Immunological test kit.

0001The invention relates to an immunological test kit for the detection of autoantibodies against collagen in serum and in synovial fluids. This detection of antibodies against the body's own collagens, i.e. autoantibodies, allows statements to be made for the diagnosis of diseases, especially those of the rheumatic type (e.g. chronic polyarthritis, rheumatoid arthritis, lupus erythematosus, Sjörgen syndrome, Bechterew's disease, scleroderma, etc.)

0002A common detection method for autoantibodies to date is the antiglobulin consumption test (AGK test). This method is technical and very complex to implement. Therefore, it often leads to untrustworthy results.

0003Further rheumatism tests currently being carried out are based on the detection of rheumatoid factors (RF). This is a heterogeneous group of autoantibodies against various endogenous gamma globulins. However, these rheumatoid factors do not occur in all rheumatic affections (seropositive / seronegative). In contrast, they can also be detected in healthy people and in non-rheumatic diseases. A large number of qualitative and quantitative methods are known for the diagnostic determination of the rheumatoid factors, which are often very complex and not very specific (Waaler-Rose test, Waller test, latex fixation test, passive hemaglutination test (K. Fehr, A. Böni, " Joints - Chronic Polyarthritis ", in" Immunology ", edited by KO Vorländer, 1983, pp. 465-499)).

0004For differential diagnosis, especially in chronic polyarthritis (cP), antinuclear factors, autoantibodies against cell nucleus components (DNA, histones, nucleolus RNA, etc.) are determined. Tests for C-reactive protein and for determining the antistreptolysin titer are also used in rheumatism diagnostics.

0005It is known from the literature that autoantibodies against the body's own collagens occur in diseases of the rheumatic type, both in serum and in synovial fluids (C.Steffen et al., Klin. Wschr. 51, 222-229 (1973); C. Steffen, Z. Rheumatol. 37, 131-147 (1978)). These autoantibodies therefore represent a medically interesting diagnostic parameter.

0006The invention is based on the object of providing an immunological test kit for the diagnosis of diseases of the rheumatic type, in which an assay can be carried out in a technically simple manner. The required amounts of serum or synovial fluids should be small. The sensitivity of the detection must be as high as possible and the specificity must be directly verifiable. In addition, both a simple qualitative and a quantitative analysis should be possible using this assay.

0007This object is achieved with an immunological test kit of the aforementioned type, comprising<ul id="ul0001" list-style="none"><li>a) a carrier on which the collagen is immobilized,</li><li>b) an enzyme-labeled anti-antibody reagent</li><li>c) a substrate which is capable of enzyme-catalyzed dye formation.</li></ul>

0008The immunological test kit according to the invention is based on the following test method:<ul id="ul0002" list-style="none"><li>a) An antigen (collagen) is adsorbed and immobilized on a suitable carrier material.</li><li>b) Autoantibodies contained in the patient's serum or synovial fluid are bound to the immobilized antigen (collagen).</li><li>c) The autoantibody bound to the antigen is detected.</li></ul>

0009According to the invention, the immunological test kit is suitable for different test variants. Different collagens can be immobilized on the carriers. Human collagens of types I to VI are particularly preferred. Calf collagen (predominantly type I) can also be used. An examination of the type of collagen against which the autoantibody is directed allows additional statements to be made for medical diagnostics.

0010Commercial microtiter plates (for example from Greiner, Nunc, Dynatech, Miles, Millipore) are particularly suitable as carriers in the immunological test kit according to the invention. These are preferably made of a polystyrene material. Commercially available special tubes can also be used. Carrier materials made of nitrocellulose or other suitable materials which are capable of binding the antigen can also be used, for example Diazobenzyloxymethyl (DBM) or diazophenylthioether materials (DPT). All commercially available carriers are suitable for carrying out the ELISA method; the last-mentioned carriers are particularly preferred for the bloting method.

0011The detection of the autoantibody bound to the immobilized antigen is carried out by means of an anti-antibody which is additionally provided with an enzyme. This enzyme catalyzes the conversion of initially colorless substrate molecules into a colored end product. The dye formed here can be determined visually, which gives a purely qualitative yes-no statement. However, it can also be determined quantitatively by means of suitable measuring methods, as a result of which a semi-quantitative or quantitative statement is obtained.

0012According to the invention, the immunological test kit is suitable for different test variants. For the type of detection system, the enzyme-linked immunosorbent assay (ELISA) as well as the protein blot assay and the protein dot blot assay come into consideration.

0013The ELISA is based on the following principles (E. Engvall; Methods in Enzymology 70, 419-439 (1980)): An antigen is adsorbed on a suitable surface. The immobilized antigen is incubated with the solutions to be tested for antibodies. Any antibodies that are present bind to the antigen. With the help of an enzyme-labeled anti-antibody, which in turn recognizes the bound antibody, the antibody directed against the antigen can then be detected with an enzyme reaction by a color change. Positive and negative controls carried out in parallel guarantee the accuracy of the evidence.

0014However, the ELISA method does not allow to see directly which antigen an antibody is directed against and therefore only allows certain statements to be made about the specificity of the detection reaction. With the help of the protein blot technique, the specificity of the reaction measured in the ELISA can be examined. Proteins are separated according to their molecular weight by means of polyacrylamide gel electrophoresis and then transferred electrophoretically to nitrocellulose paper, to which they bind with high affinity. The remaining free binding sites on the nitrocellulose are then saturated with a solution containing a carrier protein (for example bovine serum albumin). The nitrocellulose is then incubated in an antibody-containing solution. The antibodies bind to the antigen. With the help of the enzyme-labeled anti-antibody already mentioned and a precipitable enzyme substrate, the antigen is detected in a color reaction.

0015In the protein dot blot assay, the protein is adsorbed directly onto nitrocellulose, to which all proteins strongly bind, without prior gel-electrophoretic separation. Then proceed as for the protein blot assay. The substrate can be selected so that it results in a water-soluble or a water-insoluble, precipitating dye in the enzyme-catalyzed reaction. The positive reaction can then be recognized either by the color change of the substrate solution or by the staining protein stain on the paper (test strips for a simple yes-no statement):<ul id="ul0003" list-style="none"><li>Blot variants can also be used according to the invention. Here, a test strip with immobilized antigen, autoantibody bound to it and attached enzyme-labeled antibody is introduced into a suitable substrate solution. This creates a soluble dye that does not attach to the test strips. The resulting dye solution can then be quantitatively evaluated without any problems.</li></ul>

0016The substrate for the enzyme-catalyzed dye reaction and thus the resulting dye can be varied. Preferred substrates are those which are capable of forming soluble dyes, for example 3, 3 ', 5, 5'-tetramethylbenzidine, 0.004% H₂0₂ in 100 mM sodium acetate pH 6 when using horseradish peroxidase or preferably 0.1% p-nitrophenyl phosphate, 10% diethanolamine-HC1, pH 9.8, 5 mM MgCl₂ when using alkaline phosphatase. In the same way, substrates are suitable which form precipitating dyes, for example 10% diethanolamine-HCl, pH 9.8, 0.01% nitro blue tetrazolium, 0.005% 5-bromo-4-chloro-3-indolyl phosphate, 4 mM MgCl; 0.25 mg / ml 3, 3^1-Diaminobenzidine, 0.015% H₂0₂ in 50 mM Tris-HCl pH 7. The various substrates used are all commercially available.

0017Soluble dyes are normally preferred in the ELISA technique, since they can easily be quantified photometrically. Blot techniques, on the other hand, use insoluble dyes so that the individual "bands" remain recognizable and the dye is not distributed over the entire support. In this case, quantitative determinations are more complex.

0018Antibodies of the IgM class are basically first formed by the immune system, which are then replaced by IgG antibodies. The immunoglobulin class of the autoantibody can be determined with the help of appropriate anti-antibodies (anti-IgG, anti-IgM). It may be possible to make statements about the course of the disease. Antibodies, against human IgG (y chain specific; IgG_1-4) or IgM (u chain-specific) are commercially available, for example from Sigma, Dako, Behringwerken and Boehringer, Mannheim.

0019The test substances for positive and negative controls are the substances customary in the present field. In particular, a human serum which does not contain antibodies against collagens is used as a negative control in the two test systems used here, so that there should be no change in color.

0020A mixture of patient sera containing autoantibodies serves as a positive control. This serum mixture is adjusted so that it causes a defined color change. This color change then serves as a reference value for the sera to be measured with an unknown autoantibody content.

0021The carrier can be provided with any amount of collagen; when using microtiter plates, it is preferred to use at least 400 ng protein per cavity, preferably 400 ng - 1000 ng protein / cavity.

0022A test system according to the invention is manufactured and used as follows:<ul id="ul0004" list-style="none"><li>To coat a carrier with collagens, this is in a solution of collagens in a buffer 8 to</li><li>16 Hours, preferably 12 hours, incubated at 37 ° C. Buffers suitable for this purpose are, for example</li><li>15 mM Na₂CO₃, 34 mM NaHCO₃, pH 9.6; or 0.12 M NaCl, 0.01 M Na₂HPO₄, 0.004 M KH₂P0₄, ^pH 7.4.</li></ul>

0023Then the cavities of the carrier are washed at least once with a washing buffer, for example 0.12 m NaCl, 0.01 m Na₂HPO₄, 0.004 m KH₂P0₄, pH 7.8 (PBS solution), or 50 mM Tris-HCl, pH 7.4, 100 mM NaCl.

0024The wash buffer solutions preferably contain Tween 20 ^R, for example 0.05% by weight. The effect of the washing buffer can be improved even further by adding sodium chloride. Tween 20^R can be replaced by 0.1% NP40, 0.1% Triton X-100 or Brij 59, for example. The former buffer is preferred.

0025After the washing process, incubation with patient sera at room temperature is carried out for several hours, preferably 1 to 4 hours. The patient sera are used diluted in a buffer, the dilution is 1:10 - 1: 2000, preferably 1:20 - 1: 100. Suitable dilution buffers are the above-mentioned washing buffers in combination with 1% bovine serum albumin or 10% fetal calf serum, 100 mM NaCl, 10 mM Tris-HCl, pH 7.4; or 3% gelatin, 100mM NaCl, 10mM Tris-HCl, pH 7.4. After the incubation, wash again with one of the wash buffers mentioned above.

0026This is followed by incubation for several hours, preferably 2 hours, with an antibody conjugate diluted in a buffer, eg anti-human IgG or IgM conjugated with alkaline phosphatase or a-human IgG or IgM conjugated with horseradish peroxidase. Incubation takes place at room temperature, the conjugate dilution is 1: 250-1: 1000, preferably 1: 400-1: 600, most preferably 1: 500.

0027It is washed again with one of the aforementioned washing buffers. The reaction then develops, using the substrates and dyes mentioned above. The substrates and dyes are incubated in 10% diethanolamine-HC1 pH 9.8; 5 mM NaCL₂were dissolved with the antigen-antibody complexes formed in the previous steps for 20-30 minutes at 37 ° C.

0028With the test kit according to the invention, the detection of autoantibodies against collagen and the titer determination, for example using the ELISA technique, can be carried out easily and reproducibly. With this test one can differentiate between immunoglobulins of the class IgG and IgM. The specificity of this procedure can be checked directly using protein blot technology.

0029The test kit according to the invention is also suitable for the rapid detection of autoantibodies with a test strip. Here you get a yes-no statement. However, the test kit is also ideal for semi-quantitative and quantitative determinations.

Example 1 1

0030A test system according to the invention was produced. Highly pure human collagens (type I - VI), which are in buffer A (15 mM Na₂ C0₃; 34 mM NaHC0₃; pH 9.6) were incubated for 12 hours at 37 ° C. in Greiner microtiter plates (microtiter plate, F-form 655101). The wells of the microtiter plate were then triple with buffer B (0.12 M NaCL, 0.01 M Na₂HP0₄; 0,004 _M KH₂ PO₄; 0.05% Tween 20^R; pH 7.8). The washing process was followed by incubation for two hours with patient sera, diluted in buffer C (buffer B, 1% bovine serum albumin) at room temperature. After subsequent washing of the wells with buffer B, the two-hour incubation was carried out with the anti-antibody conjugate diluted in buffer C (here anti-human-IgG or -IgM conjugated with alkaline phosphatase) at room temperature. After washing again, the reaction was carried out with buffer D (0.1% p-nitrophenyl phosphate; 10% diethanolamine HC1; pH 9.8; 5 mM MgCl₂) developed. Both the amount of collagen adsorbed on the cavity and the dilution of the anti-antibody conjugate are crucial parameters for the detection reaction. It could be demonstrated that 400 ng collagen adsorbed on the cavity and a conjugate dilution of 1: 500 lead to good results in the test system described here.

Example 2

0031The tests for autoantibodies against collagens of human origin described in Example 1 can also be carried out using the protocol described under 1 in a polystyrene tube (eg Nunc Star Tubes). The results are identical to those mentioned in Example 1.

Example 3

0032After the preparation of the ELISA protocol using the test system according to Example 1, human sera were tested for autoantibodies against the various collagens I to VI. The serum dilution was 1:50. The results of the tests are shown in FIGS. 1 and 2. Duplicate determinations were carried out to check the reproducibility of the assay. There were significant differences in the reactions of the individual patients to human collagens I, II and III (Fig.l). The reactions were weaker against collagens IV, V and VI (Fig. 2). In these experiments, the antibodies of individual patients against bacterial membranes are measured as a positive control, the reactions against bovine serum albumin and the reactions with normal patient serum against the collagens as negative controls.

0033The sera were titrated against the collagens. This resulted in very different serum titers in the individual patients. Logarithmic serum dilutions with collagen I-VI were tested in an ELISA. All of these experiments served for the detection of autoantibodies of the IgG class.

0034The level of the serum titer can be important for diagnostic purposes. An assessment of the status of the disease is then possible.

Example 4

0035The detection of autoantibodies is not only possible with the help of human collagens. Calf collagens also allow the display of autoantibodies in the ELISA test system described in Example 1. For this purpose, 400 ng of calf collagen in buffer A were incubated for 12 hours at 37 ° C. in the cavities of the microtiter plate referred to in example 1 and proceeded according to the protocol. The results thus obtained were in good agreement with those listed in Example 3.

Example 5

0036In order to check the specificity of the reactions measured in the ELISA, protein blots according to the method described by Towbin et al. (PNAS 76, 4350-4354 (1979)). The detection was carried out with the aid of a dye solution (10% diethanolamine-HCl; pH 9.8; 0.01% nitro blue tetrazolium; 0.005% 5-bromo-4-chloro-3-indolyl phosphate; 4 mM MgCl₂). In fact, the autoantibodies could be shown to react directly with the collagen protein band. This is direct proof of the correctness of the results measured in the ELISA.

Example 6

0037Autoantibodies against collagens were also successfully detected in the form of a protein dot blot assay by applying the collagens directly to nitrocellulose paper strips. After the remaining protein binding sites of the nitrocellulose were saturated with the aid of a 1% bovine serum albumin solution, the test strips were incubated in serum for 30 minutes. After washing twice in buffer E (10 mM Tris-HCl; 0.9% NaCl). the test strip was incubated in conjugate solution and washed again. The mixture was then incubated for 5 minutes in the dye solution described in Example 5. A positive result was shown in a punctiform staining of the nitrocellulose paper. As a control, a test strip was incubated with normal serum and proceeded as described above.

Example 7

0038All of the application examples described so far can easily be transferred to the detection of immunoglobulins that do not belong to the IgG class. To do this, it is necessary to work with an antibody conjugate that recognizes the corresponding immunoglobulin class. Investigations were carried out here using the methods presented in Examples 1 to 6 for the detection of autoantibodies of the IgM class. The corresponding results are shown in FIG. 3. Indeed, there are those who do not have IgG-class autoantibodies against collagen, but do have IgM-class autoantibodies.

0039The results of the experiments explained in Examples 1 to 7 are summarized in the following table (AI patient sera; J, K negative control sera)<tables id="tabl0001" num="0001"><img file="EP0199261A2_D0001.tif" /></tables>