard in the reliability of the diagnostic method, which is a great advantage compared to previously known diagnostic methods which have been introduced for this purpose. This is also not the case, for example, in the<patcit id="pcit0003" dnum="EP0806667A"><text>EP-A 0 806 667</text></patcit> Always need new immunizations and subsequent testing of experimental animals. Another advantage of the method according to the invention is its design as a direct and non-invasive method, which increases the convenience mentioned for the patient and the reliability in determining the stage of the disease.

In a preferred embodiment, the acid-resistant microorganism is an acid-resistant bacterium.

A number of acid-resistant bacteria are known in the prior art. In a particularly preferred embodiment, the acid-resistant bacterium is a bacterium of the genus Helicobacter or of the genus Mycobacterium or of the genus Campylobacter.

In another particularly preferred embodiment, the bacterium is a bacterium of the species <i>Helicobacter pylori, Helicobacter hepaticum</i> or a bacterium of the species <i>Mycobacterium tuberculosis</i> or a bacterium of the species <i>Campylobacter jejuni</i> or <i>Campylobacter pylori.</i>

In a further preferred embodiment, the epitope of the first antigen is an epitope of a urease, for example an α-urease or a β-urease, and the epitope of the second antigen is an epitope of a heat shock protein, an alkyl hydroperoxide reductase or the 20kDa protein (3- Dehydro-Quinase Type II), the 16.9kDa protein (neutrophil-activating protein) or the 33.8 kDa protein (fructose bisphosphate aldolase).

Surprisingly, it was found according to the invention that in a population of mammals, in particular human patients, whose stool has been tested for infections with acid-resistant bacteria, essentially all members of this population have at least one of the abovementioned Epitopes were present in the stool, so that a relatively reliable diagnosis can be made with a set of only two corresponding monoclonal antibodies, fragments or derivatives thereof or aptamers.

It is particularly preferred that the urease is the β-urease of <i>H. pylori</i> is.

In a further particularly preferred embodiment, the heat shock protein is an Hsp60.

Furthermore, it is particularly preferred that the Hsp60 is the Hsp 60 from <i>H. pylori</i> acts.

In an additionally particularly preferred embodiment, the alkyl hydroperoxide reductase is the 26kDa protein of <i>H. pylori.</i>

It is also preferred that the other proteins mentioned above are proteins of <i>H</i>. <i>pylori</i> are.

In a further (preferred) embodiment, the method according to the invention for detecting an infection of a mammal with an acid-resistant microorganism comprises the following steps, wherein (a) a stool sample of the mammal with three different monoclonal antibodies, fragments or derivatives thereof or aptamers under conditions incubated, which form a complex formation of antigens from the acid-resistant microorganism with the antibodies, Allow fragments or derivatives thereof or aptamers, and wherein (aa) the first monoclonal antibody or the fragment or derivative thereof or the first aptamer specifically binds an epitope of the first antigen which, at least in part of the mammals, has a structure after passage through the intestine which corresponds to the native structure or the structure, against which a mammal produces antibodies after infection or immunization with the acid-resistant microorganism or an extract or lysate thereof or a protein thereof or a fragment thereof or a synthetic peptide; (ab) the second monoclonal antibody or the fragment or derivative thereof or the second aptamer specifically binds an epitope of a second antigen which differs from the epitope of the first antigen and which, at least in part of the mammals after the intestine passage, has a structure which corresponds to the native structure or corresponds to the structure, against which a mammal produces antibodies after infection or immunization with the acid-resistant microorganism or an extract or lysate thereof or a protein thereof or a fragment thereof or a synthetic peptide; and (ac) the third monoclonal antibody or the fragment or derivative thereof or the third aptamer specifically binds an epitope of a third antigen different from the epitope of the first and second antigen, which at least in a part of the mammals has a structure after passage through the intestine which the corresponds to the native structure or the structure, against which a mammal produces antibodies after infection or immunization with the acid-resistant microorganism or an extract or lysate thereof or a protein thereof or a fragment thereof or a synthetic peptide, wherein the parts of the mammals according to (aa), according to (ab) and can overlap according to (ac) and make up the total essentially the total number of infected mammals, and detects the formation of at least one antigen-antibody complex / antigen-aptamer complex according to (aa), (ab) or (ac).

With regard to the definitions and preferred embodiments of this method, reference is made to the embodiments explained above. The explanations for the combination of different types of detection reagents when using at least two detection reagents apply here correspondingly to three detection reagents. In this way, a first monoclonal antibody can be combined with a second fragment and a third aptamer in the test. Of course, this combination does not mean that two more aptamers are used in the test. The term "third" thus refers to the third detection reagent, regardless of its possible structure. The same applies to the terms "first" and "second".

The epitope of the first antigen is preferably an epitope of a urease, preferably the β-urease of <i>H. pylori,</i> the epitope of the second antigen is an epitope of a heat shock protein, preferably of Hsp60, preferably from <i>H. pylori,</i> and the epitope of the third antigen is an epitope of an alkyl hydroperoxide reductase, preferably the 26kDa protein of <i>H. pytori.</i> In other preferred embodiments, the epitopes of the second and third antigens are epitopes of the 20kDa protein (3-dehydro-quinase type II), the 16.9kDa protein (neutrophil activating protein) or the 33.8 kDa protein (fructose Bisphosphate aldolase), in combination with the urease as the first and possibly the Hsp60 or the 26kDa protein as the second or third antigen. In a further preferred embodiment, the epitopes of the second antigen are epitopes of Hsp60 and the epitopes of the third antigen are epitopes of the 20kDa protein (3-dehydro-quinase type II), the 16.9kDa protein (neutrophil-activating protein) or the 33rd , 8 kDa protein (fructose bisphosphate aldolase). Further embodiments with detection of combinations of the abovementioned proteins or their epitopes, even without urease, are possible.

When screening stool samples with <i>H. pylori</i> infected patients to different <i>H. pylori</i>-specific proteins (urease, HSP60, alkyl hydroperoxide reductase) by means of ELISA using monoclonal antibodies, it was found that the detectability of different antigens in stool samples from different patients is very inhomogeneous. It was also found, surprisingly, that combinations of monoclonal antibodies, which partially recognize overlapping epitopes, were very suitable for the detection of these antigens by means of sandwich ELISA (see Table 3).

According to the invention, the monoclonal antibodies, fragments or derivatives thereof or the aptamers can recognize and specifically bind linear or conformation epitopes. In a further preferred embodiment, at least one of the monoclonal antibodies or one of the fragments, derivatives or aptamers binds a conformation epitope.

In a particularly preferred embodiment, all monoclonal antibodies etc. bind conformation epitopes.

In epitope mapping, 5 of the murine MAK (monoclonal antibodies) that are particularly suitable in the stool ELISA were examined (Table 3). Surprisingly, no linear epitope could be determined for three of these antibodies. It was therefore assumed that these mAbs recognize conformational epitopes. This statement is confirmed for the anti-β-urease antibodies by the immunoblot results, which show that the denatured urease is no longer recognized or only very weakly (see Table 2).

In another embodiment, the invention relates to a method for detecting an infection with <i>Helicobacter pylori</i> in a mammalian stool, wherein (a) a stool sample is incubated with at least two different monoclonal antibodies, fragments, derivatives thereof or aptamers under conditions that allow antigen-antibody / antigen-aptamer complex formation, wherein (aa) the first monoclonal antibody , the fragment, derivative thereof or the first aptamer specifically binds β-urease or a fragment thereof; (ab) the second monoclonal antibody, the fragment, derivative thereof or the second aptamer specifically binds the 26kDa antigen or a fragment thereof or specifically binds Hsp60 or a fragment thereof; and (b) detects the formation of at least one antigen-antibody complex / antigen-aptamer complex according to (aa) or (ab).

The invention further relates to a method for detecting an infection with <i>Helicobacter pylori</i> in the stool of a mammal, (a) incubating a stool sample with three different monoclonal antibodies, fragments, derivatives thereof or aptamers under conditions which allow antigen-antibody / antigen-aptamer complex formation, where (aa) the first monoclonal antibody, the fragment, derivative thereof or the first aptamer specifically binds β-urease or a fragment thereof; (ab) the second monoclonal antibody, the fragment, derivative thereof or the second aptamer specifically binds Hsp60 or a fragment thereof; and (ac) the third monoclonal antibody, the fragment, derivative thereof or the third aptamer specifically binds the 26kDa antigen or a fragment thereof; and (b) detects the formation of at least one antigen-antibody / antigen-aptamer complex according to (aa), (ab) or (ac).

With regard to the definitions used and applicable preferred embodiments, reference is made to the previous explanations.

The proteins mentioned in the two above embodiments are self-evident <i>H. pylori</i>-Proteins.

In another preferred embodiment, the Hsp60-specific antibody is that of the hybridoma HP16m / 2A5-E6-E5 deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) on June 23, 1998 according to the statutes of the Budapest Treaty under the deposit number DSM ACC2356 produced antibodies.

In a further preferred embodiment, the 26kDa antigen-specific antibody is that of the hybridoma HP15m / 3E8-D9 deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) on June 23, 1998 according to the statutes of the Budapest Treaty under the deposit number DSM ACC2355 -D6 produced antibodies.

In another preferred embodiment, the β-urease-specific antibody is that of one of the hybridomas HP8m deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) on June 23, 1998 according to the statutes of the Budapest Treaty under the deposit numbers DSM ACC2360 or DSM ACC2362 / 4H5-D4-C9 or HP9.1m / 3C2-F8-E2 produced antibodies. The β-urease-specific antibody HP8m / 1H5-G2-B4 described in the examples / figures is produced by a daughter clone of the deposited hybridoma HP8m / 4H5-D4-C9. The two antibodies produced by the mother and daughter clones are encoded by identical DNA sequences and have the same properties.

In a particularly preferred embodiment, the heavy chain of the antibody binding an Hsp60 epitope has at least one of the following CDRs, preferably CDR3 and more preferably all three of the following CDRs:<dl id="dl0001"><dt>CDR1:</dt><dd>GFSLSRYSVH</dd><dt>CDR2:</dt><dd>MIWGGGSTDYNSGLKS</dd><dt>CDR3:</dt><dd>NMGGRYPDYFDY</dd></dl>

In a further particularly preferred embodiment, the DNA sequence coding the heavy chain of the antibody binding an Hsp60 epitope has at least one of the following CDRs, preferably the CDR3 and more preferably all three of the following CDRs:<dl id="dl0002"><dt>CDR1:</dt><dd>GG GTTCTCATTA TCCAGATATA GTGTACAC</dd><dt>CDR2:</dt><dd>ATGATATGGG GTGGTGGAAG CACAGACTAT AATTCAGGTC TCAAATCC</dd><dt>CDR3:</dt><dd>AATATG GGGGGTAGGT ACCCGGACTA CTTTGACTAC</dd></dl>

In another particularly preferred embodiment, the light chain of the antibody binding an Hsp60 epitope has at least one of the following CDRs, preferably the CDR3 and more preferably all three of the following CDRs<dl id="dl0003"><dt>CDR1:</dt><dd>RASKSVSTSGYSYIH</dd><dt>CDR2:</dt><dd>LASNLES</dd><dt>CDR3:</dt><dd>QHSRELPLT</dd></dl>

Furthermore, in a particularly preferred embodiment, the DNA sequence encoding the light chain of this antibody has at least one of the following CDRs, preferably CDR3 and more preferably all three of the following CDRs:<dl id="dl0004"><dt>CDR1:</dt><dd>A GGGCCAGCAA GAGTGTCAGT ACATCTGGCT ATAGTTACAT ACAC</dd><dt>CDR2:</dt><dd>C TTGCATCCAA CCTAGAATCT</dd><dt>CDR3:</dt><dd>CAGC ACAGTAGGGA GCTTCCGCTC ACG.</dd></dl>

In a further particularly preferred embodiment of the method according to the invention, the heavy chain of the antibody binding a 26kDa protein has at least one of the following CDRs, preferably CDR3 and more preferably all three of the following CDRs:<dl id="dl0005"><dt>CDR1:</dt><dd>GFTFNSYAMY</dd><dt>CDR2:</dt><dd>RIRSKSDNYATYYANSVKD</dd><dt>CDR3:</dt><dd>DHDKFPFYYALDY</dd></dl>

In another particularly preferred embodiment, the DNA sequence encoding the heavy chain of the antibody binding an epitope of the 26kDa protein has at least one of the following CDRs, preferably the CDR3 and more preferably all three of the following CDRs:<dl id="dl0006"><dt>CDR1:</dt><dd>GG TTTCACCTTC AATTCCTATG CCATGTAC</dd><dt>CDR2:</dt><dd>CGCATAAGAA GTAAAAGTGA TAATTATGCA ACATATTATG CCAATTCAGT GAAAGAC</dd><dt>CDR3:</dt><dd>GATCATG ATAAGTTTCC TTTTTACTAT GCTCTGGACT AC</dd></dl>

In another particularly preferred embodiment of the method according to the invention, an antibody, fragment or derivative thereof is used whose light chain of the antibody binding a 26kDa protein has at least one of the following CDRs, preferably the CDR3 and more preferably all three of the following CDRs:<dl id="dl0007"><dt>CDR1:</dt><dd>TASSSVSSSYLH</dd><dt>CDR2:</dt><dd>STSNLAS</dd><dt>CDR3:</dt><dd>HQYHRSPPT</dd></dl>

Furthermore, in another particularly preferred embodiment, the DNA sequence encoding the light chain of this antibody has at least one of the following CDRs, preferably CDR3 and more preferably all three of the following CDRs:<dl id="dl0008" compact="compact"><dt>CDR1:</dt><dd>A CTGCCAGCTC AAGTGTGAGT TCCAGTTACT TGCAC</dd><dt>CDR2:</dt><dd>AGCACTTCCA ACCTGGCTTC T</dd><dt>CDR3:</dt><dd>CAC CAGTATCATC GTTCCCCACC GACG</dd></dl>

In another particularly preferred embodiment of the method according to the invention, the heavy chain of the antibody binding an epitope of the β-urease has at least one of the following CDRs, preferably CDR3 and more preferably all three of the following CDRs:<dl id="dl0009"><dt>CDR1:</dt><dd>GFTFSSHFMS</dd><dt>CDR2:</dt><dd>SISSGGDSFYPDSLKG</dd><dt>CDR3:</dt><dd>DYSWYALDY</dd></dl>or:<dl id="dl0010"><dt>CDR1:</dt><dd>GYAFSTSWMN</dd><dt>CDR2:</dt><dd>RIYPGDGDTNYNGKFKG</dd><dt>CDR3:</dt><dd>EDAYYSNPYSLDY</dd></dl>

In a further particularly preferred embodiment, the DNA sequence coding the epitope of the β-urease-binding antibody has at least one of the following CDRs, preferably CDR3 and more preferably all three of the following CDRs:<dl id="dl0011"><dt>CDR1:</dt><dd>GG CTACGCATTC AGTACCTCCT GGATGAAC</dd><dt>CDR2:</dt><dd>CGGATTTATC CTGGAGATGG AGATACTAAC TACAATGGGA AGTTCAAGGG C</dd><dt>CDR3:</dt><dd>GAG GATGCCTATT ATAGTAACCC CTATAGTTTG GACTAC</dd></dl>or:<dl id="dl0012"><dt>CDR1:</dt><dd>GG ATTCACTTTC AGTAGCCATT TCATGTCT</dd><dt>CDR2:</dt><dd>TCCATTAGTA GTGGTGGTGA CAGTTTCTAT CCAGACAGTC TGAAGGGC</dd><dt>CDR3:</dt><dd>GACTAC TCTTGGTATG CTTTGGACTA C</dd></dl>

In another particularly preferred embodiment of the method according to the invention, the light chain of the antibody binding an epitope of the β-urease has at least one of the following CDRs, preferably CDR3 and more preferably all three of the following CDRs:<dl id="dl0013"><dt>CDR1:</dt><dd>RASQSIGTRIH</dd><dt>CDR2:</dt><dd>YGSESIS</dd><dt>CDR3:</dt><dd>QQSNTWPLT</dd></dl>or:<dl id="dl0014"><dt>CDR1:</dt><dd>HASQNINVWLS</dd><dt>CDR2:</dt><dd>KASNLHT</dd><dt>CDR3:</dt><dd>QQGRSYPLT</dd></dl>

Furthermore, the DNA sequence encoding the light chain of this antibody preferably has the following CDRs:<dl id="dl0015"><dt>CDR1:</dt><dd>A GGGCCAGTCA GAGCATTGGC ACAAGAATAC AC</dd><dt>CDR2:</dt><dd>TAT GGTTCTGAGT CTATCTCT</dd><dt>CDR3:</dt><dd>CAACAA AGTAATACCT GGCCGCTCAC G</dd></dl>or:<dl id="dl0016"><dt>CDR1:</dt><dd>C ATGCCAGTCA GAACATTAAT GTTTGGTTAA GC</dd><dt>CDR2:</dt><dd>AAG GCTTCCAACT TGCACACA</dd><dt>CDR3:</dt><dd>CAACAG GGTCGAAGTT ATCCTCTCAC G</dd></dl>

It is furthermore particularly preferred that the heavy and light chains which have the CDRs indicated above occur together in an antibody, fragment or derivative thereof, the Hsp60, the 26kDa protein or the β-urease, or a fragment thereof, preferably from <i>H. pylori</i> binds specifically. However, the invention also encompasses embodiments in which these heavy or light chains are combined with other light or heavy chains, whereby the binding properties can essentially be maintained or improved. Corresponding methods are known in the prior art. Particularly preferred antibodies in the variable regions of the light and heavy chains have the amino acid sequences shown in FIGS. 1 and 2, FIGS. 3 and 4, FIGS. 5 and 6 or FIGS. 7 and 8, or the regions are represented by those shown there DNA sequences encoded.

In a preferred embodiment, the following steps are carried out with the stool sample before incubation with the antibodies: The stool sample is resuspended 1: 3 to 1:25, preferably about 1:10 in a resuspending buffer and then mixed on a vortex mixer. An example of a resuspend buffer is 150 mM PBS, 0.1% SDS.

In a further preferred embodiment, the formation of the at least one antigen-antibody complex / ahtigen-aptamer complex is detected in step (b) by means of an immunological method.

In another preferred embodiment, the formation of the at least one antigen-antibody complex / antigen-aptamer complex is detected in step (b) by means of ELISA, RIA, Western blot or an immunochromatographic method.

Such methods are known per se in the prior art; see. Harlow and Lane, loc. Cit

In a particularly preferred embodiment of the method according to the invention, the same antibody or its fragment or derivative or the aptamer is used in the immunological method, in particular in the RIA or in the ELISA, for binding to the solid phase and for detecting the epitope. While the catcher antibody / catcher aptamer can be bound in unmodified form to the solid phase, for example a microtiter plate, the antibody used for detection, the fragment or derivative thereof or the aptamer is optionally provided with a label. On the other hand, this antibody, the fragment or derivative thereof or this aptamer can likewise not be labeled and thus the epitope of the microorganism, preferably the bacterial epitope, can also be detected via a third labeled antibody, the fragment or derivative thereof a third labeled aptamer, this antibody , the fragment or derivative thereof or this aptamer can be a species-specific or Ig-class-specific antibody or a corresponding aptamer. Labeling of antibodies, for example with radioactive or fluorescent markers, is known in the prior art; see. Harlow and Lane loc. The same applies to aptamers. The embodiment described above is particularly favorable for the detection of urease, preferably β-urease, which may even after the intestinal passage as a dimer, possibly in a multimeric version. Of course, combinations of antibodies, fragments, derivatives and aptamers can also be used in this embodiment, for example combinations of antibodies, etc., which bind to different epitopes of the same antigen.

In another preferred embodiment of the method according to the invention, the monoclonal antibody is a mouse antibody.

Furthermore, in a preferred embodiment, the antibodies, fragments or derivatives thereof or the aptamers are fixed to a support.

The fixation of the antibodies, fragments or derivatives thereof or the aptamers to a carrier is particularly advantageous for carrying out routine checks. The combination of antibody carrier / aptamer carrier can also be packaged well as a test kit or in kit form.

In a particularly preferred embodiment, the carrier material is a porous carrier material.

In a further particularly preferred embodiment, the carrier material is a test strip.

In addition, in a preferred embodiment, the carrier material consists of cellulose or a cellulose derivative.

The mammal, whose stool can be examined with the method according to the invention, can be an animal, for example a pet such as a cat or a dog, a farm animal, for example a pig or another animal such as a mouse, a tiger or a ferret.

In a preferred embodiment, the mammal is a human.

Furthermore, the invention relates to a monoclonal antibody, a fragment or derivative thereof, which has a V region, which has a combination of the CDRs shown above or which is produced by one of the hybridomas shown above.

A monoclonal antibody, fragment or derivative thereof is preferred which has at least one of the V regions shown in FIGS. 1 to 8. This antibody preferably has two of the V regions shown in FIGS. 1 and 2, 3 and 4, 5 and 6 or FIGS. 7 and 8. It is also preferred that these V regions are encoded by the DNA sequences shown in FIGS. 1 to 8.

In a particularly preferred embodiment of the invention, the monoclonal antibody, the fragment or derivative thereof is a mouse antibody or a fragment or derivative thereof or a chimeric, preferably a humanized antibody or a fragment or derivative thereof. The derivative can also be a fusion protein. It is further preferred that the antibody is labeled, for example with a colloid, with a radioactive, fluorescent, phosphorescent or chemiluminescent label. The production of chimerized humanized and human antibodies and the other derivatives is well known in the art (e.g. Vaughan et al., 1998; Orlandi et al., 1989, Harlow and Lane, op. Cit.).

The invention also relates to an aptamer that specifically binds the same epitope as the monoclonal antibody, fragment or derivative thereof. Such aptamers can be prepared using methods known in the art.

The invention further relates to an epitope which is specifically bound by one of the monoclonal antibodies, fragment or derivative thereof or aptamer described above.

In addition, the invention relates to further antibodies, derivatives or fragments thereof which specifically bind the epitope according to the invention. These antibodies can be, for example, monoclonal antibodies, which can be produced using the epitope as a hapten / component of an antigen by conventional methods.

The present invention also relates to a diagnostic composition containing at least two monoclonal antibodies, fragments or derivatives thereof or aptamers as defined above, optionally fixed to a carrier material.

Furthermore, the present invention relates to a test device for the detection of at least one epitope as defined above, comprising (a) at least two monoclonal antibodies, fragments or derivatives thereof or aptamers as defined above, fixed to a support material; (b) a device for the preparation and analysis of stool samples and optionally (c) a mixture of at least two monoclonal antibodies, fragments or derivatives thereof or aptamers.

The invention further relates to a test device comprising (a) at least two monoclonal antibodies, fragments or derivatives thereof or aptamers as defined above, the antibodies, fragments or derivatives thereof or aptamers being conjugated to colloidal gold, latex particles or other coloring particles, the thereof Size is typically in the range between 5nm and 100nm, preferably between 20nm and 60nm; (b) a device for the preparation and analysis of stool samples and optionally (c) a mixture of at least two monoclonal antibodies, fragments or derivatives thereof or aptamers.

In addition, the present invention relates to a kit containing (a) at least two monoclonal antibodies, fragments or derivatives thereof or aptamers as defined above, optionally fixed to a support material; optionally (b) a device for the preparation and analysis of stool samples and optionally (c) a mixture of at least two monoclonal antibodies, fragments or derivatives thereof or aptamers.

The invention also relates to a composition comprising at least one of the above-described antibodies or one of the above-described fragments, derivatives or aptamers, optionally in combination with a pharmaceutically acceptable carrier and / or diluent. The composition is preferably a drug.

Examples of suitable pharmaceutically acceptable carriers are known to those skilled in the art and include phosphate buffered saline, water, emulsions such as oil / water emulsions, various types of detergents, sterile solutions, etc. Drugs comprising such carriers can be made by known conventional methods be formulated. These drugs can be administered to an individual in a suitable dose, e.g. in a range from 1µg to 100 mg per day and patient. Administration can take place in various ways, for example intravenously, introperitoneally, subcutaneously, intramuscularly, locally or intradermally. The treating doctor determines the type of dosage according to the clinical factors. It is known to the person skilled in the art that the type of dosage depends on various factors, such as, for example the size, body surface area, age, sex or general health of the patient, but also the specific agent to be administered, the duration and type of administration and other medications which may be administered in parallel.

Finally, the invention relates to a package containing the diagnostic composition according to the invention, the test device according to the invention or the kit according to the invention. The components of the diagnostic composition according to the invention, the test device according to the invention and / or the kit according to the invention can be packaged in containers, such as vials or tubes, optionally in buffers and / or solutions. Under certain circumstances, one or more of the components can be packaged in one and the same container.

The figures show:<ul id="ul0003" list-style="none"><li>1: DNA sequence coding for a light chain of a monoclonal antibody specific for HSP60 (DMS ACC2356). The DMSZ deposit number refers here, as elsewhere in the application, to the hybridoma producing the monoclonal antibody. The encoded amino acid sequence is also shown.</li><li>2: DNA sequence coding for a heavy chain of a monoclonal antibody specific for HSP60 (DMS ACC2356). The encoded amino acid sequence is also shown.</li><li>3: DNA sequence coding for a light chain of a monoclonal antibody (DMS ACC2355) specific for 26kDa protein. The encoded amino acid sequence is also shown.</li><li>4: DNA sequence coding for a heavy chain of a monoclonal antibody (DMS ACC2355) specific for 26kDa protein. The encoded amino acid sequence is also shown.</li><li>Fig. 5: DNA sequence coding for a light chain of a first urease-specific monoclonal antibody (DMS ACC2360). The encoded amino acid sequence is also shown.</li><li>Fig. 6: DNA sequence coding for a heavy chain of a first urease-specific monoclonal antibody (DMS ACC2360). The encoded amino acid sequence is also shown.</li><li>Fig. 7: DNA sequence coding for a light chain of a second urease-specific monoclonal antibody (DMS ACC2362). The encoded amino acid sequence is also shown.</li><li>Figure 8: DNA sequence coding for a heavy chain of a second urease-specific monoclonal DMS ACC2362). The encoded amino acid sequence is also shown.</li></ul>

The examples illustrate the invention.

Example 1: Isolation of H. <i>pylori</i>-Antigen

Cultivation of <i>H. pylori</i>

<i>H. pylori</i> (Strain NCTC 11637) was streaked in Petri dishes on Wilkins-Chalcese agar with the addition of 10% horse blood and amphotericin B, vancomycin and cefsoludine (Sigma Chemicals) and incubated for 3-4 days in a microaerophilic atmosphere (Anaerocult GasPAk, Merck) at 37 ° C . The contents of 2 dishes were suspended in 350 ml BHIB medium with the addition of antibiotics as above in a 1 liter bottle (Schott), the medium for 10 minutes with a gas mixture of 10% CO₂, 5% O₂, 85% N₂ fumigated and the bottle closed. The culture was shaken on a rotary shaker at 37 ° C for 2 days, after 24 hours the bottle was opened. The contents of the bottle were then placed sterile in a 10l bottle, filled with 4.7l BHIB medium, the medium for 10min with a gas mixture of 10% CO₂, 5% O₂, 85% N₂ fumigated and the bottle closed. Again it was shaken on a rotary shaker at 37 ° C for 2 days, after 24 hours the bottle was opened. The entire volume is then centrifuged at 11000 g for 10 min, the supernatant decanted and the bacterial pellet weighed. For storage, the pellet was resuspended in a physiological saline solution with the addition of 15% glycerol in a ratio of 2: 1 (w / v) and frozen at -80 ° C. In order to verify the identity of the cultivated bacteria, a visual inspection of the bacteria and tests for urease, oxidase and catalase activity were carried out. The freedom from contamination was demonstrated by culture of some suspension taken before freezing at 37 ° C in air for 48 hours.

Example 2: General procedure for selecting suitable antigens for detection on microorganisms

After rearing, a lysate of the microorganism is produced. The lysate is separated by means of gel filtration. Then those proteins are isolated which are present in large quantities in the lysate. These proteins are identified by means of protein sequencing and comparison with sequences from suitable databases. In this way, characteristic or specific proteins for the respective microorganism can be determined. A suitable purification scheme is then developed for these selected antigens.

Example 3: Preparation of <i>H. pylori</i> Urease, alkyl hydroperoxide reductase and HSP60

The preparation of the three <i>H. pylori</i>Antigenic urease, alkyl hydroperoxide reductase and HSP60 corresponds to a modification of a known method (Eschweiler et al., 1993). Frozen bacterial pellet was mixed in a ratio of 1: 2 (w / v) with 10% butanol, shaken on an overhead mixer until thawed completely and continued for about 15 minutes. After sedimentation for 20 min at 20,000 g, 4 ° C., the supernatant was decanted and the pellet extracted again with 10% butanol for 30 min as above. After renewed sedimentation analogously to the above, the supernatants were combined and the pellet was frozen for further use.

Isolation of urease and alkyl hydroperoxide reductase (as oligomers)

The combined clear supernatants were filtered through 0.8 μm filters and immediately transferred to Source Q 16/10 column (with 20 mM HEPES, pH 7.0, equilibrated). Fractions eluted with 20mM HEPES, pH7.0, 2M NaCl were immediately checked for urease activity (4ml 1M urea, 1ml 0.2M HEPES, pH7.0 and 80µl of a solution of 5g / l phenol red were mixed and with aqua distilled to 20ml. 100 μl of this solution were then placed in a test tube with the same amount of a fraction. The color change from yellow to red indicated the presence of urease). Urease-positive fractions were placed on a chromatography column filled with Sephacryl S200 50/100 (Pharmacia) and separated (buffer: 50mM Tris, 100mM NaCl, 0.05% NaN₃, pH 7.3). The protein concentration in the outlet was monitored by measuring the optical density at 280nm. The fractions of the first protein peak contained oligomeric urease (molecular weight approx. 550-600 kDa), followed by a second peak, which was attributable to the alkyl hydroperoxide reductase (also as an oligomer). Overlapping fractions were discarded, pure fractions (checking by electrophoresis on polyacrylamide gel) were combined and concentrated in vacuo (Speed-Vac) or by ultrafiltration. Alkyl hydroperoxide reductase fractions, which contained even larger amounts of urease, were subsequently purified using a β-urease affinity column (antibody Hp8m / 1H5-G2B4, Connex, on CNBr-activated Sepharose 4B matrix, Amersham Pharmacia, coupling according to the manufacturer) .

Isolation of HSP60 (as an oligomer)

The HSP60 antigen could not be obtained in sufficient quantity using the butanol extraction method described above. That is why the bacteria were disrupted using ultrasound. Fresh bacterial pellet or the pellet obtained after extraction with 10% butanol was thawed, resuspended 1:10 in 150mM PBS, pH 7.5 and in a Falcon tube on ice water in a Sonikator (Sonifire) at step 25-30% (Level 7) Ultrasound-treated (4x90s with a 90s break each). After centrifugation at 20,000 g for 40 min, the supernatant was applied to a chromatography column filled with Sephacryl S200 50/100 (Pharmacia) and separated (buffer: 50mM Tris, 100mM NaCl, 0.05% NaN₃, pH 7.3). HSP60 co-eluted with urease, therefore urease-positive (see above) fractions were pooled and cleaned on a β-urease affinity column (see above).

Example 4: Production of polyclonal and monoclonal antibodies (PAH; mAb) against <i>H. pylori</i>-specific antigens

Polyclonal antisera were produced by pab Productions (Hebertshausen) using standard procedures against antigens purified as above. Monoclonal antibodies are produced by methods known to the person skilled in the art (Harlow &amp; Lane, 1988; Peters &amp; Baumgarten, 1990).

immunization

The purified proteins urease, HSP60 and alkyl hydroperoxide reductase as well as recombinant α- and β-urease (Austral Biologics) were used as immunogen. 4-6 mice (BALB / cx C57 Black, F1 generation, 8-12 weeks old) were immunized (priming) per immunogen and boosted 3-4x at intervals of approximately four weeks. 200-300µl antigen solutions (25-50µg antigen / animal) in a ratio of 1: 1 with complete (priming) or Incomplete (Boost) Freund's adjuvant (Difco) emulsified and 100µl / mouse injected intraperitoneally. Before the fusion, blood was taken retroorbitally from the mice and the antibody titer was determined from the antiserum obtained therefrom by means of an enzyme-linked immunosorbent assay (ELISA, see below).

Fusion and fusion screening

2-3 days after the last boost, the spleen cells of the immunized mice were fused with the myeloma cells P3x63Ag8.653 (ATCC CRL-1580; Kearney et al., 1979) in a 5: 1 ratio with polyethylene glycol 4000. The fused cells were suspended in cloning medium (RPMI 1640 medium + 20% FCS + 200U IL-6 / ml) with hypoxanthine-aminopterin-thymidine supplement (100x concentrate, Sigma) and with a cell density of 2-6x10⁴ Cells / well in culture on 96-well microtiter plates (37 ° C., 6% CO₂ and 95% relative humidity). After about 10 days, the culture supernatant was screened in the ELISA (see below) for the presence of antibodies of the desired specificity.

Establishment and cultivation of hybridomas

Clones that produce antigen-specific antibodies were cloned twice according to the principle of limiting dilution (Coller &amp; Coller, 1983) in cloning medium with hypoxanthine-thymidine supplement (100x concentrate, Sigma). The monoclonal end clone was then adapted to the flat bottle culture in RPMI 1640 medium with 10% FCS and for the cryopreservation of 5-10 aliquots with 2-5x10 each⁶ Cells and the production of antibody-containing culture supernatants expanded.

ELISA

The direct ELISA was chosen for the titer determination and the test of the culture supernatants for antigen-specific antibodies. The ELISA plates (MaxiSorb; Nunc) were coated above sea level. at 5 ° C with an antigen suspension (2-6 µg antigen / ml carbonate buffer, 0.1M, pH9.5). To block the binding sites that were still free, 200 .mu.l of PBS with 2% skim milk powder (weight: volume) per well were pipetted and incubated for 1 hour at room temperature. The incubation of 100µl culture supernatant was carried out for 1-2 hours at room temperature. The bound antibodies are detected by adding a secondary antibody (rabbit anti-mouse IgG-POD, DAKO) conjugated with horseradish peroxidase (POD). The next step in the POD is to convert the colorless substrate tetramethylbenzidine (TMB, Sigma) into a blue product. After 5 to 10 minutes, or as soon as the negative control also showed a slight blue color, the reaction was stopped by adding 1N sulfuric acid (100 μl / well). The strength of the color reaction was measured in the ELISA reader (MWG spectral). The measurement is carried out at 455 nm against the reference wavelength of 620 nm. Between the individual work steps, the ELISA plate was washed 2-3 times with 250 μl PBS with 0.025% Tween 20 (vol.:vol.).

Results

A total of 24 mAbs against β-urease, 6 mAbs against α-urease, 8 mAbs against 26kDa protein and 10 mAbs against HSP60 were generated. From this antibody repertoire, those mAbs with the lowest detection limits for the respective antigens were selected.

Example 5: Purification of MAK from hybridoma culture supernatants and preparation of conjugates

The purification of mAb from hybridoma culture supernatants is carried out using protein-G affinity chromatography (modified from: Pharmacia Biotech, 1994). The culture supernatants were filtered (0.8 µm) and passed directly over the Protein G matrix. Washing was carried out with Tris / HCl until the signal at the detector had returned to the background. Elution was carried out with 0.1M glycine / HCl, pH 3.0, and the protein concentration was detected in the eluate via optical density at 280 nm. All fractions in the range of the single elution signal can be used.

A possible contamination with bovine IgG from the serum additive can be classified as uncritical, since the detection antibodies found do not cross-react with bovine Ig. Monoclonal antibodies were coupled to biotin and POD using literature methods (Harlow &amp; Lane, 1988).

Example 6: Characterization of the monoclonal antibodies

Isotyping

The murine mAbs were isotyped using the IsoStrip isotyping kit from Boehringer Mannheim (Mannheim).

Immunoblot

For the immunoblot analysis, 30-50µg purified antigen or 300µg were used per gel <i>H. pylori</i>-Lysate boiled in reducing sample buffer (Laemmli, 1970) and applied to a 12% preparative SDS-poplyacrylamide mini gel (8.6x7.7x0.1cm, Biometra). The electrophoretic separation was carried out at 25-30 mA / gel.

The proteins (antigens) separated in the gel were then immobilized on a nitrocellulose membrane using the semidry blot method.

The membrane was blocked for 30 min at room temperature with 2% skim milk powder in PBS and washed three times for 5 min with TBS / Tween 20 (0.2%). For the following incubation step, the membrane was clamped into the Accutran cross-blot screening unit (Schleicher and Schuell) using a grid plate with 34 cross channels. 250 μl of TBS / Tween 20 were placed in each of the transverse channels and 250 μl of the hybridoma culture supernatants to be tested were added. The incubation was carried out for 2 hours at room temperature with agitation.

After washing three times (see above), the membrane was incubated for 1 hour with the POD-conjugated secondary antibody (rabbit-anti-mouse IgG-POD, DAKO). The membrane was washed three times and the immune complex was visualized by adding the 3,3-diaminobenzidine substrate solution (DAB, Sigma).

Characterization of antibody-antigen interactions using epitope mapping

In order to be able to identify the antibody binding site on a protein, the <i>H. pylori</i>-Proteins α- and β-urease, HSP60 and 26kDa protein overlapping peptide sequences (two amino acids shifted against each other) with 12 amino acid residues synthesized on a solid phase and covalently coupled to a cellulose acetate membrane (Jerini GmbH, Berlin). These membranes were incubated with hybridoma culture supernatant and the bound antibodies were then blotted on nitrocellulose membranes using the semidry blot method. The membrane was blocked and the immobilized antibodies were detected as described under 5.2 with a POD-labeled secondary antibody (rabbit anti mouse IgG-POD, DAKO).

Determination of the detection limit for <i>H. pylori</i>-Antigens in ELISA

Between the individual work steps, the ELISA plate was washed 2-3 times with 300 µl PBS with 0.025% Tween (wash buffer) (v: v). The ELISA plates (MaxiSorb; Nunc) were coated for 1 hour at 37 ° C. with 100 μl of a solution of a polyclonal rabbit anti<i>H</i>. <i>pylori</i>-Antigen antibody (pAK; approx. 10µg antibody / ml carbonate buffer, 0.1M, pH9.5). To block the binding sites that were still free, 200 μl of 150 mM PBS with 2% skim milk powder (weight: volume) per well were pipetted and incubated for 30 minutes at room temperature. 50ng / ml cleaned<i>H. pylori</i>-Antigens dissolved in 150mM PBS with the addition of 0.1% skimmed milk powder were diluted in 1: 2 steps. Buffer served as a negative control. Then 100 μl of 1:10 diluted culture supernatant of the mac to be examined was added against the same antigen and incubated for 30-60 min at RT. The bound antibody was detected as described under 3.4. The lowest concentration at which an absorbance greater than or equal to twice the control was detected was accepted as the detection limit.<tables id="tabl0001" num="0001"><table frame="all"><title><b>Table 1:</b> Results for the characterization of the MAK</title><tgroup cols="6" colsep="1" rowsep="1"><colspec colnum="1" colname="col1" colwidth="40mm" colsep="1" /><colspec colnum="2" colname="col2" colwidth="41mm" colsep="1" /><colspec colnum="3" colname="col3" colwidth="17mm" colsep="1" /><colspec colnum="4" colname="col4" colwidth="15mm" colsep="1" /><colspec colnum="5" colname="col5" colwidth="25mm" colsep="1" /><colspec colnum="6" colname="col6" colwidth="30mm" colsep="1" /><thead><row><entry namest="col1" nameend="col1" align="left" valign="top"><b>Fusion / clone</b></entry><entry namest="col2" nameend="col2" align="left" valign="top"><b>Ag specificity</b></entry><entry namest="col3" nameend="col3" align="left" valign="top"><b>Isotype</b></entry><entry namest="col4" nameend="col4" align="left" valign="top"><b>IB (Ag)</b></entry><entry namest="col5" nameend="col5" align="left" valign="top"><b>IB (HP lysate)</b></entry><entry namest="col6" nameend="col6" align="left" valign="top"><b>NWG (ng Ag / ml)</b></entry></row></thead><tbody><row rowsep="0"><entry namest="col1" nameend="col1" align="left" valign="top">HP8m / 4H5-D4-C9</entry><entry namest="col2" nameend="col2" align="left" valign="top">β-urease</entry><entry namest="col3" nameend="col3" align="left" valign="top">IgG1, κ</entry><entry namest="col4" nameend="col4" align="left" valign="top">+/-</entry><entry namest="col5" nameend="col5" align="left" valign="top">+/-</entry><entry namest="col6" nameend="col6" align="left" valign="top">0,6</entry></row><row rowsep="0"><entry namest="col1" nameend="col1" align="left" valign="top">HP9.1m / 2D1-F6-G1</entry><entry namest="col2" nameend="col2" align="left" valign="top" /><entry namest="col3" nameend="col3" align="left" valign="top">IgG1, κ</entry><entry namest="col4" nameend="col4" align="left" valign="top">-</entry><entry namest="col5" nameend="col5" align="left" valign="top">+/-</entry><entry namest="col6" nameend="col6" align="left" valign="top">0,3</entry></row><row rowsep="0"><entry namest="col1" nameend="col1" align="left" valign="top">HP9.1m / 3C2-F8-E2</entry><entry namest="col2" nameend="col2" align="left" valign="top" /><entry namest="col3" nameend="col3" align="left" valign="top">IgG1, κ</entry><entry namest="col4" nameend="col4" align="left" valign="top">+/-</entry><entry namest="col5" nameend="col5" align="left" valign="top">+/-</entry><entry namest="col6" nameend="col6" align="left" valign="top">1,2</entry></row><row><entry namest="col1" nameend="col1" align="left" valign="top">HP16.1m / 3G1-H2-D7</entry><entry namest="col2" nameend="col2" align="left" valign="top" /><entry namest="col3" nameend="col3" align="left" valign="top">IgG2a, κ</entry><entry namest="col4" nameend="col4" align="left" valign="top">+</entry><entry namest="col5" nameend="col5" align="left" valign="top">+</entry><entry namest="col6" nameend="col6" align="left" valign="top">0,6</entry></row><row rowsep="0"><entry namest="col1" nameend="col1" align="left" valign="top">HP15m / 3E8-D9-D6</entry><entry namest="col2" nameend="col2" morerows="1" rowsep="1" align="left" valign="top">Alkyl hydroperoxide reductase</entry><entry namest="col3" nameend="col3" align="left" valign="top">IgG1, κ</entry><entry namest="col4" nameend="col4" align="left" valign="top">+</entry><entry namest="col5" nameend="col5" align="left" valign="top">+</entry><entry namest="col6" nameend="col6" align="left" valign="top">0,6</entry></row><row><entry namest="col1" nameend="col1" align="left" valign="top">HP15m / 3F5-D5-B6</entry><entry namest="col3" nameend="col3" align="left" valign="top">IgG1, κ</entry><entry namest="col4" nameend="col4" align="left" valign="top">+</entry><entry namest="col5" nameend="col5" align="left" valign="top">+</entry><entry namest="col6" nameend="col6" align="left" valign="top">0,3</entry></row><row rowsep="0"><entry namest="col1" nameend="col1" align="left" valign="top">HP16m / 2A5-E6-E5</entry><entry namest="col2" nameend="col2" align="left" valign="top">HSP60</entry><entry namest="col3" nameend="col3" align="left" valign="top">IgG1, κ</entry><entry namest="col4" nameend="col4" align="left" valign="top">+</entry><entry namest="col5" nameend="col5" align="left" valign="top">+</entry><entry namest="col6" nameend="col6" align="left" valign="top">3</entry></row><row rowsep="0"><entry namest="col1" nameend="col1" align="left" valign="top">HP18.1m / 3F11-G11-H11</entry><entry namest="col2" nameend="col2" align="left" valign="top" /><entry namest="col3" nameend="col3" align="left" valign="top">IgG1, κ</entry><entry namest="col4" nameend="col4" align="left" valign="top">+</entry><entry namest="col5" nameend="col5" align="left" valign="top">+</entry><entry namest="col6" nameend="col6" align="left" valign="top">3</entry></row><row><entry namest="col1" nameend="col1" align="left" valign="top">HP18.1m / 4D9-B9-A2</entry><entry namest="col2" nameend="col2" align="left" valign="top" /><entry namest="col3" nameend="col3" align="left" valign="top">IgG1, κ</entry><entry namest="col4" nameend="col4" align="left" valign="top">+</entry><entry namest="col5" nameend="col5" align="left" valign="top">+</entry><entry namest="col6" nameend="col6" align="left" valign="top">3</entry></row></tbody></tgroup><tgroup cols="6" rowsep="0"><colspec colnum="1" colname="col1" colwidth="40mm" /><colspec colnum="2" colname="col2" colwidth="41mm" /><colspec colnum="3" colname="col3" colwidth="17mm" /><colspec colnum="4" colname="col4" colwidth="15mm" /><colspec colnum="5" colname="col5" colwidth="25mm" /><colspec colnum="6" colname="col6" colwidth="30mm" /><tbody><row><entry namest="col1" nameend="col6" align="justify" valign="top">Abbreviations: Ag Antigen; IB immunoblot; NWG detection limit</entry></row></tbody></tgroup></table></tables>

Results

Table 1 summarizes the results of isotyping, immunoblot analysis and detection limit determination for the MAK listed in Table 1.<tables id="tabl0002" num="0002"><table frame="all"><title><b>Table 2:</b> Results of epitope mapping against monoclonal antibodies <i>H</i>. <i>pylori</i>-Antigens</title><tgroup cols="4" colsep="1" rowsep="1"><colspec colnum="1" colname="col1" colwidth="44mm" colsep="1" /><colspec colnum="2" colname="col2" colwidth="33mm" colsep="1" /><colspec colnum="3" colname="col3" colwidth="37mm" colsep="1" /><colspec colnum="4" colname="col4" colwidth="33mm" colsep="1" /><thead><row><entry namest="col1" nameend="col1" align="left" valign="top"><b>Ag specificity</b></entry><entry namest="col2" nameend="col2" align="left" valign="top"><b>Fusion / clone</b></entry><entry namest="col3" nameend="col3" align="left" valign="top"><b>Epitope (AS position)</b></entry><entry namest="col4" nameend="col4" align="left" valign="top"><b>AS sequence</b></entry></row></thead><tbody><row rowsep="0"><entry namest="col1" nameend="col1" align="left" valign="top">β-urease</entry><entry namest="col2" nameend="col2" align="left" valign="top">HP8m / 1H5-G2-B4</entry><entry namest="col3" nameend="col3" align="left" valign="top">negative</entry><entry namest="col4" nameend="col4" align="left" valign="top" /></row><row rowsep="0"><entry namest="col1" nameend="col1" align="left" valign="top">(large subunit)</entry><entry namest="col2" nameend="col2" align="left" valign="top">HP8m / 4H5-D4-C9</entry><entry namest="col3" nameend="col3" align="left" valign="top">negative</entry><entry namest="col4" nameend="col4" align="left" valign="top" /></row><row rowsep="0"><entry namest="col1" nameend="col1" align="left" valign="top" /><entry namest="col2" nameend="col2" align="left" valign="top">HP9m / 2B12-G7-</entry><entry namest="col3" nameend="col3" align="left" valign="top">369-375</entry><entry namest="col4" nameend="col4" align="left" valign="top">VGEVITR</entry></row><row><entry namest="col1" nameend="col1" align="left" valign="top" /><entry namest="col2" nameend="col2" align="left" valign="top">B12</entry><entry namest="col3" nameend="col3" align="left" valign="top" /><entry namest="col4" nameend="col4" align="left" valign="top" /></row><row rowsep="0"><entry namest="col1" nameend="col1" align="left" valign="top">α-urease</entry><entry namest="col2" nameend="col2" align="left" valign="top">HP9m / 2E7-B8-G10</entry><entry namest="col3" nameend="col3" align="left" valign="top">negative</entry><entry namest="col4" nameend="col4" align="left" valign="top" /></row><row><entry namest="col1" nameend="col1" align="left" valign="top">(small subunit)</entry><entry namest="col2" nameend="col2" align="left" valign="top">HP9m / 1H7-C6-D5</entry><entry namest="col3" nameend="col3" align="left" valign="top">negative</entry><entry namest="col4" nameend="col4" align="left" valign="top" /></row><row rowsep="0"><entry namest="col1" nameend="col1" align="left" valign="top">Alkyl hydroperoxide</entry><entry namest="col2" nameend="col2" align="left" valign="top">HP15m / 3E8-D9-D6</entry><entry namest="col3" nameend="col3" align="left" valign="top">negative</entry><entry namest="col4" nameend="col4" align="left" valign="top" /></row><row rowsep="0"><entry namest="col1" nameend="col1" align="left" valign="top">Reductase</entry><entry namest="col2" nameend="col2" align="left" valign="top">HP15m / 3F5-D5-B6</entry><entry namest="col3" nameend="col3" align="left" valign="top">negative</entry><entry namest="col4" nameend="col4" align="left" valign="top" /></row><row rowsep="0"><entry namest="col1" nameend="col1" align="left" valign="top" /><entry namest="col2" nameend="col2" align="left" valign="top">HP15m / 4H12-A4-</entry><entry namest="col3" nameend="col3" align="left" valign="top">negative</entry><entry namest="col4" nameend="col4" align="left" valign="top" /></row><row rowsep="0"><entry namest="col1" nameend="col1" align="left" valign="top" /><entry namest="col2" nameend="col2" align="left" valign="top">D5</entry><entry namest="col3" nameend="col3" align="left" valign="top">143-149</entry><entry namest="col4" nameend="col4" align="left" valign="top">LPLGRNA</entry></row><row><entry namest="col1" nameend="col1" align="left" valign="top" /><entry namest="col2" nameend="col2" align="left" valign="top">HP15m / 1H7-H3-B7</entry><entry namest="col3" nameend="col3" align="left" valign="top" /><entry namest="col4" nameend="col4" align="left" valign="top" /></row><row rowsep="0"><entry namest="col1" nameend="col1" align="left" valign="top">HSP60</entry><entry namest="col2" nameend="col2" align="left" valign="top">HP16m / 2A5-E6-E5</entry><entry namest="col3" nameend="col3" align="left" valign="top">negative</entry><entry namest="col4" nameend="col4" align="left" valign="top" /></row><row><entry namest="col1" nameend="col1" align="left" valign="top" /><entry namest="col2" nameend="col2" align="left" valign="top">HP16m / 1D10-A8</entry><entry namest="col3" nameend="col3" align="left" valign="top">negative</entry><entry namest="col4" nameend="col4" align="left" valign="top" /></row></tbody></tgroup><tgroup cols="4" rowsep="0"><colspec colnum="1" colname="col1" colwidth="44mm" /><colspec colnum="2" colname="col2" colwidth="33mm" /><colspec colnum="3" colname="col3" colwidth="37mm" /><colspec colnum="4" colname="col4" colwidth="33mm" /><tbody><row><entry namest="col1" nameend="col4" align="justify" valign="top">Abbreviations: Ag Antigen; AS amino acid; negative: epitope could not be mapped</entry></row></tbody></tgroup></table></tables>

In two anti-α-urease macs and two anti-HSP60 macs, no epitope could be mapped using the immunoblot method. The epitope could only be determined from one of the three MAs directed against the β-urease and four against the alkyl hydroperoxide reductase (see Table 2). This method therefore did not appear to be suitable for predicting the possible overlap of the epitopes of antibodies of the same specificity.

Example 7: Characterization of antibody-antigen interactions by means of surface plasmon resonance spectroscopy

The epitope overlap measurement with SPR spectroscopy provides information about the simultaneous accessibility of antibody epitopes. Suitable pairs of antibodies can thus be found for the development of ELISA and rapid test (Fägerstam LG et al., 1990; Malmqvist M., 1996).

Carrying out surface plasmon resonance spectroscopy at the Pharmacia BlAcore

All steps were carried out on a Pharmacia Biacore Processing Unit CA 186 according to standard protocols (NHS / EDC, BlAcore Methods Manual). For this purpose, a polyclonal rabbit anti-mouse antibody was immobilized on the dextran matrix of the BIAcore CM5 glass carrier chip. By adding the first<i>H. pylori</i>-antigen-specific monoclonal antibody (1st mAb; 100µg / ml; 10µl) was reacted with the immobilized capture antibody and the corresponding change measured on the detector (ΔRU₁; RU Resonance Units). After adding<i>H</i>. <i>pylori</i>-Antigen (45µg / ml; 5µl) reacted with the 1st MAK. The remaining free catcher antibody was then blocked with an unspecific mouse antibody (1 mg / ml; 10µl) and oligomeric epitopes by adding a further 1st mAb (100µg / ml; 10µl). After the reaction of the second<i>H. pylori</i>-antigen-specific antibody (2nd mAb; 100µg / ml; 10µl) with the bound antigen, the change in the signal of the detector was determined again (ΔRU₂) and the change in the detector after adding the 1st mAb in a percentage ratio (ΔRU₂/ ΔRU₁). A ΔRU₂/ ΔRU₁&lt;10% indicated overlapping epitopes. (The epitopes of an antigen are described as overlapping when a first antibody has occupied all the epitopes of an antigen to be bound by it and the subsequent binding of a second antibody is prevented or prevented). Kinetic measurements can also be used to calculate the values ​​for the rate constants for the adsorption and desorption of antibodies.

Results

Twelve monoclonal anti-urease antibodies and five antibodies against alkyl hydroperoxide reductase were tested. By comparing the 144 and 25 possible combinations with each other, those combinations were determined which showed simultaneous accessibility of the epitopes and whose affinity constants appeared favorable for use in the stool ELISA. Table 3 shows a selection of the results from the combination of different antibodies against urease and compares the kinetic constants with the suitability of these antibody combinations in the stool ELISA (see Example 9). The suitability of antibody pairs for the ELISA need not be limited to independent epitopes. Also a combination of antibodies, some of which Recognizing overlapping epitopes (such as HP9.1m-2D1 and HP8m-4H5) can be explained by the fact that oligomeric proteins such as urease present the same epitope several times. This enables the binding of a capture antibody to an epitope in the ELISA and the simultaneous binding of one or more detection antibodies to one or more identical epitopes of the oligomeric protein.<tables id="tabl0003" num="0003"><table frame="all"><title><b>Table 3:</b> Four mAK-pair relationships for urease determined using surface plasmon resonance and their behavior in the ELISA</title><tgroup cols="6" colsep="1" rowsep="1"><colspec colnum="1" colname="col1" colwidth="27mm" colsep="1" /><colspec colnum="2" colname="col2" colwidth="28mm" colsep="1" /><colspec colnum="3" colname="col3" colwidth="29mm" colsep="1" /><colspec colnum="4" colname="col4" colwidth="28mm" colsep="1" /><colspec colnum="5" colname="col5" colwidth="28mm" colsep="1" /><colspec colnum="6" colname="col6" colwidth="27mm" colsep="1" /><thead><row><entry namest="col1" nameend="col1" align="left" valign="top"><b>Catcher</b></entry><entry namest="col2" nameend="col2" align="left" valign="top"><b>k_off/ k_on</b></entry><entry namest="col3" nameend="col3" align="left" valign="top"><b>detector</b></entry><entry namest="col4" nameend="col4" align="left" valign="top"><b>k_off/ k_on</b></entry><entry namest="col5" nameend="col5" align="left" valign="top"><b>Epitopes</b></entry><entry namest="col6" nameend="col6" align="left" valign="top"><b>Evaluation in the ELISA</b></entry></row></thead><tbody><row><entry namest="col1" nameend="col1" align="left" valign="top">HP9.1m -2B11</entry><entry namest="col2" nameend="col2" align="left" valign="top">5,9*10^-4/7,3*10⁴</entry><entry namest="col3" nameend="col3" align="left" valign="top">HP9.1m -2D1</entry><entry namest="col4" nameend="col4" align="left" valign="top">1,1*10^-4/3,7*10⁴</entry><entry namest="col5" nameend="col5" align="left" valign="top">independently</entry><entry namest="col6" nameend="col6" align="left" valign="top">-</entry></row><row><entry namest="col1" nameend="col1" align="left" valign="top">HP9.1m -2B11</entry><entry namest="col2" nameend="col2" align="left" valign="top">5,9*10^-4/7,3*10⁴</entry><entry namest="col3" nameend="col3" align="left" valign="top">HP9.1m -3G 1</entry><entry namest="col4" nameend="col4" align="left" valign="top">4,9*10^-4/7,6*10⁴</entry><entry namest="col5" nameend="col5" align="left" valign="top">partly overlapping</entry><entry namest="col6" nameend="col6" align="left" valign="top">-</entry></row><row><entry namest="col1" nameend="col1" align="left" valign="top">HP9.1m -2D1</entry><entry namest="col2" nameend="col2" align="left" valign="top">1,1*10^-4/3,7*10⁴</entry><entry namest="col3" nameend="col3" align="left" valign="top">HP8m -4H5</entry><entry namest="col4" nameend="col4" align="left" valign="top">4,5*10^-6/4,8*10⁴</entry><entry namest="col5" nameend="col5" align="left" valign="top">partly overlapping</entry><entry namest="col6" nameend="col6" align="left" valign="top">+++</entry></row><row><entry namest="col1" nameend="col1" align="left" valign="top">HP9.1m -XG1</entry><entry namest="col2" nameend="col2" align="left" valign="top">3,0*10^-4/1,5*10⁵</entry><entry namest="col3" nameend="col3" align="left" valign="top">HP9.1m -3G1</entry><entry namest="col4" nameend="col4" align="left" valign="top">4,9*10^-4/7,6*10⁴</entry><entry namest="col5" nameend="col5" align="left" valign="top">independently</entry><entry namest="col6" nameend="col6" align="left" valign="top">++</entry></row><row><entry namest="col1" nameend="col1" align="left" valign="top">HP8m -4H5</entry><entry namest="col2" nameend="col2" align="left" valign="top">4,5*10^-6/4,8*10⁴</entry><entry namest="col3" nameend="col3" align="left" valign="top">HP9.1m -3C2</entry><entry namest="col4" nameend="col4" align="left" valign="top">2,6*10^-4/7,4*10⁴</entry><entry namest="col5" nameend="col5" align="left" valign="top">independently</entry><entry namest="col6" nameend="col6" align="left" valign="top">+++</entry></row></tbody></tgroup></table></tables>

Example 8: Selection of antibody pairs for use in ELISA on human stool

For those antibodies which showed the lowest detection limits when measured from the culture supernatant, epitope overlaps were determined by means of surface plasmon resonance. The combinations that showed promising results in these measurements (epitope overlap as low as possible, high rate constant for adsorption, low rate constant for desorption) were tested for their detection limit in the stool ELISA.

Example 9: Detection of <i>H, pylori</i> in human stool using ELISA

Abbreviations: Detector: detection antibody; Captor: capture antibody

Stool samples from patients from the Großhadern Clinic, Munich, were available as test samples <i>H. pylori</i> (Categories 0 and 4) was determined using serological and histological examinations. Patients were classified in category 0 in whom the clinical result from serology and histology was clearly negative, in category 4 patients the clinical result from serology and histology was clearly positive. Between the individual work steps, the ELISA plate was 2-3x with 250µl PBS with the addition of 0.025% (wash buffer 1) or 0.2% Tween 20 (Wash Buffer 2; v: v) washed. The ELISA plates (MaxiSorb; Nunc) were coated for 1 h at 37 ° C. with 100 μl of a mac solution (2.5 μg antibody / ml carbonate buffer, 0.1 M, pH 9.5). To block the binding sites that were still free, 200 .mu.l of 150 mM PBS with 0.2% fish gelatin or 1% skim milk powder (weight: volume) per well were pipetted and incubated for 30 minutes at room temperature. Was washed with wash buffer 1. Human stool was suspended in a ratio of 1:10 (w / v) with 150 mM PBS with the addition of 2% skim milk powder, cleaned <i>H. pylori</i>-Antigens dissolved in 150mM PBS added in known concentrations. 100 µl of the suspensions were incubated for 1 hour per well. The plate was first rinsed by hand and washed 4x with wash buffer 2. Then 100 .mu.l of a solution with biotin-coupled mAK (0.5 .mu.g antibody / ml PBS) against the same antigen were added and incubated for 30-60 min at RT. The bound antigens are detected by adding a conjugate of streptavidin with POD (DAKO). The POD converts the colorless substrate TMB (Sigma) into a blue product. After 5 to 10 minutes, or as soon as the negative control also showed a slight blue color, the reaction was stopped by adding 1N sulfuric acid (100 μl / well). The strength of the color reaction was measured in the ELISA reader (MWG spectral). The measurement takes place at 455 nm against the reference wavelength 620 nm.

<b>Table 4:</b> proof of <i>H. pylori</i>Antigens from the stool of safely negative (category 0) or safely positive (category 4) patients by means of ELISA using monoclonal antibodies:<tables id="tabl0004" num="0004"><table frame="all"><title>Table 4a: Category 0 samples: 0 means false positive, 1 means true negative</title><tgroup cols="8" colsep="1" rowsep="1"><colspec colnum="1" colname="col1" colwidth="16mm" colsep="1" /><colspec colnum="2" colname="col2" colwidth="16mm" colsep="1" /><colspec colnum="3" colname="col3" colwidth="15mm" colsep="1" /><colspec colnum="4" colname="col4" colwidth="16mm" colsep="1" /><colspec colnum="5" colname="col5" colwidth="16mm" colsep="1" /><colspec colnum="6" colname="col6" colwidth="15mm" colsep="1" /><colspec colnum="7" colname="col7" colwidth="16mm" colsep="1" /><colspec colnum="8" colname="col8" colwidth="29mm" colsep="1" /><thead><row><entry namest="col1" nameend="col1" align="center" valign="top" /><entry namest="col2" nameend="col4" colsep="1" rowsep="1" align="center" valign="top"><b>[µg Ag / g stool]</b></entry><entry namest="col5" nameend="col8" colsep="1" rowsep="1" align="center" valign="top"><b>resulting truth value</b></entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top"><b>sample</b></entry><entry namest="col2" nameend="col2" align="center" valign="top"><b>Urease</b></entry><entry namest="col3" nameend="col3" align="center" valign="top"><b>26kDa</b></entry><entry namest="col4" nameend="col4" align="center" valign="top"><b>HSP60</b></entry><entry namest="col5" nameend="col5" align="center" valign="top"><b>Urease</b></entry><entry namest="col6" nameend="col6" align="center" valign="top"><b>26kDa</b></entry><entry namest="col7" nameend="col7" align="center" valign="top"><b>HSP60</b></entry><entry namest="col8" nameend="col8" align="center" valign="top"><b>3 combination</b></entry></row></thead><tbody><row><entry namest="col1" nameend="col1" align="center" valign="top"><b>cut-off</b></entry><entry namest="col2" nameend="col2" align="center" valign="top" /><entry namest="col3" nameend="col3" align="center" valign="top" /><entry namest="col4" nameend="col4" align="center" valign="top" /><entry namest="col5" nameend="col5" align="center" valign="top"><b>1</b></entry><entry namest="col6" nameend="col6" align="center" valign="top"><b>15</b></entry><entry namest="col7" nameend="col7" align="center" valign="top"><b>5</b></entry><entry namest="col8" nameend="col8" align="center" valign="top" /></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0047</entry><entry namest="col2" nameend="col2" align="center" valign="top">0,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,3</entry><entry namest="col4" nameend="col4" align="center" valign="top" /><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">1</entry><entry namest="col7" nameend="col7" align="center" valign="top" /><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0048</entry><entry namest="col2" nameend="col2" align="center" valign="top">0,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,3</entry><entry namest="col4" nameend="col4" align="center" valign="top" /><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">1</entry><entry namest="col7" nameend="col7" align="center" valign="top" /><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0051</entry><entry namest="col2" nameend="col2" align="center" valign="top">2,1</entry><entry namest="col3" nameend="col3" align="center" valign="top">22</entry><entry namest="col4" nameend="col4" align="center" valign="top" /><entry namest="col5" nameend="col5" align="center" valign="top">0</entry><entry namest="col6" nameend="col6" align="center" valign="top">0</entry><entry namest="col7" nameend="col7" align="center" valign="top" /><entry namest="col8" nameend="col8" align="center" valign="top">0</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0057</entry><entry namest="col2" nameend="col2" align="center" valign="top">0,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,3</entry><entry namest="col4" nameend="col4" align="center" valign="top" /><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">1</entry><entry namest="col7" nameend="col7" align="center" valign="top" /><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0069</entry><entry namest="col2" nameend="col2" align="center" valign="top">0,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,3</entry><entry namest="col4" nameend="col4" align="center" valign="top" /><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">1</entry><entry namest="col7" nameend="col7" align="center" valign="top" /><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0074</entry><entry namest="col2" nameend="col2" align="center" valign="top">0,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,3</entry><entry namest="col4" nameend="col4" align="center" valign="top" /><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">1</entry><entry namest="col7" nameend="col7" align="center" valign="top" /><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0087</entry><entry namest="col2" nameend="col2" align="center" valign="top">0,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,3</entry><entry namest="col4" nameend="col4" align="center" valign="top" /><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">1</entry><entry namest="col7" nameend="col7" align="center" valign="top" /><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0099</entry><entry namest="col2" nameend="col2" align="center" valign="top">0,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,3</entry><entry namest="col4" nameend="col4" align="center" valign="top" /><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">1</entry><entry namest="col7" nameend="col7" align="center" valign="top" /><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0185</entry><entry namest="col2" nameend="col2" align="center" valign="top">0,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,3</entry><entry namest="col4" nameend="col4" align="center" valign="top" /><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">1</entry><entry namest="col7" nameend="col7" align="center" valign="top" /><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0186</entry><entry namest="col2" nameend="col2" align="center" valign="top">0,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,3</entry><entry namest="col4" nameend="col4" align="center" valign="top" /><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">1</entry><entry namest="col7" nameend="col7" align="center" valign="top" /><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0189</entry><entry namest="col2" nameend="col2" align="center" valign="top">0,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,3</entry><entry namest="col4" nameend="col4" align="center" valign="top" /><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">1</entry><entry namest="col7" nameend="col7" align="center" valign="top" /><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0265</entry><entry namest="col2" nameend="col2" align="center" valign="top">0,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,3</entry><entry namest="col4" nameend="col4" align="center" valign="top" /><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">1</entry><entry namest="col7" nameend="col7" align="center" valign="top" /><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0298</entry><entry namest="col2" nameend="col2" align="center" valign="top">0,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,3</entry><entry namest="col4" nameend="col4" align="center" valign="top" /><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">1</entry><entry namest="col7" nameend="col7" align="center" valign="top" /><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0305</entry><entry namest="col2" nameend="col2" align="center" valign="top">0,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,3</entry><entry namest="col4" nameend="col4" align="center" valign="top" /><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">1</entry><entry namest="col7" nameend="col7" align="center" valign="top" /><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0373</entry><entry namest="col2" nameend="col2" align="center" valign="top">0,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,3</entry><entry namest="col4" nameend="col4" align="center" valign="top" /><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">1</entry><entry namest="col7" nameend="col7" align="center" valign="top" /><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0074</entry><entry namest="col2" nameend="col2" align="center" valign="top">0,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,3</entry><entry namest="col4" nameend="col4" align="center" valign="top">2,3</entry><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">1</entry><entry namest="col7" nameend="col7" align="center" valign="top">1</entry><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0089</entry><entry namest="col2" nameend="col2" align="center" valign="top">0,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,3</entry><entry namest="col4" nameend="col4" align="center" valign="top">2,3</entry><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">1</entry><entry namest="col7" nameend="col7" align="center" valign="top">1</entry><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0090</entry><entry namest="col2" nameend="col2" align="center" valign="top">0,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,3</entry><entry namest="col4" nameend="col4" align="center" valign="top">2,3</entry><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">1</entry><entry namest="col7" nameend="col7" align="center" valign="top">1</entry><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0091</entry><entry namest="col2" nameend="col2" align="center" valign="top">0,76</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,3</entry><entry namest="col4" nameend="col4" align="center" valign="top">2,3</entry><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">1</entry><entry namest="col7" nameend="col7" align="center" valign="top">1</entry><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0097</entry><entry namest="col2" nameend="col2" align="center" valign="top">1,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,3</entry><entry namest="col4" nameend="col4" align="center" valign="top">2,3</entry><entry namest="col5" nameend="col5" align="center" valign="top">0</entry><entry namest="col6" nameend="col6" align="center" valign="top">1</entry><entry namest="col7" nameend="col7" align="center" valign="top">1</entry><entry namest="col8" nameend="col8" align="center" valign="top">0</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0103</entry><entry namest="col2" nameend="col2" align="center" valign="top">1</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,4</entry><entry namest="col4" nameend="col4" align="center" valign="top">15</entry><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">1</entry><entry namest="col7" nameend="col7" align="center" valign="top">0</entry><entry namest="col8" nameend="col8" align="center" valign="top">0</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0107</entry><entry namest="col2" nameend="col2" align="center" valign="top">1</entry><entry namest="col3" nameend="col3" align="center" valign="top">12</entry><entry namest="col4" nameend="col4" align="center" valign="top">2,3</entry><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">1</entry><entry namest="col7" nameend="col7" align="center" valign="top">1</entry><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0114</entry><entry namest="col2" nameend="col2" align="center" valign="top">4</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,3</entry><entry namest="col4" nameend="col4" align="center" valign="top">44</entry><entry namest="col5" nameend="col5" align="center" valign="top">0</entry><entry namest="col6" nameend="col6" align="center" valign="top">1</entry><entry namest="col7" nameend="col7" align="center" valign="top">0</entry><entry namest="col8" nameend="col8" align="center" valign="top">0</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0126</entry><entry namest="col2" nameend="col2" align="center" valign="top">0,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">12</entry><entry namest="col4" nameend="col4" align="center" valign="top">2,3</entry><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">1</entry><entry namest="col7" nameend="col7" align="center" valign="top">1</entry><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0130</entry><entry namest="col2" nameend="col2" align="center" valign="top">0,76</entry><entry namest="col3" nameend="col3" align="center" valign="top">67</entry><entry namest="col4" nameend="col4" align="center" valign="top">2,3</entry><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">0</entry><entry namest="col7" nameend="col7" align="center" valign="top">1</entry><entry namest="col8" nameend="col8" align="center" valign="top">0</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0141</entry><entry namest="col2" nameend="col2" align="center" valign="top">20</entry><entry namest="col3" nameend="col3" align="center" valign="top">220</entry><entry namest="col4" nameend="col4" align="center" valign="top">105</entry><entry namest="col5" nameend="col5" align="center" valign="top">0</entry><entry namest="col6" nameend="col6" align="center" valign="top">0</entry><entry namest="col7" nameend="col7" align="center" valign="top">0</entry><entry namest="col8" nameend="col8" align="center" valign="top">0</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0142</entry><entry namest="col2" nameend="col2" align="center" valign="top">1,8</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,3</entry><entry namest="col4" nameend="col4" align="center" valign="top">2,3</entry><entry namest="col5" nameend="col5" align="center" valign="top">0</entry><entry namest="col6" nameend="col6" align="center" valign="top">1</entry><entry namest="col7" nameend="col7" align="center" valign="top">1</entry><entry namest="col8" nameend="col8" align="center" valign="top">0</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0144</entry><entry namest="col2" nameend="col2" align="center" valign="top">0,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,3</entry><entry namest="col4" nameend="col4" align="center" valign="top">2,3</entry><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">1</entry><entry namest="col7" nameend="col7" align="center" valign="top">1</entry><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0146</entry><entry namest="col2" nameend="col2" align="center" valign="top">0,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,3</entry><entry namest="col4" nameend="col4" align="center" valign="top">2,3</entry><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">1</entry><entry namest="col7" nameend="col7" align="center" valign="top">1</entry><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0148</entry><entry namest="col2" nameend="col2" align="center" valign="top">0,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,3</entry><entry namest="col4" nameend="col4" align="center" valign="top">2,3</entry><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">1</entry><entry namest="col7" nameend="col7" align="center" valign="top">1</entry><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0168</entry><entry namest="col2" nameend="col2" align="center" valign="top">0,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,3</entry><entry namest="col4" nameend="col4" align="center" valign="top">2,3</entry><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">1</entry><entry namest="col7" nameend="col7" align="center" valign="top">1</entry><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0193</entry><entry namest="col2" nameend="col2" align="center" valign="top">0,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,3</entry><entry namest="col4" nameend="col4" align="center" valign="top">20</entry><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">1</entry><entry namest="col7" nameend="col7" align="center" valign="top">0</entry><entry namest="col8" nameend="col8" align="center" valign="top">0</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0207</entry><entry namest="col2" nameend="col2" align="center" valign="top">0,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,3</entry><entry namest="col4" nameend="col4" align="center" valign="top">20</entry><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">1</entry><entry namest="col7" nameend="col7" align="center" valign="top">0</entry><entry namest="col8" nameend="col8" align="center" valign="top">0</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0220</entry><entry namest="col2" nameend="col2" align="center" valign="top">0,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,3</entry><entry namest="col4" nameend="col4" align="center" valign="top">2,2</entry><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">1</entry><entry namest="col7" nameend="col7" align="center" valign="top">1</entry><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0225</entry><entry namest="col2" nameend="col2" align="center" valign="top">0,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,3</entry><entry namest="col4" nameend="col4" align="center" valign="top">2,2</entry><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">1</entry><entry namest="col7" nameend="col7" align="center" valign="top">1</entry><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0231</entry><entry namest="col2" nameend="col2" align="center" valign="top">0,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,3</entry><entry namest="col4" nameend="col4" align="center" valign="top">2,2</entry><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">1</entry><entry namest="col7" nameend="col7" align="center" valign="top">1</entry><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0235</entry><entry namest="col2" nameend="col2" align="center" valign="top">0,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,3</entry><entry namest="col4" nameend="col4" align="center" valign="top">2,2</entry><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">1</entry><entry namest="col7" nameend="col7" align="center" valign="top">1</entry><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0251</entry><entry namest="col2" nameend="col2" align="center" valign="top">0,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,3</entry><entry namest="col4" nameend="col4" align="center" valign="top" /><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">1</entry><entry namest="col7" nameend="col7" align="center" valign="top" /><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0258</entry><entry namest="col2" nameend="col2" align="center" valign="top">0,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">95</entry><entry namest="col4" nameend="col4" align="center" valign="top" /><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">0</entry><entry namest="col7" nameend="col7" align="center" valign="top" /><entry namest="col8" nameend="col8" align="center" valign="top">0</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0274</entry><entry namest="col2" nameend="col2" align="center" valign="top">0,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,3</entry><entry namest="col4" nameend="col4" align="center" valign="top" /><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">1</entry><entry namest="col7" nameend="col7" align="center" valign="top" /><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row></tbody></tgroup></table></tables><tables id="tabl0005" num="0005"><table frame="all"><title>Table 4b: Category 4 samples: 0 means false-negative, 1 means true-positive</title><tgroup cols="8" colsep="1" rowsep="1"><colspec colnum="1" colname="col1" colwidth="16mm" colsep="1" /><colspec colnum="2" colname="col2" colwidth="16mm" colsep="1" /><colspec colnum="3" colname="col3" colwidth="15mm" colsep="1" /><colspec colnum="4" colname="col4" colwidth="16mm" colsep="1" /><colspec colnum="5" colname="col5" colwidth="16mm" colsep="1" /><colspec colnum="6" colname="col6" colwidth="15mm" colsep="1" /><colspec colnum="7" colname="col7" colwidth="16mm" colsep="1" /><colspec colnum="8" colname="col8" colwidth="29mm" colsep="1" /><thead><row><entry namest="col1" nameend="col1" align="center" valign="top" /><entry namest="col2" nameend="col4" colsep="1" rowsep="1" align="center" valign="top"><b>[µg Ag / g stool]</b></entry><entry namest="col5" nameend="col8" colsep="1" rowsep="1" align="center" valign="top"><b>resulting truth value</b></entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top"><b>sample</b></entry><entry namest="col2" nameend="col2" align="center" valign="top"><b>Urease</b></entry><entry namest="col3" nameend="col3" align="center" valign="top"><b>26kDa</b></entry><entry namest="col4" nameend="col4" align="center" valign="top"><b>HSP60</b></entry><entry namest="col5" nameend="col5" align="center" valign="top"><b>Urease</b></entry><entry namest="col6" nameend="col6" align="center" valign="top"><b>26kDa</b></entry><entry namest="col7" nameend="col7" align="center" valign="top"><b>HSP60</b></entry><entry namest="col8" nameend="col8" align="center" valign="top"><b>3 combination</b></entry></row></thead><tbody><row><entry namest="col1" nameend="col1" align="center" valign="top"><b>cut-off</b></entry><entry namest="col2" nameend="col2" align="center" valign="top" /><entry namest="col3" nameend="col3" align="center" valign="top" /><entry namest="col4" nameend="col4" align="center" valign="top" /><entry namest="col5" nameend="col5" align="center" valign="top"><b>1</b></entry><entry namest="col6" nameend="col6" align="center" valign="top"><b>15</b></entry><entry namest="col7" nameend="col7" align="center" valign="top"><b>5</b></entry><entry namest="col8" nameend="col8" align="center" valign="top" /></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0006</entry><entry namest="col2" nameend="col2" align="center" valign="top">1,9</entry><entry namest="col3" nameend="col3" align="center" valign="top">25</entry><entry namest="col4" nameend="col4" align="center" valign="top">34</entry><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">1</entry><entry namest="col7" nameend="col7" align="center" valign="top">1</entry><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0010</entry><entry namest="col2" nameend="col2" align="center" valign="top">5,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">91,5</entry><entry namest="col4" nameend="col4" align="center" valign="top">170</entry><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">1</entry><entry namest="col7" nameend="col7" align="center" valign="top">1</entry><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">22</entry><entry namest="col2" nameend="col2" align="center" valign="top">7,8</entry><entry namest="col3" nameend="col3" align="center" valign="top">45</entry><entry namest="col4" nameend="col4" align="center" valign="top">25</entry><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">1</entry><entry namest="col7" nameend="col7" align="center" valign="top">1</entry><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0350</entry><entry namest="col2" nameend="col2" align="center" valign="top">15,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">198</entry><entry namest="col4" nameend="col4" align="center" valign="top">95</entry><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">1</entry><entry namest="col7" nameend="col7" align="center" valign="top">1</entry><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0288</entry><entry namest="col2" nameend="col2" align="center" valign="top">18</entry><entry namest="col3" nameend="col3" align="center" valign="top">34</entry><entry namest="col4" nameend="col4" align="center" valign="top">65,9</entry><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">1</entry><entry namest="col7" nameend="col7" align="center" valign="top">1</entry><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0332</entry><entry namest="col2" nameend="col2" align="center" valign="top">29</entry><entry namest="col3" nameend="col3" align="center" valign="top">360</entry><entry namest="col4" nameend="col4" align="center" valign="top">650</entry><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">1</entry><entry namest="col7" nameend="col7" align="center" valign="top">1</entry><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0218</entry><entry namest="col2" nameend="col2" align="center" valign="top">7</entry><entry namest="col3" nameend="col3" align="center" valign="top">24</entry><entry namest="col4" nameend="col4" align="center" valign="top">3</entry><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">1</entry><entry namest="col7" nameend="col7" align="center" valign="top">0</entry><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0285</entry><entry namest="col2" nameend="col2" align="center" valign="top">7,4</entry><entry namest="col3" nameend="col3" align="center" valign="top">115</entry><entry namest="col4" nameend="col4" align="center" valign="top">3</entry><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">1</entry><entry namest="col7" nameend="col7" align="center" valign="top">0</entry><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0004</entry><entry namest="col2" nameend="col2" align="center" valign="top">9,5</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,3</entry><entry namest="col4" nameend="col4" align="center" valign="top">350</entry><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">0</entry><entry namest="col7" nameend="col7" align="center" valign="top">1</entry><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">11</entry><entry namest="col2" nameend="col2" align="center" valign="top">21</entry><entry namest="col3" nameend="col3" align="center" valign="top">15</entry><entry namest="col4" nameend="col4" align="center" valign="top">26</entry><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">0</entry><entry namest="col7" nameend="col7" align="center" valign="top">1</entry><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">8</entry><entry namest="col2" nameend="col2" align="center" valign="top">105</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,3</entry><entry namest="col4" nameend="col4" align="center" valign="top">89</entry><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">0</entry><entry namest="col7" nameend="col7" align="center" valign="top">1</entry><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0003</entry><entry namest="col2" nameend="col2" align="center" valign="top">1,5</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,3</entry><entry namest="col4" nameend="col4" align="center" valign="top">10</entry><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">0</entry><entry namest="col7" nameend="col7" align="center" valign="top">1</entry><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">15</entry><entry namest="col2" nameend="col2" align="center" valign="top">2,3</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,3</entry><entry namest="col4" nameend="col4" align="center" valign="top">15</entry><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">0</entry><entry namest="col7" nameend="col7" align="center" valign="top">1</entry><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">3005</entry><entry namest="col2" nameend="col2" align="center" valign="top">1,2</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,3</entry><entry namest="col4" nameend="col4" align="center" valign="top">2,3</entry><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">0</entry><entry namest="col7" nameend="col7" align="center" valign="top">0</entry><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0002</entry><entry namest="col2" nameend="col2" align="center" valign="top">1,8</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,3</entry><entry namest="col4" nameend="col4" align="center" valign="top">2,3</entry><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">0</entry><entry namest="col7" nameend="col7" align="center" valign="top">0</entry><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0206</entry><entry namest="col2" nameend="col2" align="center" valign="top">9,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,9</entry><entry namest="col4" nameend="col4" align="center" valign="top">3</entry><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">0</entry><entry namest="col7" nameend="col7" align="center" valign="top">0</entry><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0135</entry><entry namest="col2" nameend="col2" align="center" valign="top">11</entry><entry namest="col3" nameend="col3" align="center" valign="top" /><entry namest="col4" nameend="col4" align="center" valign="top">3</entry><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">0</entry><entry namest="col7" nameend="col7" align="center" valign="top">0</entry><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0175</entry><entry namest="col2" nameend="col2" align="center" valign="top">16</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,9</entry><entry namest="col4" nameend="col4" align="center" valign="top">3</entry><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">0</entry><entry namest="col7" nameend="col7" align="center" valign="top">0</entry><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0005</entry><entry namest="col2" nameend="col2" align="center" valign="top">25</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,3</entry><entry namest="col4" nameend="col4" align="center" valign="top">2,3</entry><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">0</entry><entry namest="col7" nameend="col7" align="center" valign="top">0</entry><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0278</entry><entry namest="col2" nameend="col2" align="center" valign="top">25,7</entry><entry namest="col3" nameend="col3" align="center" valign="top">4</entry><entry namest="col4" nameend="col4" align="center" valign="top">3</entry><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">0</entry><entry namest="col7" nameend="col7" align="center" valign="top">0</entry><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0001</entry><entry namest="col2" nameend="col2" align="center" valign="top">127</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,3</entry><entry namest="col4" nameend="col4" align="center" valign="top">2,3</entry><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">0</entry><entry namest="col7" nameend="col7" align="center" valign="top">0</entry><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0004</entry><entry namest="col2" nameend="col2" align="center" valign="top">127</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,9</entry><entry namest="col4" nameend="col4" align="center" valign="top">3</entry><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">0</entry><entry namest="col7" nameend="col7" align="center" valign="top">0</entry><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0037</entry><entry namest="col2" nameend="col2" align="center" valign="top">127</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,9</entry><entry namest="col4" nameend="col4" align="center" valign="top">3</entry><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">0</entry><entry namest="col7" nameend="col7" align="center" valign="top">0</entry><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0294</entry><entry namest="col2" nameend="col2" align="center" valign="top">127</entry><entry namest="col3" nameend="col3" align="center" valign="top">14</entry><entry namest="col4" nameend="col4" align="center" valign="top">2,3</entry><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top">0</entry><entry namest="col7" nameend="col7" align="center" valign="top">0</entry><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0108</entry><entry namest="col2" nameend="col2" align="center" valign="top">17,5</entry><entry namest="col3" nameend="col3" align="center" valign="top" /><entry namest="col4" nameend="col4" align="center" valign="top">3</entry><entry namest="col5" nameend="col5" align="center" valign="top">1</entry><entry namest="col6" nameend="col6" align="center" valign="top" /><entry namest="col7" nameend="col7" align="center" valign="top">0</entry><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">28</entry><entry namest="col2" nameend="col2" align="center" valign="top">0,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">69</entry><entry namest="col4" nameend="col4" align="center" valign="top">330</entry><entry namest="col5" nameend="col5" align="center" valign="top">0</entry><entry namest="col6" nameend="col6" align="center" valign="top">1</entry><entry namest="col7" nameend="col7" align="center" valign="top">1</entry><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0012</entry><entry namest="col2" nameend="col2" align="center" valign="top">0,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">23</entry><entry namest="col4" nameend="col4" align="center" valign="top">8,6</entry><entry namest="col5" nameend="col5" align="center" valign="top">0</entry><entry namest="col6" nameend="col6" align="center" valign="top">1</entry><entry namest="col7" nameend="col7" align="center" valign="top">1</entry><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0013</entry><entry namest="col2" nameend="col2" align="center" valign="top">0,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">27,7</entry><entry namest="col4" nameend="col4" align="center" valign="top">3</entry><entry namest="col5" nameend="col5" align="center" valign="top">0</entry><entry namest="col6" nameend="col6" align="center" valign="top">1</entry><entry namest="col7" nameend="col7" align="center" valign="top">0</entry><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">6c</entry><entry namest="col2" nameend="col2" align="center" valign="top">0,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,8</entry><entry namest="col4" nameend="col4" align="center" valign="top">35</entry><entry namest="col5" nameend="col5" align="center" valign="top">0</entry><entry namest="col6" nameend="col6" align="center" valign="top">0</entry><entry namest="col7" nameend="col7" align="center" valign="top">1</entry><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">18c</entry><entry namest="col2" nameend="col2" align="center" valign="top">0,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,3</entry><entry namest="col4" nameend="col4" align="center" valign="top">15</entry><entry namest="col5" nameend="col5" align="center" valign="top">0</entry><entry namest="col6" nameend="col6" align="center" valign="top">0</entry><entry namest="col7" nameend="col7" align="center" valign="top">1</entry><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">53</entry><entry namest="col2" nameend="col2" align="center" valign="top">0,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,3</entry><entry namest="col4" nameend="col4" align="center" valign="top">17</entry><entry namest="col5" nameend="col5" align="center" valign="top">0</entry><entry namest="col6" nameend="col6" align="center" valign="top">0</entry><entry namest="col7" nameend="col7" align="center" valign="top">1</entry><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0164</entry><entry namest="col2" nameend="col2" align="center" valign="top">0,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,9</entry><entry namest="col4" nameend="col4" align="center" valign="top">5,9</entry><entry namest="col5" nameend="col5" align="center" valign="top">0</entry><entry namest="col6" nameend="col6" align="center" valign="top">0</entry><entry namest="col7" nameend="col7" align="center" valign="top">1</entry><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">3007</entry><entry namest="col2" nameend="col2" align="center" valign="top">0,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,3</entry><entry namest="col4" nameend="col4" align="center" valign="top">2,3</entry><entry namest="col5" nameend="col5" align="center" valign="top">0</entry><entry namest="col6" nameend="col6" align="center" valign="top">0</entry><entry namest="col7" nameend="col7" align="center" valign="top">0</entry><entry namest="col8" nameend="col8" align="center" valign="top">0</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">9c</entry><entry namest="col2" nameend="col2" align="center" valign="top">0,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,3</entry><entry namest="col4" nameend="col4" align="center" valign="top">2,3</entry><entry namest="col5" nameend="col5" align="center" valign="top">0</entry><entry namest="col6" nameend="col6" align="center" valign="top">0</entry><entry namest="col7" nameend="col7" align="center" valign="top">0</entry><entry namest="col8" nameend="col8" align="center" valign="top">0</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">13c</entry><entry namest="col2" nameend="col2" align="center" valign="top">0,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,3</entry><entry namest="col4" nameend="col4" align="center" valign="top">2,3</entry><entry namest="col5" nameend="col5" align="center" valign="top">0</entry><entry namest="col6" nameend="col6" align="center" valign="top">0</entry><entry namest="col7" nameend="col7" align="center" valign="top">0</entry><entry namest="col8" nameend="col8" align="center" valign="top">0</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">47</entry><entry namest="col2" nameend="col2" align="center" valign="top">0,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,3</entry><entry namest="col4" nameend="col4" align="center" valign="top">2,3</entry><entry namest="col5" nameend="col5" align="center" valign="top">0</entry><entry namest="col6" nameend="col6" align="center" valign="top">0</entry><entry namest="col7" nameend="col7" align="center" valign="top">0</entry><entry namest="col8" nameend="col8" align="center" valign="top">0</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">17</entry><entry namest="col2" nameend="col2" align="center" valign="top">0,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,3</entry><entry namest="col4" nameend="col4" align="center" valign="top">2,3</entry><entry namest="col5" nameend="col5" align="center" valign="top">0</entry><entry namest="col6" nameend="col6" align="center" valign="top">0</entry><entry namest="col7" nameend="col7" align="center" valign="top">0</entry><entry namest="col8" nameend="col8" align="center" valign="top">0</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">18</entry><entry namest="col2" nameend="col2" align="center" valign="top">0,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,3</entry><entry namest="col4" nameend="col4" align="center" valign="top">2,3</entry><entry namest="col5" nameend="col5" align="center" valign="top">0</entry><entry namest="col6" nameend="col6" align="center" valign="top">0</entry><entry namest="col7" nameend="col7" align="center" valign="top">0</entry><entry namest="col8" nameend="col8" align="center" valign="top">0</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0331</entry><entry namest="col2" nameend="col2" align="center" valign="top">0,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,3</entry><entry namest="col4" nameend="col4" align="center" valign="top">3</entry><entry namest="col5" nameend="col5" align="center" valign="top">0</entry><entry namest="col6" nameend="col6" align="center" valign="top">0</entry><entry namest="col7" nameend="col7" align="center" valign="top">0</entry><entry namest="col8" nameend="col8" align="center" valign="top">0</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0063</entry><entry namest="col2" nameend="col2" align="center" valign="top">0,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,9</entry><entry namest="col4" nameend="col4" align="center" valign="top">3</entry><entry namest="col5" nameend="col5" align="center" valign="top">0</entry><entry namest="col6" nameend="col6" align="center" valign="top">0</entry><entry namest="col7" nameend="col7" align="center" valign="top">0</entry><entry namest="col8" nameend="col8" align="center" valign="top">0</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0179</entry><entry namest="col2" nameend="col2" align="center" valign="top">0,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,9</entry><entry namest="col4" nameend="col4" align="center" valign="top">3</entry><entry namest="col5" nameend="col5" align="center" valign="top">0</entry><entry namest="col6" nameend="col6" align="center" valign="top">0</entry><entry namest="col7" nameend="col7" align="center" valign="top">0</entry><entry namest="col8" nameend="col8" align="center" valign="top">0</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0213</entry><entry namest="col2" nameend="col2" align="center" valign="top">0,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,9</entry><entry namest="col4" nameend="col4" align="center" valign="top">3</entry><entry namest="col5" nameend="col5" align="center" valign="top">0</entry><entry namest="col6" nameend="col6" align="center" valign="top">0</entry><entry namest="col7" nameend="col7" align="center" valign="top">0</entry><entry namest="col8" nameend="col8" align="center" valign="top">0</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0233</entry><entry namest="col2" nameend="col2" align="center" valign="top">0,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,9</entry><entry namest="col4" nameend="col4" align="center" valign="top">3</entry><entry namest="col5" nameend="col5" align="center" valign="top">0</entry><entry namest="col6" nameend="col6" align="center" valign="top">0</entry><entry namest="col7" nameend="col7" align="center" valign="top">0</entry><entry namest="col8" nameend="col8" align="center" valign="top">0</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">0081</entry><entry namest="col2" nameend="col2" align="center" valign="top">0,6</entry><entry namest="col3" nameend="col3" align="center" valign="top">2,9</entry><entry namest="col4" nameend="col4" align="center" valign="top">14,4</entry><entry namest="col5" nameend="col5" align="center" valign="top">0</entry><entry namest="col6" nameend="col6" align="center" valign="top" /><entry namest="col7" nameend="col7" align="center" valign="top">1</entry><entry namest="col8" nameend="col8" align="center" valign="top">1</entry></row></tbody></tgroup></table></tables><tables id="tabl0006" num="0006"><table frame="all"><title>Table 4c: Truth values ​​for the detection of urease, 26kDa protein, and HSP60</title><tgroup cols="5" colsep="1" rowsep="1"><colspec colnum="1" colname="col1" colwidth="33mm" colsep="1" /><colspec colnum="2" colname="col2" colwidth="21mm" colsep="1" /><colspec colnum="3" colname="col3" colwidth="20mm" colsep="1" /><colspec colnum="4" colname="col4" colwidth="21mm" colsep="1" /><colspec colnum="5" colname="col5" colwidth="30mm" colsep="1" /><thead><row><entry namest="col1" nameend="col1" align="center" valign="top" /><entry namest="col2" nameend="col5" colsep="1" rowsep="1" align="center" valign="top"><b>Truth value</b></entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top" /><entry namest="col2" nameend="col2" align="center" valign="top"><b>Urease</b></entry><entry namest="col3" nameend="col3" align="center" valign="top"><b>26kDa</b></entry><entry namest="col4" nameend="col4" align="center" valign="top"><b>HSP60</b></entry><entry namest="col5" nameend="col5" align="center" valign="top"><b>3 combination</b></entry></row></thead><tbody><row><entry namest="col1" nameend="col1" align="center" valign="top"><b>Threshold</b></entry><entry namest="col2" nameend="col2" align="center" valign="top"><b>1</b></entry><entry namest="col3" nameend="col3" align="center" valign="top"><b>15</b></entry><entry namest="col4" nameend="col4" align="center" valign="top"><b>5</b></entry><entry namest="col5" nameend="col5" align="center" valign="top" /></row><row><entry namest="col1" nameend="col1" align="center" valign="top"><b>Specificity</b></entry><entry namest="col2" nameend="col2" align="center" valign="top"><b>88%</b></entry><entry namest="col3" nameend="col3" align="center" valign="top"><b>90%</b></entry><entry namest="col4" nameend="col4" align="center" valign="top"><b>77%</b></entry><entry namest="col5" nameend="col5" align="center" valign="top"><b>75%</b></entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top"><b>sensitivity</b></entry><entry namest="col2" nameend="col2" align="center" valign="top"><b>57%</b></entry><entry namest="col3" nameend="col3" align="center" valign="top"><b>26%</b></entry><entry namest="col4" nameend="col4" align="center" valign="top"><b>41%</b></entry><entry namest="col5" nameend="col5" align="center" valign="top"><b>75%</b></entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">negatively correct</entry><entry namest="col2" nameend="col2" align="center" valign="top">35</entry><entry namest="col3" nameend="col3" align="center" valign="top">36</entry><entry namest="col4" nameend="col4" align="center" valign="top">17</entry><entry namest="col5" nameend="col5" align="center" valign="top">30</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">negative overall</entry><entry namest="col2" nameend="col2" align="center" valign="top">40</entry><entry namest="col3" nameend="col3" align="center" valign="top">40</entry><entry namest="col4" nameend="col4" align="center" valign="top">22</entry><entry namest="col5" nameend="col5" align="center" valign="top">40</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">positive-correct</entry><entry namest="col2" nameend="col2" align="center" valign="top">25</entry><entry namest="col3" nameend="col3" align="center" valign="top">11</entry><entry namest="col4" nameend="col4" align="center" valign="top">18</entry><entry namest="col5" nameend="col5" align="center" valign="top">33</entry></row><row><entry namest="col1" nameend="col1" align="center" valign="top">positive overall</entry><entry namest="col2" nameend="col2" align="center" valign="top">44</entry><entry namest="col3" nameend="col3" align="center" valign="top">42</entry><entry namest="col4" nameend="col4" align="center" valign="top">44</entry><entry namest="col5" nameend="col5" align="center" valign="top">44</entry></row></tbody></tgroup><tgroup cols="5" rowsep="0"><colspec colnum="1" colname="col1" colwidth="33mm" /><colspec colnum="2" colname="col2" colwidth="21mm" /><colspec colnum="3" colname="col3" colwidth="20mm" /><colspec colnum="4" colname="col4" colwidth="21mm" /><colspec colnum="5" colname="col5" colwidth="30mm" /><tbody><row><entry namest="col1" nameend="col5" align="justify" valign="top">Abbreviations: 26kDa means 26 kDa protein; 3 combination means combination of all three antigens (urease, 26kDa protein and HSP60)</entry></row></tbody></tgroup></table></tables>

HP8m / 4H5 + HP16m / XG1 were used as capture antibodies and detection antibodies in urease. The detection limit was 0.075 ng / ml. In the case of 26 kDa protein, HP15m / 4H12 were used as capture antibodies and HP15m / 3E8 as detection antibodies. The detection limit was 1.5 ng / ml. In the HSP60, capture antibodies HP18.1m / 3F11 and detection antibodies HP16m / 2A5 + HP18.1m / 4D9 were used. The detection limit was 6 ng / ml.

Results

Tables 4a-4c show the results of the examination of stool samples for urease, alkyl hydroperoxide reductase and HSP60. The last column (combination of 3) shows the values ​​for the resulting truth value of the combination of all three antigens (urease, 26kDa protein, HSP60). A total of 40 category 0 control samples (Table 4a) examined identified 30 as negative (specificity 75%), of 44 Category 4 samples (Table 4b) 33 were identified as positive (sensitivity 75%). Detection of all three antigens was possible in 6 out of 44 samples, urease in 25 out of 44 samples, alkyl hydroperoxide reductase in 11 out of 42 samples and HSP60 in 18 out of 44 samples. For the combination of all three antigens (urease, 26kDa, HSP60), a specificity of 75% and a sensitivity of 75% were achieved (Table 4c).

Table 5 shows the result of examining 3 samples taken from a patient's stool at three different locations. The figures correspond to the measured absorbance at 650nm divided by the absorbance of a zero sample, i.e. the multiple of the background (H). There was an inhomogeneous distribution of the detected antigens.<tables id="tabl0007" num="0007"><table frame="all"><title><b>Table 5:</b> distribution <i>H. pylori</i>-specific antigens in stool samples taken from different sites in the stool of an infected person</title><tgroup cols="4" colsep="1" rowsep="1"><colspec colnum="1" colname="col1" colwidth="41mm" colsep="1" /><colspec colnum="2" colname="col2" colwidth="42mm" colsep="1" /><colspec colnum="3" colname="col3" colwidth="42mm" colsep="1" /><colspec colnum="4" colname="col4" colwidth="42mm" colsep="1" /><thead><row><entry namest="col1" nameend="col1" align="left" valign="top"><b>Detected antigen</b></entry><entry namest="col2" nameend="col2" align="left" valign="top"><b>β-urease</b></entry><entry namest="col3" nameend="col3" align="left" valign="top"><b>26kDa protein</b></entry><entry namest="col4" nameend="col4" align="left" valign="top"><b>HSP60</b></entry></row></thead><tbody><row><entry namest="col1" nameend="col1" align="left" valign="top">Sample 1</entry><entry namest="col2" nameend="col2" align="left" valign="top">8.6 x H</entry><entry namest="col3" nameend="col3" align="left" valign="top">2.5 x H</entry><entry namest="col4" nameend="col4" align="left" valign="top">1 x H</entry></row><row><entry namest="col1" nameend="col1" align="left" valign="top">Sample 2</entry><entry namest="col2" nameend="col2" align="left" valign="top">11 x H</entry><entry namest="col3" nameend="col3" align="left" valign="top">1 x H</entry><entry namest="col4" nameend="col4" align="left" valign="top">1 x H</entry></row><row><entry namest="col1" nameend="col1" align="left" valign="top">Sample 3</entry><entry namest="col2" nameend="col2" align="left" valign="top">3.5 x H</entry><entry namest="col3" nameend="col3" align="left" valign="top">1 x H</entry><entry namest="col4" nameend="col4" align="left" valign="top">1 x H</entry></row></tbody></tgroup></table></tables>

Example 10: Cloning and sequence determination of the functional variable regions of immunoglobulins from hybridoma cell lines

Total RNA was isolated from Chomczynski antibody-producing hybridoma cell lines (Chomczynski, 1987).

The corresponding cDNA was then synthesized using standard methods (Sambrook et al., 1989)

The DNA regions which encode the kappa light chain and the heavy chain Fd segment (VH + CH1) of the respective antibodies were amplified by means of PCR. The oligunucleotide primer set listed in Table 6 was used. The cDNA isolated from the individual hybridoma cell lines served as a template.

The primer set used results in a 5 '<i>-Xhol</i> and a 3'-<i>Spe</i>l Interface in the heavy chain Fd fragments as well as a 5'-Sacl and a 3'-Xbal interface in the kappa-light chains. For PCR amplification of the heavy chain Fd-coding DNA fragments, 11 different 5'-VH primers (MVH 1-8 and MULH1-3) were each combined with the 3'-VH primer MlgG1. To amplify the DNA fragments which code for the kappa light chains, 11 different 5'-VK primers (MUVK 1-7 and MULK1-4) were combined with the 3'-VK primer 3'MUCK.

The following temperature program was used for all PCR amplifications: initial denaturation at 94 ° C for 3 min, denaturation at 94 ° C for 25 s, priming at 52 ° C for 60 s, polymerization at 72 ° C for 90 s . This program was maintained for 40 cycles followed by a 10 min. final completion of the fragments at 72 ° C.

The results of the PCR amplifications were separated by means of agarose gel electrophoresis and DNA bands of the expected molecular weight were isolated. The isolated bands were then subjected to restriction digestion using the enzymes<i>Xhol</i> and <i>Spel</i> (heavy chains) or <i>Sacl</i> and <i>Xbal</i> (light chains) and the fragments obtained were cloned into the plasmid vector Bluescript KS (Stratagene) after this first with the restriction enzymes <i>Xhol</i> and <i>Spel</i> or. <i>Sacl</i> and <i>Xbal</i> had been split.

Plasmid preparations of the cloned heavy and light chain fragments were then sequence analyzed. For each hybridoma cell line, sequences were selected that code heavy and light chain (VH and VL) for functional variable regions of the immunoglobulin. In this way, exactly one functional VH and one functional VL region could be identified for each hybridoma cell line. The functional VH and VL sequences are shown in Figures 1-8. Cloning and sequencing were carried out according to standard methods (Sambrook et al., 1989).<tables id="tabl0008" num="0008"><table frame="none"><title><b>Table 6: List of those used for the PCR amplification of the functional variable regions of heavy and light immunoglobulin chains</b></title><tgroup cols="2" colsep="0" rowsep="0"><colspec colnum="1" colname="col1" colwidth="83mm" colsep="0" /><colspec colnum="2" colname="col2" colwidth="83mm" colsep="0" /><tbody><row><entry namest="col1" nameend="col1" align="left" valign="top">MVH1</entry><entry namest="col2" nameend="col2" align="left" valign="top">(GC) AG GTG CAG CTC GAG GAG TCA GGA CCT</entry></row><row><entry namest="col1" nameend="col1" align="left" valign="top">MVH2</entry><entry namest="col2" nameend="col2" align="left" valign="top">GAG GTC CAG CTC GAG CAG TCT GGA CCT</entry></row><row><entry namest="col1" nameend="col1" align="left" valign="top">MVH3</entry><entry namest="col2" nameend="col2" align="left" valign="top">CAG GTC CAA CTC GAG CAG CCT GGG GCT</entry></row><row><entry namest="col1" nameend="col1" align="left" valign="top">MVH4</entry><entry namest="col2" nameend="col2" align="left" valign="top">GAG GTT CAG CTC GAG CAG TCT GGG GCA</entry></row><row><entry namest="col1" nameend="col1" align="left" valign="top">MVH5</entry><entry namest="col2" nameend="col2" align="left" valign="top">GA (AG) GTG AAG CTC GAG GAG TCT GGA GGA</entry></row><row><entry namest="col1" nameend="col1" align="left" valign="top">MVH6</entry><entry namest="col2" nameend="col2" align="left" valign="top">GAG GTG AAG CTT CTC GAG TCT GGA GGT</entry></row><row><entry namest="col1" nameend="col1" align="left" valign="top">MVH7</entry><entry namest="col2" nameend="col2" align="left" valign="top">GAA GTG AAG CTC GAG GAG TCT GGG GGA</entry></row><row><entry namest="col1" nameend="col1" align="left" valign="top">MVH8</entry><entry namest="col2" nameend="col2" align="left" valign="top">GAG GTT CAG CTC GAG CAG TCT GGA GCT</entry></row><row><entry namest="col1" nameend="col1" align="left" valign="top">MULK1</entry><entry namest="col2" nameend="col2" align="left" valign="top">GGG GAG CTC CAC CAT GGA GAC AGA CAC ACT CCT GCT AT</entry></row><row><entry namest="col1" nameend="col1" align="left" valign="top">MULK2</entry><entry namest="col2" nameend="col2" align="left" valign="top">GGG GAG CTC CAC CAT GGA TTT TCA AGT GCA GAT TTT CAG</entry></row><row><entry namest="col1" nameend="col1" align="left" valign="top">MULK3</entry><entry namest="col2" nameend="col2" align="left" valign="top">GGG GAG CTC CAC CAT GGA GWC ACA KWC TCA GGT CTT TRT A</entry></row><row><entry namest="col1" nameend="col1" align="left" valign="top">MULK4</entry><entry namest="col2" nameend="col2" align="left" valign="top">GGG GAG CTC CAC CAT GKC CCC WRC TCA GYT YCT KGT</entry></row><row><entry namest="col1" nameend="col1" align="left" valign="top">MlgG1</entry><entry namest="col2" nameend="col2" align="left" valign="top">TAT GCA ACT AGT ACA ACC ACA ATC CCT GGG</entry></row><row><entry namest="col1" nameend="col1" align="left" valign="top">MUVK1</entry><entry namest="col2" nameend="col2" align="left" valign="top">CCA GTT CCG AGC TCG TTG TGA CTC AGG AAT CT</entry></row><row><entry namest="col1" nameend="col1" align="left" valign="top">MUVK2</entry><entry namest="col2" nameend="col2" align="left" valign="top">CCA GTT CCG AGC TCG TGT TGA CGC AGC CGC CC</entry></row><row><entry namest="col1" nameend="col1" align="left" valign="top">MUVK3</entry><entry namest="col2" nameend="col2" align="left" valign="top">CCA GTT CCG AGC TCG TGC TCA CCC AGT CTC CA</entry></row><row><entry namest="col1" nameend="col1" align="left" valign="top">MUVK4</entry><entry namest="col2" nameend="col2" align="left" valign="top">CCA GTT CCG AGC TCC AGA TGA CCC AGT CTC CA</entry></row><row><entry namest="col1" nameend="col1" align="left" valign="top">MUVK5</entry><entry namest="col2" nameend="col2" align="left" valign="top">CCA GAT GTG AGC TCG TGA TGA CCC AGA CTC CA</entry></row><row><entry namest="col1" nameend="col1" align="left" valign="top">MUVK6</entry><entry namest="col2" nameend="col2" align="left" valign="top">CCA GAT GTG AGC TCG TCA TGA CCC AGT CTC CA</entry></row><row><entry namest="col1" nameend="col1" align="left" valign="top">MUVK7</entry><entry namest="col2" nameend="col2" align="left" valign="top">CCA GTT CCG AGC TCG TGA TGA CAC AGT CTC CA</entry></row><row><entry namest="col1" nameend="col1" align="left" valign="top">MULH1</entry><entry namest="col2" nameend="col2" align="left" valign="top">GGG CTC GAG CAC CAT GGR ATG SAG CTG KGT MAT SCT CTT</entry></row><row><entry namest="col1" nameend="col1" align="left" valign="top">MULH2</entry><entry namest="col2" nameend="col2" align="left" valign="top">GGG CTC GAG CAC CAT GRA CTT CGG GYT GAG CTK GGT TTT</entry></row><row><entry namest="col1" nameend="col1" align="left" valign="top">MULH3</entry><entry namest="col2" nameend="col2" align="left" valign="top">GGG CTC GAG CAC CAT GGC TGT CTT GGG GCT GCT CTT CT</entry></row><row><entry namest="col1" nameend="col1" align="left" valign="top">3'MUCK</entry><entry namest="col2" nameend="col2" align="left" valign="top">GCG CCG TCT AGA ATT AAC ACT CAT TCC TGT TGA A</entry></row></tbody></tgroup></table></tables>

Literature:

<ul id="ul0004" list-style="none"><li><b>Coller &amp; Coller,</b><nplcit id="ncit0013" npl-type="s"><text>1983: Coller, HA, Coller, BS, Meth. Enzymol. 121: 412-417</text></nplcit></li><li><b>Harlow &amp; Lane,</b><nplcit id="ncit0014" npl-type="b"><text>1988: Harlow, E., Lane, D., Antibodies: A laboratory manual, Cold Spring Harbor Laboratory, New York</text></nplcit></li><li><b>Kearney et al.,</b><nplcit id="ncit0015" npl-type="s"><text>1979: Kearney, J. Immunol. 123: 1548-1550</text></nplcit></li><li><b>Laemmli,</b><nplcit id="ncit0016" npl-type="s"><text>1970: Laemmli, EK, Nature 227: 680-685</text></nplcit></li><li><b>Peters &amp; Baumgarten,</b><nplcit id="ncit0017" npl-type="b"><text>1990: Peters, JH, Baumgarten, H. (ed.), Monoclonal Antibodies, Springer Verlag, Berl</text></nplcit>in</li><li><b>Fägerstam et al.,</b><nplcit id="ncit0018" npl-type="s"><text>1990: Fägerstam, LG et al., J. Mol. Recognit. 3: 208-214</text></nplcit></li><li><b>Malmqvist,</b><nplcit id="ncit0019" npl-type="s"><text>1996: Malmqvist, M., Methods 9: 525-532</text></nplcit></li><li><b>Eschweiler et al.,</b><nplcit id="ncit0020" npl-type="s"><text>1993: Eschweiler, B., et al., Zentralbl. F. Bakt. 280: 73-85</text></nplcit></li><li><nplcit id="ncit0021" npl-type="b"><text>Pharmacia Biotech, 1994: Monoclonal Antibody Purification Handbook</text></nplcit></li><li><nplcit id="ncit0022" npl-type="s"><text>Chomczynski, 1987: Anal. Biochem. 162: 156-159</text></nplcit>.</li><li><nplcit id="ncit0023" npl-type="b"><text>Sambrook et al., 1989: Molecular cloning: A laboratory manual, Cold Spring Harbor Laboratory Press, second editi</text></nplcit>on</li><li><nplcit id="ncit0024" npl-type="s"><text>Vaughan et al., 1998: Nature Biotechnology 16: 535-539</text></nplcit></li><li><nplcit id="ncit0025" npl-type="s"><text>Orlandi et al., 1989: Proc. Natl. Acad. Sci USA 86: 3833-3837</text></nplcit></li><li><nplcit id="ncit0026" npl-type="b"><text>Janeway &amp; Travers, 1997: Immunology, 2nd edition, Spektrum Akademischer Verlag GmbH, Heidelberg</text></nplcit></li><li><nplcit id="ncit0027" npl-type="s"><text>Osborne et al., 1997: Curr. Opin. Chem. Biol. 1: 5-9</text></nplcit></li><li><nplcit id="ncit0028" npl-type="s"><text>Stull and Szoka, 1995: Pharm. Res. 12: 465-483</text></nplcit></li></ul>

Annex to the registration documents - subsequently submitted sequence listing

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