Nucleic acid hybridization assay

The present invention relates to reagent systems for detecting a particular polynucleotide sequence in an experimental medium by nucleic acid hybridization, and to the use of the reagent system.

The principle of nucleic acid hybridization assays was developed by those working in the field of recombinant DNA as a means of determining and isolating particular polynucleotide base sequences of interest. It was found that single chain nucleic acids, e.g. DNA and RNA, as present by denaturing the double-chain forms, will hybridize or recombine under appropriate conditions with complementary single-chain nucleic acids. By labeling such complementary probe nucleic acids with an easily detectable chemical group, it was made possible to detect the presence of any polynucleotide sequence of interest, with an experimental medium, which contains sample nucleic acids in single chain form.

In addition to the field of recombinant DNA, the analytical hybridization technique can be used in the detection of important polynucleotides in the field of human and veterinary medicine, agriculture, and food technology, among others. In particular, the technique can be used to detect and identify etiological agents such as e.g. bacteria and viruses, and examine bacteria for antibiotic resistance; to facilitate the diagnosis of genetic disorders such as sickle cell anemia and thalassemia, and to detect cancer cells. A general overview of the technique and its current and assumed future significance can be found in Biotechnology (August 1983), pages 471-478.

The following information is provided to make known information which is believed to be relevant in connection with the present invention.

The prior art regarding methods for nucleic acid hybridization analysis generally involves immobilizing sample nucleic acid on a solid bar. Hybridization between particular base sequences or genes of interest, in the sample nucleic acid, is determined by separating the solid support from the remainder of the reaction mixture containing unbound labeled probe, followed by detecting the label on the solid support /

The necessity of immobilizing sample nucleic acids to perform the hitherto known hybridization assays presents two significant problems. First, the procedures that must be performed to achieve immobilization are generally time consuming and constitute an additional step that is undesirable for routine use in a clinical laboratory. Second, proteins and other materials in the heterogeneous sample, especially in the case of clinical samples, can affect the immobilization of the sample nucleic acids.

As alternatives to immobilization of sample nucleic acids and addition of labeled probe, one can use an immobilized probe and label sample nucleic acid in situ, or one can use a dual hybridization technique that requires two probes, one of which is mobilized and the other labeled (Methods in Enzymology 65: 468 (1968): and

Gene 21: 77-86 (1983)). However, the first alternative is even less desirable because in-situ labeling of the sample nucleic acids places greater demands on technical skills than might be expected from clinical laboratory staff, and there is no simple, reliable method for monitoring labeling yields. This can be a significant problem if the labeling medium contains different amounts of inhibitory preparation for labeling reaction. The dual hybridization technique has the disadvantage that it requires an additional reagent and an additional incubation step, and the kinetics of the hybridization16630I reaction may be slow and inefficient. The accuracy of the assay can also be variable if the complementarity of the two probes with the sample sequence is variable.

Techniques for direct detection of the polynucleotide duplex formed as a result of the hybridization between the sample and the polynucleotides, thereby avoiding chemical labeling and immobilization of sample or probe polynucleotides, have generally not been satisfactory. Attempts to generate antibodies that will selectively bind the double-stranded DNA'DNA hybrid over the sing le-stranded DNA have failed (Parker and Halloran, Nucleic Acids in Immunology, ed. Plescia and Braun, Springer-Verlag, NY (1969) p. 18 et seq). Some progress has been made in generating antibodies that bind DNA-RNA blended hybrids or RNA'RNA hybrids and have low affinity for the single chain polynucleotides (see, e.g., Rudkin and Stollar, Nature 265: 472 (1977)). Rudkin and Stollar, attached whole cells to slides, and exposed DNA in the nucleus. It was hybridized with an RNA probe and the hybrid was detected by fluorescence by microscopy with fluorescein-labeled antibody to DNA · RNA. However, these methods described, as in the case of the hybridization techniques discussed above, using labeled probes, require immobilization and the sample nucleic acids. Immobilization of cellular DNA for in situ hybridization is particularly difficult because DNA must remain attached to sensitive cellular residues during hybridization and the immunochemical detection steps. The results observed by fluorescence microscopy do not provide quantitative data regarding the amount of hybrid formed.

Accordingly, there is a need for a nucleic acid hybridization assay that does not require immobilization or labeling of sample nucleic acids, and that does not require dual probes. Furthermore, such a technique should allow the use of a number of brands, especially of it not_tradioisotope types. A method of nucleic acid hybridization analysis and a reagent system having these and other advantages are the main objects of the present invention.

A method has now been developed for nucleic acid hybridization analysis which eliminates the need to immobilize or label sample nucleic acids and which requires only a single probe element.

The present invention provides: a reagent system for detecting a particular polynucleotide sequence in a test medium by nucleic acid hybridization, including combining the test medium with a polynucleotide. probe comprising at least one single-chain base sequence which is substantially complementary to the sequence to be determined, under conditions which promote hybridization between the sequence to be determined and the complementary probe sequence, the complementary probe sequence (i) being substantially composed of RNA when the sequence to be determined is RNA or DNA, or (ii) essentially consisting of DNA or RNA, when the sequence to be determined is RNA, and hybridized probe is detected by the addition of an antibody reagent capable of binding to DNA-RNA duplexes formed between the sequence to be determined and the complementary probe sequence and determination of the antibody reagent bound to such duplexes.

The reagent system is characterized in that the probe is in an immobilized form or includes a binding site for a specific binding substance, and when the latter is the case, the reagent system further comprises an immobilized form of a specific binding substance.

The present invention further encompasses the use of the reagent system for detecting a particular polynucleotide sequence in nucleic acid hybridization.

The resulting hybrids can then be detected, after, or simultaneously with, immobilization of the probe where it was combined with the test medium in an immobilized form, with the addition of an antibody reagent capable of binding to the DNA 'RNA or RNA' RNA duplexes which are formed, and determining the antibody reagent that binds to such duplexes. A variety of methods and combinations of reagents can be used to implement the principles of the present method. Important features of the present invention are that the sample nucleic acids are not immobilized or must be labeled before being brought into contact with the probe.

The antibody reagent is the key to specific and sensitive detection of hybridization between the probe and the sample nucleic acids. Of course, whole antibodies or suitable fragments and polyfunctional forms thereof can be used as described below, and it should be noted that when the term antibody reagent is used in the present specification and claims, it means both whole antibodies and their polyfunctional and fragmented forms, unless otherwise indicated. .

Determination of binding between antibody reagents and hybridization duplexes can be achieved by any convenient means. It is preferred that the antibody reagent be labeled with a detectable chemical group such as e.g. an enzymatically active group, a fluorescent group, a chromophoric group, a luminescent group. A specifically binding ligand, or a radioisotope, non-radioisotope labels are especially preferred. The labeled antibody reagent that binds to the resulting immobilized hybrid duplexes can be easily separated from the antibody that does not bind, and the detectable chemical group or label is measured in one of the separate fractions, usually in the former.

By eliminating the need to immobilize or label the sample nucleic acids, the present invention provides a very advantageous hybridization technique for analysis. A high degree of technical skill is not required of the person performing the analysis, further saving the time that would have been required to perform the immobilization or the marking procedures. Furthermore, the possibility of interference between the sample and the immobilization procedure is completely eliminated. The test kit provided to the clinical user includes the probe in an already immobilized, or in an easily immobilizable, form, such as e.g. by binding to an immobilized binding partner.

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In the prior art systems, interference from foreign proteins and other materials in the sample can be a serious problem, whether the sample nucleic acids to be immobilized are RNA or DNA.

In the prior art methods, immobilization is achieved by absorption on a microporous membrane, such as e.g. nitrocellulose, or by covalent attachment to reactive positions, on a solid support. In the first case, proteins from the sample can cover the surface and block the absorption of nucleic acids. Furthermore, many methods require heating to elevated temperatures, usually higher than 80 ° C, in vacuo, to attach absorbed nucleic acids to the support. If mucus or other materials endogenous to the sample are present, they can be dried on the support to form a film which can absorb the labeled probe during hybridization and increase the background signal and consequently decrease the sensitivity. Furthermore, if an enzyme or other protein is involved in this section of the label, it can often bind non-specifically to the film and further contribute to the background signal problem. If covalent immobilization is used, proteins and others can

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materials from the sample are expected to have available reactive groups that will take part in the coupling reaction and neutralize the coupling of the desired nucleic acids.

₅ Since the present invention provides the probe in preferred embodiments in an already immobilized form, or in a form that can be easily immobilized by binding to an immobilized binding partner, the disadvantages associated with the previously known IQ known immobilization methods are overcome and consequently the detection limits of the assay are maintained.

A further claim is that non-specific binding of sample RNA or DNA to the solid support will not be recognized by the antibody reagent. Consequently, the background signal will be low and the detection limit will be improved accordingly. In the dual hybridization method using both a labeled probe and an immobilized probe, the labeled nucleotide may bind non-specifically to the solid support, contributing to the background signal. This is not possible with the present method since no label probe is used.

The figures are schematic representations of preferred methods for carrying out the present method. The use of nucleic acid hybridization as an analytical tool is fundamentally based on the double-chain, duplex structure of DNA. The hydrogen bonds between the purine and pyrimidine30 bases in the respective chains of the double-stranded DNA can be reversibly broken. The two complementary single chains of DNA that result from this melting or denaturation of DNA will be associated (also referred to as recombination or hybridization) so that the duplex structure is restored. It is now well known that contacting a first single chain nucleic acid, either DNA or RNA, which includes a base sequence sufficiently complementary to a second single chain nucleic acid under suitable dissolution conditions will result in the formation of DNA-DNA, DNA- RNA, or RNA * RNA hybrids, depending on the zone.

In the embodiment shown in Figure 1, the single chain probe nucleic acids are contacted with the immobilized probe under advantageous hybridization conditions. The resulting immobilized, hybridized duplexes are contacted, optionally after separation of the duplexes from the rest of the reaction mixture, with a labeled form of antibodies specific for the DNA 'RNA or RNA' RNA duplexes. After washing to remove unbound labeled antibody, the label present on the solid support is measured.

In the embodiment shown in Figure 2 of the drawings, the single chain sample nucleic acids are contacted with a soluble form of the probe which has been chemically modified to include binding biotin moieties. To the resulting soluble hybrids formed, an immobilized form of avidin, a binding partner for biotin, is added, resulting in the formation of immobilized hybrids. The duplexes immobilized in this way, optionally after being separated from the rest of the reaction mixture, are contacted with labeled anti-hybrid antibodies._lfand after washing, the mark present on the solid support is measured in the same manner as above.

The probe includes at least one single-chain base sequence that is substantially complementary to the sequence to be detected. However, such a base sequence need not be a single continuous polynucleotide segment, but may consist of two or more individual segments interrupted by non-complementary sequences. These non-hybridizable sequences may be linear, or they may be self-complementary and form hairpin loops. In addition, the complementary region of the probe may be flanked at the 3 'and 5' terminals by non-hybridizable sequences, such as e.g. those that include DNA or RNA of a vector into which the complementary sequence was introduced for proliferation.

In each case, the probe, as present, as an analytical reagent, will show detectable hybridization at one or more points with sample nucleic acids of interest. Linear or circular single-chain polynucleotides can be used as probe elements, with larger or smaller parts duplexed with a complementary polynucleotide chain or chains, provided that the critical homologous segment or segments are in single-chain form and available for hybridization with sample DNA or RNA, and provided that the antibody reagent selected for use with the probe does not significantly cross-react with the double-stranded strands of the probe (i.e. where the antibody reagent is specific for the DNA-RNA hybrids and the probe includes RNA-RNA double-stranded regions, or vice versa). The complementary probe sequence may be of any suitable or desired length, ranging from as few as a dozen to as many as 10,000 bases, and including oligonucleotides having less than 50 bases.

The RNA or DNA probe can be obtained in a number of conventional ways. When, for example. In the case of RNA probes, RNA can be isolated as the natural product from cells, such as 5s, 16s, and 23s ribosomal RNAs from bacteria or cellular tRNAs. It is also convenient to isolate specific mRNAs from cells that s pecialize in the production of large amounts of a protein that mRNA encodes.

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In vitro synthesis of RNA probes can be achieved with a vector containing the highly active Salmonella typhimurium bacteriophage SP6 transcriptional promoter (Green et al. (1983) Cell 32: 681). A vector with multiple restriction endonuclease sites near the promoter is available from Promega Biotec, Madison, WI. A DNA probe is cloned into the vector which is then grown in a bacterial host. Multiple RNA copies of the cloned DNA probe can be synthesized in vitro using DNA-dependent RNA polymerase from bacteriophage SP6. '

DNA probes can be prepared from a variety of sources. An entire bacterial genome can be immobilized for a hybridization assay designed to detect bacteria in a typical sterile sample. The assays will be able to detect large amounts of bacterial RNAs such as e.g. ribosomal RNAs and tRNAs. Alternatively, specific DNA sequences complementary to cellular RNA s can be cloned into well-known plasmids or viral vectors and used as hybridization probes.

It should be noted that in the use of the terms RNA probe¹'and DNA probe, it is not suggested that all the nucleotides included in the probe must be ribonucleotides or 2'-deoxyribonucleotides. The fundamental feature of the RNA or DNA probe for the purposes of the present invention is that it is of such a nature that it allows the stimulation of antibodies to DNA 'RNA or RNA' RNA hybrids including an RNA or DNA probe which does not cross-react. to an analytically significant degree with the individual individual chains constituting such hybrids. Therefore, one or more of the 2 'positions can be on

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06301 the nucleotides included in the sample must be chemically modified, provided that the antibody binding properties required for the present assay are substantially maintained. Similarly, in addition to or alternatively to such limited 2 'deoxy modification, a probe may generally have any other modification along the ribose phosphate backbone provided that there is no significant interference with the specificity of the antibody for the double chain hybridization product, as compared to its individual single chains.

Where such modification exists in an RNA or DNA probe, the immunogen used to grow the antibody reagent should preferably include a chain having substantially similar modification and another chain that is substantially unmodified RNA or DNA, depending on whether sample RNA or DNA must be detected. Preferably, the modified chain of the immunogen should be identical to the modified chain of an RNA or DNA probe. An example of an immunogen is hybrid poly (2'-O-methyladenyl acid) * poly (2'-deoxythymidyl acid). Another example is poly (2'-O-ethylinosinic acid) 'poly (ribocytidyl acid). The following are further examples of modified nucleotides that may be included in a modified probe:

2' -O-methylribonucleotide,

2'-O-ethylribonucleotide,

2'-azidodeoxyribonucleotide,

2'-chlorodeoxyribonucleotide,

2'-O-acetylribonucleotide, and the phosphorothiolates or methylphosphonates of ribonucleotides or deoxyribonucleotides. Modified nucleotides

2 may appear in probes as a result of introduction, during enzymatic synthesis, of the probe from a template. For example. are adenosine 5'-O- (1-thiotriphosphate) (ATPaS) and dATPaS substrates for DNA-dependent RNA polymerases and DNA polymerases, respectively. Alternatively, the chemical modification may be introduced after the probe has been prepared. For example. For example, an RNA probe 2'0-acetylated with acetic anhydride under mild conditions in an aqueous solvent (Steward, DL et al., (1972) Biochim. Biophys. Acta 262: 227).

The critical property of an RNA or DNA probe to be used here is that antibodies grown against the probe duplexed with a complemen tary RNA or DNA chain, if desired, must have binding properties different from the duplexed form of the probe and single chain. nucleic acids. It is this property that enables the detection of hybridized probe in the assay mixture without significant background binding to the unhybridized single chain form of the probe or any non-specifically bound single chain sample nucleic acids. As described above, certain modifications along the ribonucleotide or deoxyribonucleotide chain can be tolerated without loss of the ability of the antibody to distinguish the duplex form from single chains. However, it is generally preferred to use RNA probes consisting exclusively of ribonucleotides when the sample nucleotide is an RNA or DNA. DNA probes can be used to advantage when the sample is RNA.

As described above, the probe will be hybridized to sample nucleic acids in either immobilized, or an immobilizable, form.

An immobile isert form of the probe will be one in which the probe can be conveniently rendered immobile after the hybridization reaction. The method by which the probe is ultimately immobilized is not critical to the present invention, and any available method can be used, as long as the hybrid formed between the probe and the sequence of interest is rendered immobile by a property of the probe. Consequently, sample nucleic acids are not subjected to direct immobilization.

When present in a hybridization reaction on an immobilized form, the probe may be in any suitable form that allows the probe, and any components of the reaction mixture that has been associated with it, by hybridization and / or by binding of the anti-hybrid reagent. , can then be isolated or separated from the rest of the mixture as e.g. by centrifugation, filtration, chromatography, or decantation. Accordingly, a variety of compositions and configurations for an immobilized sample will be apparent and available to those skilled in the art. Virtually any form of probe that is insoluble in the reaction mixture can be used. For example. the probe may be aggregated or otherwise precipitated, associated with an insoluble material, polymer, or carrier, or be in a gel such as e.g. agarose or polyacrylamide (see Meth.

Enzymol. 12B: 635 (1968) and PNAS 67: 807 (1970)).

It is especially preferred to use a solid support to which the probe is attached or attached with covalent or non-covalent bonds, the latter including absorption methods which provide a suitable stable and strong compound. The solid support can take a variety of forms and compositions, including microparticles, beads, porous and impermeable strips and membranes, the inner surface of reaction vessels such as e.g. test tubes and microtiter plates and the like Devices for attaching a desired reaction partner to a selected solid support are a matter of routine skill for those skilled in the art.

One method of absorbing the probe on nitrocell ulose membranes involves saturating a solution of the probe with sodium iodide and spraying or filtering on the membrane (Bresser et al. (1983) DNA 2: 243). The sodium iodide facilitates the denaturation of the probe and improves the absorption on the membrane. Alternatively, the probe can be treated with glyoxal, usually at concentrations of approx. 1 molar (M) and then absorbed on the membrane. The probe is attached by heating to approx. 80 ° C under vacuum, for a period of time ranging from 2 to 4 hours. (Thomas, PS, (1983) Meth, in En2ymol. 100: 255).

Covalent immobilization of RNA or DNA probes can also be achieved. A wide variety of carrier materials and coupling techniques can be used. For example. the probe can be linked to phosphorus cellulose, through phosphate groups activated with carbodiimide or carbonylimidazole (Bautz, EKF, and Hall, BD, (1962) Proc. Nat'l. Acad. Sci. USA 48: 400-408; Shih, TY, and Martin, MA, (1974) Biochem. 13: 3411-3418).

Fu rthermore, diazo groups on m-diazobenzoyloxymethylcellulose can be reacted with quanine and thymidine residues on the polynucleotide (Noyes, BE, and Stark, GR, (1975) Cell 5: 301-310; Reiser, J., et al., (1978 ) Biochem. Biophys. Res. Commun. 85: 1104-1112). Polysaccharide carriers can also be used by coupling through phosphorus diester chains formed between the terminal phosphate on the polynucleotide and the carrier hydroxyl15 ί 06301 groups by water-soluble carbodiimide activation (Richwood, D., (1972) Biochim. Biophys. Acta 269: 47-50; Gilham, PT, (1968) Biochem. 7: 2809-2813), or by coupling nucleophilic positions on the polynucleotide with a cyanogen bromide-activated carrier (Arndt-Jovin, DJ, et al., (1975) Eur.

J. Biochem. 54: 411-418; Linberg, U., and Eriksson, S., (1971) Eur. J. Biochem. 18: 474-479).

Furthermore, the 3 'hydroxyl terminal of the probe can be oxidized with periodate and coupled by Shiff base formation with carriers containing admiral or hydrazide groups (Gilham, PT, (1971) Method. Enzymol. 21: 191-197; Hansske, HD, et al., ( 1979) Method Enzymol 59: 172-181). Carriers having nucleophilic positions can be reacted with cyanuric chloride and then with the polynucleotide (Hunger, HDm et al., (1981) Biochim. Biophys. Acta 653: 344-349).

In general, any method can be used to immobilize the probe, provided that the complementary single chain sequence is available for hydrolysis to sample nucleic acid. Particular methods or materials are not critical to the present invention.

A particularly advantageous alternative to using a directly immobilized probe is to use an immobilizable form of the probe which allows the hybridization to proceed in solution where the kinetics are faster. Normally, in such an embodiment, a probe will be used which includes a reactive position capable of forming a stable covalent or non-covalent bond with a reactant and achieving immobilization by exposure to an immobilize d form of such a reactant. Preferably, such a reactive position of the probe is a binding position such as e.g. a biotin or hapten moiety capable of specific non-covalent bonding with a binding substance, such as e.g. avidin or an antibody that serves as a reaction partner.

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Virtually any pair of substances can constitute the pair reactive position / reactive partner which shows a suitable affinity for interaction so that a stable bond is formed, i.e. a compound or link between the two which remains substantially intact during the subsequent analysis steps , mainly the separation and detection steps. The bond formed may be a covalent bond or a non-covalent interaction, the latter being preferred especially when characterized by a degree of selectivity or specificity. In the case of such preferred bond formation, the reactive position on the probe will be referred to as a binding position and the reaction partner as a binding substance with which it forms a non-covalent, usually specific, bond or entanglement.

In such a preferred embodiment, the binding site may be present in a single chain hybridizable portion or in a single or double-chain non-hybridizable portion of the probe, or it may be present as a result of a chemical modification of the probe. Examples of binding sites found in the polynucleotide sequence where the probe includes a promoter sequence, (e.g. lac promoter, trp promoter), which is binding to a promoter protein (e.g. bacteriophage promoter, RNA polymerase), or includes an operator sequence (e.g., lac operatives) that is binding to a repressor protein (e.g., lac repressor), or that includes rare antigenic nucleotides or sequences (e.g., -bromo- or 5-iododeoxyuridine, Z-DNA) which are binding by specific antibodies (see also British Patent No. 2,125,964). Binding positions introduced by chemical modification of the polynucleotide included in the probe are particularly useful and normally involve attaching a portion of a specific binding pair to the probe nucleic acid. Useful binding pairs to choose from include biotin / avidin (including egg white avidin and streptavidin), haptens and antigens / antibodies, carbohydrates / lectins, enzymes / inhibitors, etc. Where the binding pair consists of a proteinaceous moiety and a non-proteinaceous moiety, it is normally preferred to attach the non-proteinaceous moiety to the probe, since the proteinaceous moiety may be unstable under the denaturing conditions for hybridization of the probe. Preferred systems include coupling the probe to biotin or a hapten and using immobilized avidin or anti-hapten antibody reagent, respectively.

Where the probe is hybridized to the sequence of interest in an immobilizable form, the subsequent steps of immobilizing the duplexes formed by a property of the probe and adding the anti-hybrid antibody reagent may take place in any order. Immobilization and anti-hybrid batch can be performed by simultaneous addition of the included reagents and materials, or one may follow one after the other, with or without washing or separation steps between the main steps, in any order. When using stepwise addition, one will of course take into account the concentrations of the added reagents so that one does not supersaturate the hybrids formed and inhibit their interaction with the subsequently added materials.

Although immobilized probes or immobilizable probes that bind to solid supports by specific binding processes described above are preferred, the immobilizable probes can bind to carriers by processes of relatively low specificity.

In this case, the carrier will bind the hybridized probe, not the unhybridized form. The amount of the hybrid can then be measured with the antibody reagent. An example of a carrier of this type is hydroxyapitite which binds DNA'RNA and RNA'RNA duplexes, but not single-chain species (Brenner and Falkow, Adv. In Genet., 16:81 (1973)).

Furthermore, a chemically active, or activatable, group can be introduced into the probe and reacted with the solid support after hybridization. This system will yield a covalently immobilized probe and the amount of hybrid attached to the carrier can be determined by the antibody reagent.

The antibody reagent used in the present invention is mainly characterized by its ability to bind to the DNA 'RNA or RNA' RNA hybrids formed between the probe and the complementary sample nucleic acids, substantially excluding single chain polynucleotides. As indicated above, the antibody reagent may consist of whole antibodies, antibody fragments, polyfunctional antibody aggregates, or generally any substance that includes one or more specific binding sites for an antibody to RNA * RNA or DNA'RNA, as the case may be. When present as a complete antibody, it may belong to any of the classes and subclasses of known immunoglobulins, e.g. IgG, IgM, etc. Any fragment of any such antibody which retains the specific binding affinity of the hybridized probe may also be used, e.g. the fragments of IgG, conventionally known as Fab »F (ab '), and F (ab')₂. In addition, aggregates, polymers, derivatives and conjugates of immunoglobulins or their fragments may be used where appropriate.

The immunoglobulin source of the antibody reagent can be obtained by any available means, such as e.g. conventional antiserum or monoclonal techniques. Antiserum can be obtained by well-established techniques which include immunizing an animal, e.g. a mouse, rabbit, guinea pig or goat, with a suitable immunogen. The immunoglobulins can also be obtained by somatic cell hybridization techniques, this results in what is commonly referred to as monoclonal antibodies, and also this includes the use of a suitable immunogen.

Immunogens for the stimulation of antibodies specific for DNA * RNA hybrids may include homopolymers or heteropolymeric polynucleotide duplexes. Among the possible homopolymeric duplexes, poly (rA) * poly (dT) is especially preferred (Kitagawa and Stollar (1982) Mol. Immunol. 19: 413).

In general, however, it is preferred to use heteropolymeric duplexes, and these can be prepared in a variety of ways, including transcription of ΨΧ174 virion DNA with RNA polymerase (Nakazato (1980) Biochem. 19: 2835). The selected RNA'DNA duplexes are absorbed onto a methylated protein, or otherwise bound to a conventional immunogen carrier material, such as e.g. bovine serum albumin, and is injected into the desired host animal (see also Stollar (1980) Meth.

Enzymol. 70:70).

Antibodies to RNA RNA duplexes can be grown against double-stranded RNAs from viruses such as e.g. reovirus or Fiji disease virus that infects sugar cane, i.a. Furthermore, homopolymeric duplexes, such as poly (rl) · poly (rC) or poly (rA) · poly (rU), among others, are used for immunization as above.

The binding of the antibody reagent to the hybridized probe duplex in the context of the present invention can be detected by any conventional technique. The antibody reagent itself may advantageously be labeled with a detectable chemical group.

Such a detectable chemical group can be any material, which has a detectable physical or chemical property. Such materials are well developed: in the field of immunoassays, and in general, the vast majority of labels useful in such methods can be used in the present invention. Particularly useful are enzymatically active groups, such as enzymes (see

Clin. Chem. (1976), 22: 1243, U.S. Resumed Patent No. 31,006 and UK Patent No. 2,019,408), enzyme substrates (see U.S. Patent No. 4,492,751), cofactors (see U.S. Patent Nos. 4,230,797 and 4,238,565). ), and enzyme inhibitors (see U.S. Patent No. 4,134,792): fluorescent substance (see Clin. Chem. (1979) 25: 353);

chromophoric substance; luminescent substance such as

chemiluminescent and bioluminescent substances (see U.S. Patent No. 4,380,580); specifically binding ligands such as e.g. biotin (see European Patent Nos. 63-879) or a hapten (see PCT Publ. 83-2286); and radioisotopes such as³H, 125j _Og 14_c.

Such labels and tag pairs are detected on the basis of their physical properties (eg fluorescent substance, chromophoric substance and radioisotopes) or their reactive or binding properties (eg enzymes, substrates, cofactors and inhibitors). For example. For example, a cofactor-labeled antibody can be detected by the addition of the enzyme for which the label is a cofactor, and a substrate for the enzyme. A hapten or^et ligand (e.g., biotin) -labeled antibody, can be detected by the addition of an antibody to the hapten or a protein (e.g., avidin) that binds ligand effi, to which a detectable molecule is attached.

Such a detectable molecule may be a molecule with a measurable physical property (eg fluorescence or absorbance) or a participant in an enzyme reaction (eg see list above). For example. one can use an enzyme that acts on a substrate to generate a product with a measurable physical property. Examples of the latter include, but are not limited to, β-galactosidase, alkaline phosphatase, and peroxidase. Other marking procedures will be apparent to those skilled in the art.

Alternatively, the antibody reagent can be detected on the basis of an inherent property such as e.g. its own antigenicity. A labeled anti- (antibody) antibody will bind to the primary antibody reagent where the label for the second antibody is a conventional label as above. Furthermore, antibody can be detected by complement fixation or the use of labeled protein A, as well as other methods known in the art for antibody detection.

Where the antibody reagent is labeled, which is preferred, the label moiety and the antibody reagent are associated or linked to each other by direct chemical bonding such as e.g. includes covalent bonds, or by indirect bonds such as e.g. by incorporating the label into a microcapsule or liposome which in turn is bound to the antibody. Marking techniques are well known in the art, and any convenient method may be used in the present invention.

The sample to be analyzed can be any medium of interest, and will usually be a liquid sample of medical, veterinary, environmental, nutritional, or industrial significance. Samples from humans and animals, and in particular body fluids, can be analyzed by the method described, including urine, blood, (serum or plasma), milk, cerebrospinal fluid, saliva, excrement, lung aspirates, throat swabs, genital swabs, and exudates, rectal swabs, and nasopharyngeal aspirates. . Where the sample taken from the patient or another source of examination contains mainly double-chain nucleic acids, as found in cells, the sample is treated to denature nucleic acids and, if necessary, to first release nucleic acids from the cells. Denaturation of nucleic acids is preferably achieved by heating in boiling water or alkali treatment (e.g. 0.1 N sodium hydroxide), which, if desired, can be used simultaneously to lyse cells. Furthermore, the release of nucleic acids, for example, can be achieved by mechanical degradation (freezing / thawing, abrasion, ultrasonic treatment), physical / chemical degradation (detergents for eg Triton, Tween, sodium dodecyl sulphate, alkali treatment, osmotic shock, or heat), or enzymatic lysis (lysozyme, proteinase, K, pepsin). The resulting test medium will contain nucleic acids in single chain form which can then be analyzed by the hybridization method described.

As is known, different hybridization conditions can be used in the analysis. Typically, the hybridization will take place at slightly elevated temperatures, e.g. between 35 ° C and 75 ° C, and usually around 65 ° C, in a solution which includes buffer at pH between 6 and 8, and with suitable ionic strength (e.g.

2XSSC where 1XSSC = 0.15M sodium chloride and 0.015M sodium citrate, pH 7.0), protein such as bovine serum albumin, Ficoll (a trademark denoting a copolymer of sucrose and epichlorohydrin sold by Pharmacia Fine Chemicals, Piscataway, NY), polyvinylpyrrolidone, and a denatured foreign DNA (such as from calf thymus or salmon sperm).

The degree of complementarity between the sample and the probe chains required for hybridization to occur depends on the stringency of the conditions. The extent and specificity of the hybridization are affected by the following main conditions:

1. The purity of the nucleic acid preparation.

2. The base composition of the probe - GC base pairs show greater termite stability than AT or AU base pairs. Consequently, hybridizations involving higher GC contents will be very stable at higher temperatures.

3. The length of homologous base sequences Any short sequence of bases (eg less than 6 bases) has a high probability of being present in many nucleic acids.

Accordingly, little or no specificity can be obtained by hybridizations involving such short sequences. The present homologous probe sequence will be at least 10 bases, usually 20 bases or more, and preferably more than 100 bases.

From a practical point of view, the homologous probe sequence will often be in between

300 and 1000 nucleotides.

4. Ionic strength - The rate of recombination increases as the ionic strength of the incubation solution increases. The thermal stability of the hybrids also increases.

5. Incubation temperature - Optimal recombination takes place at a temperature of approx. 25-30 ° C below the melting point of a given duplex. Incubation at temperatures well below the optimum value allows fewer base sequences to hybridize.

6. Nucleic Acid Concentration and Incubation Time To drive the reaction against hybridization, either the hybridized sample nucleic acid or the probe nucleic acid is normally present in excess, usually an excess of 100-fold or greater.

7. Denaturing reagents - The presence of agents that break hydrogen bonds such as formamide and urea, increases the stringency of hybridization.

8. Incubation time - The longer the incubation time, the more complete the hybridization will be.

9. Volume Exclusion Agents - The presence of these agents, exemplified by dextran and dextran sulfate, is believed to increase the effective concentrations of the hybridization elements, thereby increasing the rate of the resulting hybridization.

Normally, the temperature conditions selected for the hybridization will be compatible with the binding of the antibody reagent to formed hybrids and detection of the label response. Accordingly, the binding step for the antibody reagent and the label detection step will proceed after the hybridization step is completed. The reaction mixture is normally brought to a temperature in the range of 3 ° C to 40 ° C, and the binding and detection steps are then performed. Dilution of the hybridization mixture prior to the addition of antibody reagent is desirable when salt and / or formamide concentrations are high enough to significantly interfere with the antibody binding reaction.

In special assay situations where the RNA probe is used, it may occur that the probe undergoes partial degradation by alkaline hydrolysis of the phosphorus 166301 diester bonds, or in the presence of ribonuclease.

In the first case, the hydrolysis can be controlled by preventing the probe from being exposed to a pH higher than 10 · Ribonuclease can be effectively inhibited in the presence of such compounds as sodium dodecyl sulfate, aurin tricarboxylic acid, ribonucleoside vanadyl complexes, heparin, diethylpyrocarbonate isolates and protein-containing inhibitors.

The reagent system is in a commercially packaged form, as a composition or mixture where the compatibility of the reagents allows, in a test device configuration, or usually as a test kit, i.e. a packaged combination of one or more containers, devices, or the like, containing the required reagents, and usually a written instruction manual for performing the assays. Reagent systems of the present invention include all configurations and compositions for carrying out the various hybridization methods described herein.

In all cases, the reagent system will include (1) an immobilized or immobilizable probe as described above, and (2) the antibody reagent, preferably labeled with a detectable chemical moiety. An experimental package of the system may additionally include auxiliary chemicals such as e.g. the components of the hybridization solution and the denaturant capable of converting double-chain nucleic acids in a sample to single-chain form. Preferably a chemical lysing or denaturing agent is included, e.g. alkali, for processing the sample so that single-chain nucleic acids are released.

The present invention will be further elucidated by reference to the following examples.

Example 1;

Hybridization assay for the detection of bacteriuria using an immobilized RNA probe.

A. Preparation of the RNA probe.

An 800 base pair fragment of the tuf A gene encoding the EF-Tu protein in Escherichia coli is derived from the bacteriophage M13-1O (ATCC 39403-131). The fragment is cloned between the Hind III and Eco RI restriction endonuclease sites at Ml3mp9 (New England Biolabs, Beverly, MA). This plasmid is cultured in an E. coli host JM103 (ålac, pro), supE, thi, strA, sbcB15, hsdR4, F'traD36, proABlak IqZM15. The fragment tuf A is cut from M13-10 and cloned inside the Hind III and Eco RI positions on the pSP64 plasmid vector available from Promega Biotec., Madison, WI.

ml of an overnight culture containing E. coli JM103 carrying the pSP64 plasmid containing the tuf A fragment is inoculated into one liter of 2xYT broth in a two liter bottle. The culture is incubated at 37 ° C for 3 hours and the cells are harvested. They are then lysed and DNA isolated by phenol / chloroform extractions.

The closed circular plasmid The DNA is purified by centrifugation in a cesium chloride-ethidium bromide gradient. (Maniatis, T., Fritsch, EF, &amp; Sambrook, J., Molecular Cloning, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982)).

The purified plasmid is chromatographed on Sephadex G-50 (Pharmacia Fine Chemicals, Piscataway, NJ) in 10 mM Tris-hydrogen chloride buffer, pH 7.5, containing 0.1 M NaCl and 1 mM EDTA. The effluent contains DNA and is collected and the DNA is precipitated with cold ethanol. The precipitate is taken up in 10 mM NaCl, 10 mM MgCl₂ and 1 mM dithiothreitol and degraded for 1 hour with one unit of EcoRI per micrograms (yg) of DNA. The reaction mixture is then extracted once with phenol / chloroform and once with chloroform, and the DNA is precipitated with cold ethanol. The precipitate is dissolved in 10 mM Tris-hydrochloride buffer, pH 7.4, to obtain 500 pg DNA / mL.

IN

A 500 microliter (pL) reaction mixture is prepared having the following composition:

pg of the EcoRI degradation element; 40 mM trishydrogen chloride buffer, pH 7.5; 6 mM MgCl 2₂; 2 mM spermine;

0.5 mM ATP, CTP, UTP and GTP; 10 mM dithiothreitol;

500 units of RNasin (Promega Biotec) and 50 units of RNA polymerase from bacteriophage SP6 (Promega Biotec). The reaction is allowed to stand for one hour at room temperature and; then another 50 units of RNA polymerase are added and the reaction is allowed to proceed for another hour.

DNA in the reaction is digested for 10 minutes at 37 ° C at 10 pg of RNase-free DNase. The reaction mixture is extracted with phenol / chloroform and chromatographed on Sephadex G.50 in 10 mM Tris-hydrogen chloride buffer, pH 7.4, 0.1 M NaCl. The RNA is collected and precipitated with cold ethanol. The precipitate is dissolved in 50 mM sodium acetate buffer, pH 5.0, containing 1 mM EDTA.

The RNA probe described above is immobilized on acrylic beads with reactive epoxide groups available under the tradename Eupergit C from Accurate Chemical and Scientific Corp., Westbury, NY. Three milliliters (3 ml) of 50 mM sodium acetate buffer, pH 4.5, containing 250 pg of RNA probe are shaken at room temperature for 10 hours with 200 mg of Eupergit C. The buffer is removed and analyzed for RNA to determine the extent of the immobilization that has found place.

The resin is then washed by brief shaking with 1 ml of 0.1 M sodium phosphate buffer, pH 6.5, containing 1.2 M NaCl, 0.5% (w / v) sodium dodecyl sulphate, 1 mg polyvinylpyrrolidone / ml, and 5 mg bovine serum albumin / ml. This hybridization solution is removed and replaced with 1 ml of new hybridization solution and the suspension is incubated at 65 ° C for 1 hour to remove the non-covalently bound RNA probe. The solution is removed and the resin-RNA probe conjugate is suspended in 50 ml of the hybridization solution.

B. Preparation of methylated thyroglobulin.

One hundred milligrams deflected. thyroglobulin (Sigma Chemical Co., St. Louis, MO), mixed with 10 ml of anhydrous methanol and 400 μl of 2.55 M HCl in methanol. This mixture is stirred on a rotary mixer at room temperature for 5 days. The precipitate is collected by centrifugation and washed twice with methanol and twice with ethanol. It is then dried under vacuum overnight. You get approx. 82 mg dry powder.

C. Production of antibody to DNA'RNA hybrid.

A DNA'RNA hybrid is prepared by transcrip tion of ΦΧ174 virion DNA with RNA polymerase as described by Nakazato (Biochem. 19: 2835 (1980)).

One hundred and fifty (150) micrograms (pg) of the hybrid in 250 μl of 20 mM tris-hydrogen chloride buffer, pH 7.4, 1 mM EDTA is mixed with 150 pg of methylated thyroglobulir in 250 μl of water. A precipitate is formed and suspended in Tris buffer. The mixture is emulsified with an equal volume of Freund's adjuvant. Mice are immunized with 0.5 ml each of the suspension and reach serum antibody titers

Until RNA DNA is developed, hybridomas are prepared and tested for monoclonal antibody specific for RNA 'DNA (Stuart et al (1981) Proc. Natl. Acad. Sci. USA 78, 3751, Galfre and Milstein, (1981) Meth. In Enzymol. 73, 1).

The cloned hybridomas multiply in the abdominal cavity of mice,

In such a way that a large amount of antibody is generated. The abdominal fluid is placed in a column of Affigel-Blue resin (Bio-Rad Laboratories, Richmond, VA) equilibrated with 10 mM Tris-hydrogen chloride buffer, pH 8.0, 0.15 M NaCl. This type of chromatography removes albumin and the leached protein containing the antibody is chromatographed on DEAE-Sepharose (Pharmacia Fine Chemicals). The chromatography is developed with a linear gradient of 10 mM Tris-hydrogen chloride, pH 8.0, to 10 mM Tris-hydrogen chloride, pH 8.0, 200 mM NaCl. The major peak of the leached protein contained the monoclonal antibody free of transferrin and albumin.

D. Preparation of 8-galactosidase-antibody-conjugate.

Sulfhydryl residues of β-galactosidase are exposed by reduction with dithiothreitol. Δ-galactosidase (30,000 units, grade VIII, Sigma Chemical

Co., St. Louis, MO) in 2 ml of 0.1 M N-2-hydroxyethylpiperazine N'2-ethane sulfonate (HEPES), pH 7.0, 0.09 M NaCl, mixed with 3.5 pmol dithiothreitol and allowed to stand at room temperature for 4 hours. Ditiotreitol is removed by chromatography on a 2.5 x 80 cm column of Sepharose 6B Cl (Pharmacia Fine Chemicals) in the buffer described above. Fractions that contain protein are collected in a container. Number of moles of sulfhydryl groups per moles of enzyme is measured by the method set forth by Ellman (Ellman (1959) Arch. Biochem. Biophys. 82, 70).

Succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) (Pierce Chemical Co., Rockford, IL), 5.3 mg, is dissolved in 250 μl of anhydrous N, N-dimethylformamide and a 40 μl portion is added to 3 ml of 0.1 M HEPES buffer, pH 7.0, 0.15 M NaCl. 2Q A 25 μl aliquot of this aqueous solution is added to 825 μl HEPES / NaCl buffer and 100 μl 1 mM glutathione. When this reaction mixture has been at room temperature for 15 minutes, the amount of unreacted glutathione is determined by Ellman's method.

Monoclonal antibody to DNA * RNA is mixed

400 pmol of SMCC in a final volume of 533 μl HEPES / 0.15 M NaCl buffer and allowed to react for 1 hour at 30 ° C. This reaction mixture is chromatographed on a 1 x 24 cm column of Biogel P-2 resin (Bio-Rad Laboratories, Rickmond, CA) and eluted with HEPES / 0.15 M NaCl buffer. Effluent containing protein is collected and the protein concentration is determined by the method set forth by Sedmack and Grossberg (Anal. Biochem. 79, 544 (1977)) and the number of maleimide166301 groups is determined by titration with glutathione as described above.

IN

A 2.8 mg portion of the antibody-maleimide addition product is mixed with 10 mg of dithiothreitol-treated S-galactosidase and allowed to react for 4 hours at room temperature. The reaction mixture is chromatographed at 4 ° C on a 2.5 x 80 cm column of Sepharose 6B; C1 in HEPES / 0.15 M NaCl at 4 ° C. The flow rate is set to 4 ml / hour and 3 ml fractions are collected. The fractions are assayed for β-galactosidase activity and antibody binding activity. Fractions that have both activities come together._;

E. Hybridization analysis.

Samples of ten milliliters of urine from patients with possible urinary tract infection are centrifuged at

10,000 xg for 10 minutes and the supernatants are decanted off and discarded. The sediments are suspended in 50 μl of 10 mM Tris-hydrogen chloride buffer, pH 8.0, containing^;20 mg egg white lysozyme / ml (Sigma Chemical Co., St.

Louis, MO), 0.1 M NaCl, and 5 mM EDTA. The reaction is allowed to stand at room temperature for 30 minutes and then 10 μl of 1 M NaOH is added. This alkaline mixture is allowed to stand at room temperature for 10 minutes to denature DNA from any bacteria in the original sample. The reaction mixture is neutralized with the addition of 250 .mu.l of the buffered suspension of resin RNA conjugate described above.

This hybridization system is included at approx. 65 ° C for 15 hours with gentle shaking.

The resin-RNA probe conjugate is allowed to sediment and the liquid to be dec anted. The resin is washed twice by suspension in 0.5 ml each time of 0.1 M sodium phosphate buffer, pH 7.4. 5 mgbovint serum albumin / ml. The resin is combined with 300 μl of 0.1 M sodium phosphate buffer, pH 7.4, containing. 10 mM MgCl 2₂»Mgbovine serum albumin (ml, and 0.4 pg 6-galactosidase antibody / ml (anti-DNA * RNA). The mixture is gently shaken for 1 hour at room temperature and the resin is washed twice, each time for 1 minute, with 5 ml 0, 1 M sodium phosphate buffer, pH 7.4, containing 0.1%.

Tween 20 detergent. Gently shake the washed resin for 30 minutes at room temperature, in 1.0 ml of 0,1 M sodium phosphate buffer, pH 7,4, containing 800 pM · 7-8-galactosyl-3- (6-aminohexylcarboxamide) coumarin (Worah et al (1981) Clin. Chem. 27: 673). At the end of this incubation, the fluorescence of the solution is recorded using 400 nanometer (nm) excitation and 450 nm emission.

Fluorescence signals developed with urine samples containing mor e than 100,000 bacteria per ml will be significantly higher than those for samples containing less than 5,000 bacteria per ml. ml.

This method can be used as a qualitative detection of bacteriuria.

Example 2:

Hybridization assay for E. coli 23s ribosomal RNA using an immobilized DNA probe.

A. DNA probe for 23s RNA.

The DNA probe is an EcoRI / BglII fragment from the rrnD operon encoding 23s RNA in E. coli (JinksRobertson et al (1983) Cell 33: 855).

The probe includes approx. 2/3 of the 23s RNA sequence from 3'-hydroxyl and is cloned into an M13 virus vector to give single-stranded virion DNA that is complementary to cellular ribosomal RNA.

The M13 virus is grown in E. coli strain JM103 and isolated from the culture medium by precipitation with polyethylene glycol. The virion DNA is purified of virus particles by phenol extraction (Maniatis, et al, supra).

The purified DNA is made 0.3 M in NaOH and incubated at 37 ° C for 4 hours to degrade contaminating RNA.

The mixture is neutralized by the addition of 30% acetic acid and the DNA is precipitated with cold ethanol.

B. Antibody to DNA'RNA.

Mice are immunized with DNA'RNA hybrid as described in Example 1, and spleen cells are fused with SP 2/0-Ag14 myeloma; cells (available from the American Type Culture Collection. Rockville, MD). Hybridoma-secreting antibodies specific for DNA'RNA are identified as indicated above. The most preferred hybridoma is that deposited with the American Type Culture Collection, Rockville, MD as ATCC HB 8730.

The antibodies are purified by HPLC using an LDC / Milton Roy liquid chromatograph equipped with a Cl ~ 10 integrator. The abdominal fluid is dialyzed against 0.01 M potassium phosphate buffer, pH 6.8, centrifuged to remove particulate matter, and passed through a 0.22 μm nitrocellulose filter. One to two milliliters of treated abdominal fluid is placed in a 10 x 250 mm anion exchange column, which is equilibrated with 0.01 M potassium phosphate, pH 6.84. The chromatography is developed with a 60 min linear gradient from 0.01 M potassium phosphate buffer, pH 6 , 84, to 0.085 M potassium phosphate, pH 6.40, at a flow rate of 1 ml / min.

The peak containing IgG is concentrated, dialyzed against phosphate buffered saline, pH 7.4, centrifuged to remove any denatured protein, and the IgG concentration is determined based on the absorbance at 280 nm using e μg / ml. ^J 1 cm

C. Immobilization of the DNA probe.

Meta-nitrophenyl groups are introduced on cellulose powder and then converted to the diazonium salt for covalent immobilization of DNA.

1 - ((m-nitrobenzyloxy) methylpyridinium chloride (690 mg, 2.46 mmol) (Aldrich Chemical Co., Milwaukee, WI) is mixed with 128 mg of sodium acetate in 7.7 ml of water. Two grams of Sigmacell, type 20, cellulose , (Sigma Chemical Co.) is added and mixed for about 15 minutes in a beaker immersed in a water bath at 60 ° C.

The cellulose becomes almost dry and is placed in an oven at 135-140 ° C for 45 minutes. Good incorporation of m-nitrophenyl residues depends on keeping the temperature as high as possible during this time.

If the temperature is too high, the cellulose caramel will be serrated.

After the heat treatment step, the cellulose is suspended in water and lumps are broken up by rubbing the cellulose in water until the particles pass through a 150 .mu.m screen. The cellulose is washed three times with 120 ml of water each time, and then twice with 50 ml of ethanol. It is then dried overnight in vacuo. in

Nitrophenyl groups on the cellulose are reduced by incubating it at 65 ° C for 1 hour, in 10 ml of 0.1 M Na₂CO 2 containing 2.0 g of sodium dithionite. Then the cellulose is washed several times with water on a sintered glass funnel, and once with 30% acetic acid. Finally, it is washed a further 3 times with water and dried in vacuo at 40 to 50 ° C overnight.

Two hundred and fifty milligrams of the reduced cellulose are added to 5.0 ml of 1.2 M HCl at 0 DEG C. and 13 .mu.l of 100 mg of NaNCl2 / ml are added. This mixture is allowed to stand for 1.0 hour, during which time the mixture is examined for the presence of NaNO₂ with starch-iodide paper. If the result is weak or negative, 20 ul of NaNO are added₂·

At the end of this reaction period, the cellulose is washed successively with 30 to 50 ml of cold (0 ° C) water on a cold sintered glass funnel, with 10 to 15 ml of cold 10 mM urea, with colder water and finally with approx.

ml cold 0.2 M sodium acetate buffer, pH 4.0. The cellulose is rapidly transferred to a vial containing 0.92 ml of cold 0.2 M sodium acetate buffer, pH 4.0, containing 69 μg of the DNA probe.

The mixture is shaken at 0-4 ° C for 15 hours, then washed with 1 x SSPE (20 mM sodium phosphate buffer, pH 7.8, 0.18 M NaCl, 1 mM EDTA), 0.1% sodium dodecyl sulfate, (SDS), on a sintered glass funnel. The cellulose is incubated at 55 ° C for 4 hours in a hybridization solution consisting of:

2.0 ml

1.5 ml

0.3 ml formamide x SSPE mg bovine serum albumin / ml mg Ficoll / ml, 10 mg polyvinyl pyrrolidone / ml

0.03 ml 10% SDS (w / v)

0.140 ml 4.25 mg salmon sperm DNA / ml.

Before use, incubate salmon sperm DNA at 37 ° C for 17 hours, in 0.3 M NaOH, neutralize with 30% acetic acid and collect by precipitation with cold (-15 ° C) ethanol.

After the incubation at 55 ° C, the cellulose is washed twice, each time with approx. 10 ml of 1 x SSPE, 0.1% SDS. The cellulose is resuspended in 5.0 ml of the hybridization solution and 0.2 ml portions of the slurry are placed in test tubes for hybridization.

D. Preparation az 23s ribosomal RNA.

Ribosomal RNA is prepared from E.coli and the 23s component is isolated by sucrose density gradient centrifugation (Takanami, M., (1967) Meth.

Enzymol., 12A: 491; McConkey, EH (1967) Meth. Enzymol., 12A: 620).

E. Hybridization assay for 23s RNA.

The hybridization solution is aspirated from test tubes containing the cellulose with the immobilized DNA probe. Then, 100 μl of hybridization solution containing 10 ng of 23s RNA / ml is added to each tube and they are incubated at 55 ° C for specified periods. At the end of the incubations, the hybridization solutions are removed and the cellulose is washed with 0.5 ml of 1 x SSPE, 0.1% SDS, incubated at 55 ° C for 30 minutes in 0.5 ml of 1 x SSPE, 0.1% SDS, and washed with once with 0.5 ml of 1 x SSPE, 0.1% SDS.

The amounts of DNARNA formed are measured by immunoassay. The cellulose in each tube is shaken at room temperature for 30 minutes at 50 μl of 20 mM sodium phosphate buffer, pH 7.4, containing 0.15 M NaCl, 1 mM EDTA, 0.5% (v / v) Tween 20. and 5.0 mg BSA / ml. Then 100 μl of this solution containing 1.0 pg of antibody to DNARNA is added to each tube and the shaking is continued for 30 minutes. The liquid is removed by suction and the cellulose is washed four times, each time with 0.5 ml of 0.1 M tris-hydrogen chloride buffer, pH 8.0, containing 5 mM.

MgCl-2 »0.5% Tween 20 and 5.0 mg bovine albumin / ml ttris / MgCl₂/ T2een^,7BSA). Then, 150 μl of alkaline phosphatase-labeled antimus IgG (Sigma Chemical Co.) diluted 200-fold in Tris / MgCl 2 / Tween / BSA is added to each tube and shaken at room temperature for 1.0 hour.

The cellulose from each assay is washed twice with 0.5 ml Tris / MgCl 2 / Tween / BSA each time, containing 0.5 M NaCl and then the cellulose is transferred to clean test tubes using 1.5 - 2.0 ml of this buffer solution. . The buffer is removed and the alkaline phosphatase label bound to the cellulose is measured.

For this purpose, 200 μl of 1.0 M diethanolamine hydrogen chloride buffer, pH 9.8, containing 1 mM MgCl is added.₂ and 1 mg p-nitrophenyl phosphate / ml and the mixture is incubated at 25 ° C for 30 minutes. Then the enzyme-catalyzed reaction is quenched by the addition of 1.5 ml of 0.1 M Na 2 PO 4 and the absorbances at 405 nm are recorded. The results are as follows:

Hybridization time (hours)

Absorbance

0,5

1,0

2,0

8,0

12,0

24,0

0,34

1,18

1,36

1,85

2,27

2,32

2,34

The absorbances increase with the hybridization time, which indicates that an increasing amount of DNA · RNA hybrid is formed.

Example 3;

Hybridization assay for 23s ribosomal RNA using immobilizable DNA probe.

The sample RNA is hybridized with a soluble DNA probe with attached biotin residues. The hybridized and unhybridized DNA probe is then bound to a solid support with the immobilized streptavidin.

The amount of DNA 'RNA on the support is measured by an immunoassay using enzyme-labeled antibody to DNA · RNA.

A. Biotinylated probe DNA.

The M-13 virus described above in Example 2, with the insert complementary to 23s RNA is propagated; in

E.coli strain JM103 and the bacterial cells are shaken to isolate the replicative form of the virus DNA. This double chain DNA is purified by cesium chloride etidium bromide density gradient centrifugation (Maniatis, et al., Supra).

Biotin residues are introduced into this double chain

DNA by nick translation using biotinylated dUTP available from Enzo Biochem. Inc., NY (Langer,

PR et al (1981) Proc. Natl. Acad. Sci., 78: 6633; Leary, JJ et al (1983) Proc. Natl. Acad. Sci., 80: 4045).

Contaminant RNA is degraded by treatment with alkali as described in Example 2. Immediately before use, the biotinylated probe is denatured and placed on a boiling water bath for 4 minut es.

B. Immobilization of streptavidin.

Streptavidin (Calbiochem-Behring Corp., La.)

Jolla, CA) is immobilized on Act-Ultrogel AcA 22 (available from LKB Instruments, Inc., Gaithersburg, MD) which is an acrylamide agarose carrier activated with glutaraldehyde (Doley, SG et al (1976) FEBS Letters, 65:87). The immobilization is carried out according to the manufacturer's instructions so that you get approx.

0.5 pg streptavidin pr. 10 pl packed ActUltrogel AcA 22 * ​​'.

C. Hybridization analysis.

Two milliliter samples of urine, suspected to contain bacterial, infection, are centrifuged at 3000 xg to sediment the bacteria, and the supernatant is decanted off. Ninety microliters of the hybridization solution described in Example 2 is added to each pellet and 5 μl of the biotinylated probe (at a concentration of 0.5 pg / ml in 20 mM sodium phosphate buffer, pH 7.0, 0.5 mM EDTA) is added. The mixtures are shaken to suspend the pellet (if present) and incubated at 55 ° C for 4 hours.

Then, 700 μl of 20 mM sodium phosphate buffer, pH 7.4, containing 5.0 mg of bovine albumin and 50 μl of Ultrogel with immobilized streptavidin are added to each mixture to dilute the hybridization solution and immobilize the biotinylated probe.

The mixtures are shaken at room temperature for two hours and the liquid is removed from the carrier.

The amount of DNA * RNA hybrid associated with

The 1Q ultrogel support is measured by immunoassay as described in Example 2 for the cellulose support.

For comparison, untreated urine samples for bacteria are examined by a microliter culture method. ₁₅ using MacConkey and blood agar plates.

The plates are incubated at 37 ° C for 36 hours and the colonies are counted.

Urine samples with a high level of bacteria in the culture2Q method give high absorbances in the hybridization analysis.