Hybridisation Assay in Which Excess Probe

Europäisches Patentamt *EP000975805B1* (19) European Patent Office

Office européen des brevets (11) EP 0 975 805 B1

(12) EUROPEAN PATENT SPECIFICATION

(45) Date of publication and mention (51) Int Cl.7: C12Q 1/68 of the grant of the patent: 05.09.2001 Bulletin 2001/36 (86) International application number: PCT/GB98/01057 (21) Application number: 98917331.5 (87) International publication number: (22) Date of filing: 09.04.1998 WO 98/46790 (22.10.1998 Gazette 1998/42)

(54) HYBRIDISATION ASSAY IN WHICH EXCESS PROBE IS DESTROYED HYBRIDISIERUNGSVERFAHREN IN DEM ÜBERSCHÜSSIGE SONDEN ABGEBAUT WERDEN TEST D’HYBRIDATION DANS LEQUEL LA SONDE EN EXCES EST DETRUITE

(84) Designated Contracting States: (56) References cited: AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU EP-A- 0 144 913 EP-A- 0 780 479 MC NL PT SE WO-A-90/01559 WO-A-98/19168 US-A- 4 833 084 (30) Priority: 14.04.1997 GB 9707531 • DATABASE WPI Derwent Publications Ltd., (43) Date of publication of application: London, GB; AN 92205018 XP002072949 "gene 02.02.2000 Bulletin 2000/05 detection method-comprises gene extn., denaturing to single strand, specifically bonding (73) Proprietor: Zetatronics Limited to labelled gene probe and reacting" & JP 04 135 Hatfield, Herts AL10 9AB (GB) 498 A (TOSHIBA) , 8 May 1992 • HARBRON S. ET AL.,: "Amplified assay of (72) Inventor: Harbron, Stuart alkaline phosphate using Flavinadenine Berkhamsted, Herts HP4 2LL (GB) dinucleotide phosphate as substrate" ANALYTICAL BIOCHEMISTRY, vol. 206, - 1 (74) Representative: Coates, Ian Harold et al October 1992 pages 119-124, XP002072948 Sommerville & Rushton, 45 Grosvenor Road St. Albans, Hertfordshire AL1 3AW (GB)

Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 0 975 805 B1

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Description ence of the label in the resulting hybrid is determined. A disadvantage of this method is that it is neither easy Background Art nor convenient to attach the single-stranded target nucleic acid to a solid support, the whole process involving [0001] Nucleic acid hybridisation is a widely used 5 a 12 - 15 hour incubation of the nucleic acid with a nitro- technique for identifying, detecting and quantitating tar- cellulose sheet, followed by a 2 hour baking step. This get polynucleotide sequences in a sample. This tech- makes the assay slow and unattractive for routine use. nique relies for its success on complementary base pair- It is also cumbersome, with the hybridisation and washing between the two halves of a double-stranded nucleic ing steps being carried out in a sealed pouch, containing acid molecule: when single-stranded nucleic acids are 10 the membrane and the buffer solution. In addition, when incubated in solution under suitable conditions of tem- very low concentrations must be detected, the ratio of perature, pH and ionic strength, complementary base specific to non-specifically bound probe can be very low sequences pair to form double-stranded stable hybrid and repeated washing under highly stringent conditions molecules. This ability of single-stranded nucleic acid is frequently required. Under these conditions the sen- molecules to form a hydrogen-bonded structure with 15 sitivity of the assay is often compromised because of their complementary nucleic acid sequences has long substantial loss of specifically bound probe. been employed as an analytical tool in recombinant [0005] Since then a many improvements have been DNA research. made, most of which employ a sandwich approach us- [0002] In most cases the sample will contain double- ing two probes: a reporter probe and a capture probe. stranded nucleic acid and must be denatured prior to 20 The reporter probe is a nucleic acid having a sequence the hybridisation assay to render it single-stranded. A complementary to at least part of the target sequence nucleic acid having a known sequence which is comple- and which is labelled with a detectable group. The cap- mentary to the target sequence is either synthesised ture probe is a nucleic acid having a sequence comple- chemically in an automated fashion with great facility, or mentary to at least part of the target sequence, but which is isolated from the appropriate organism and rendered 25 is different to that of the reporter probe, and which is single-stranded by denaturation. It is then used as a labelled with an immobilisable group. In many applica- probe to search a sample for a target complementary tions, pairs of specific binding members (sbm's) have sequence. Detection of specific target nucleic acids en- been used for this purpose. ables accurate diagnosis of bacterial, fungal and viral [0006] For example, in U. S. Pat. No. 5,273,382 to disease states in humans, animals and plants. Addition- 30 Snitman and Stroupe a capture probe complementary ally, the ability to probe for a specific nucleotide se- to part of the target nucleic acid is labelled with an anti- quence enables the diagnosis of human genetic disor- gen or antibody. After hybridisation between this capture ders. Hybridisation produces stable hybrids, and a probe and the target, the solution is introduced to a sup- number of different approaches are known to the art for port-bound antibody or antigen which immobilises the detecting these. 35 hybrid formed between the capture probe and the target. [0003] One approach involves the use of labelled Following a washing step, a second, reporter probe, probes. By labelling a probe nucleic acid with some complementary to a different region of the target nucleic readily detectable chemical group, it is possible tc detect acid, is introduced and the triple sandwich formed is de- the polynucleotide sequence of interest in a test medium tected. containing sample nucleic acids in single-stranded form. 40 [0007] Similar approaches are described by Holtke et Nucleic acids have been labelled with radioisotopes, en- al.: in U.S. Pat. No. 5,344,757 is disclosed a method in zymes and fluorescent molecules. The use of labelled which a reporter probe is labelled with digoxin or digox- nucleic acids as probes in macromolecular analysis is ygenin, and hybrids are captured using antibodies important for clinical, veterinary and environmental di- against this hapten. In this case, a capture probe is not agnostic applications. 45 used, and the method is limited either to the detection [0004] Early methods for detecting target nucleic ac- of an immobilised target, or when the assay is used for ids involved their immobilisation on a solid support such detecting PCR products, one of the primers is immobi- as nitro-cellulose paper, cellulose paper, diazotized pa- lised. In U.S. patent 5,354,657 the method is further de- per, or a nylon membrane. For example, in U. S. Pat. veloped and involves the solution hybridisation between No. 4,358,535 to Falkow a method is disclosed in which 50 the target nucleic acid and a reporter probe labelled with the target nucleic acid is rendered single-stranded and digoxin or digoxygenin. This hybrid is captured by a sol- then immobilised onto a membrane. A labelled probe id-supported capture probe, complementary to a differ- which is complementary to the target nucleic acid is ent region of the target. A detectably labelled antibody brought into contact with the solid support and hybridis- against the hapten is then added and the hybrids formed es to the target nucleic acid. The solid support is washed 55 detected. several times at a carefully controlled temperature to re- [0008] Specific binding members other than antigens move unbound and non-specifically bound probe with- or haptens and antibodies have been used. In U.S. Pat. out removing specifically bound probe, and the pres- No. 5,374,524 to Miller is described a method for the

2 3 EP 0 975 805 B1 4 solution sandwich hybridisation, capture and detection reagent is selective for DNA-RNA or RNA-RNA hybrids of amplified nucieic acids. Amplicons are denatured and over the single-stranded nucleic acids. U.S. Pat. No. treated with an enzyme-labelled reporter probe and a 5,200,313 to Carrico further discloses a nucleic acid hy- biotinylated capture probe. Hybrids formed are captured bridisation assay employing an immobilised or immobi- using streptavidin-coated chromium dioxide particles. 5 lisable polynucleotide probe selected to form DNA-RNA [0009] Disadvantages of these approaches include or RNA-DNA hybrids with the particular polynucleotide the increased cost and complexity of using two probes. sequence to be determined. Resulting hybrids are de- For example, for each assay two probes need to be syn- tected by binding of an antibody reagent, preferably la- thesised and labelled: one for use as the capture probe, belled with a detectable chemical group, selective for and the other for use as a reporter probe. In addition, 10 binding the hybrids in the presence of the single-strand- hybridisation conditions have to be carefully chosen to ed sample and probe nucleic acids. Advantageous fea- form the sandwich of target, capture probe and reporter ture of Carrico's inventions are that no immobilisation or probe. labelling of sample nucleic acids is required and hybrid- [0010] Simpler approaches which avoid the use or a isation can be performed entirely in solution. A further capture probe have been described. Atlas and Steffan 15 advantage is that a universal detection reagent may be (Biotechniques (1990) 8:316 - 318) disclose a solution used whatever the target is. hybridisation method for detecting genetically-engi- [0013] The key feature of Carrico's invention is the re- neered microorganisms in environmental samples. The quirement for antibodies specific for double-stranded detection method involves recovery of DNA from the mi- hybrids having little affinity for single-stranded nucleic crobial community of an environmental sample followed 20 acid. The generation of specific polyclonal antibodies by hybridisation in solution with a radio-labelled RNA that will bind double-stranded nucleic acid but not sin- gene probe. After nuclease digestion of non-hybridised gle-stranded nucleic acid is complicated by the fact that probe RNA, the DNA-RNA hybrids formed in the solu- polyclonal antisera may contain antibodies that will tion hybridisation are separated by column chromatog- cross-react with single-stranded nucleic acid. Polyclo- raphy and detected by liquid scintillation counting. A less 25 nal antisera may also contain naturally occurring anti- cumbersome approach is disclosed in U.S. Pat. No. bodies to single-stranded nucleic acid or antibodies to 4,978,608 to Kung and Nagainis in which DNA is detect- single-stranded nucleic acid arising as a result of the ed in a sequence non-specific manner using a high af- immunisation. Monoclonal antibody technology can pro- finity single-stranded DNA-binding protein. This ap- vide a means to select an antibody with desired affinity proach is extended in U.S. Pat. No. 5,536,648 to Kemp 30 and specificity which will overcome in part these prob- et al. who disclose an amplified DNA assay using a dou- lems. Such monoclonal antibodies which will selectively ble stranded DNA binding protein. The method uses a bind double-stranded DNA (U.S. Pat. No. 4,623,627) or PCR primer having a nucleotide sequence which is a DNA-RNA hybrids (U.S. Pat. No. 4,833,084 to Carrico) ligand for a double stranded DNA-binding protein. After have been prepared. Monoclonal antibodies are howev- amplification the amplified target is captured by the dou- 35 er more expensive to produce and generally have lower ble stranded DNA-binding protein immobilised on a solid affinities than polyclonal antibodies. surface. This method does not use a capture probe and [0014] The monoclonal antibodies disclosed by Car- will detect any amplification product containing the se- rico (U.S. Pat. No. 4,833,084) are specific for DNA-RNA quence which is a ligand for the double stranded DNA- duplexes, particularly DNA-RNA heteropolymer duplex- binding protein. A disadvantage of this approach is that 40 es, and are characterised by having cross-reactivity for it relies on the accuracy of the amplification step for its binding to single or double-stranded DNA or RNA, as specificity. measured by competitive immunoassay, of less than [0011] Another method is disclosed in U.S. Pat. No. about 1:1000, and preferably less than 1:10,000, and 4,968,602 to Dattagupta. The test sample is modified an affinity for DNA-RNA heteropolymer duplexes great- chemically to introduce a reactive site. This mixture is 45 er than 103 L/mol. then contacted with a reporter probe. After a solution [0015] Chevrier et al. (Molecular and Cellular Probes phase hybridisation step, the hybrid is brought into con- (1993) 7: 187 - 197) report that up to 200 fmol of a cap- tact with a surface having an immobilised reaction part- ture probe may be actached to Covalink NH microwells ner which reacts with the reactive site, and allows the (Nunc). The antibodies disclosed by Carrico would unhybridised material to be washed away. A disadvan- 50 therefore have a lower detection limit of approximately tage of this approach is that the initial reaction step may 1/10,000 of 200 fmol, cr 20 amol. In addition non-specific interfere with the subsequent formation of the hybrid. binding between the labelled antibody and the surface [0012] A further approach in which the hybrid itself is on which the probe is immobilised will also contribute to a hapten and which therefore only requires one probe a background signal. Carrico's method is thus not appli- is described by Carrico. In U.S. Pat. No. 4,743,535 is 55 cable to the detection of very low concentrations of tar- disclosed a nucleic acid hybridisation assay involving a get nucleic acid which are in the range of sensitive de- reporter probe which results in the formation of a hybrid tection systems such as signal amplification detection having epitopes for an antibody reagent. The antibody systems or chemiluminescence. The approach of Car-

3 5 EP 0 975 805 B1 6 rico finds utility in the detection of target amplification DNA or RNA is amplified by the polymerase chain reac- products, such as those generated by the polymerase tion (PCR). chain reaction (PCR). For example, a commercial as- [0021] This method involves the hybridisation of an ol- say, GEN-ETI-K™, from Sorin utilises probes immobi- igonucleotide primer to the 5' end of each complemen- lised on microtitre plates by means of a streptavidin-bi- 5 tary strand of the double-stranded target nucleic acid. otin bridge and antibodies against double stranded DNA The primers are extended from the 3' end in a 5'→3' di- labelled with peroxidase. Its chief application is in the rection by a DNA polymerase which incorporates free assay of nucleic acid amplification products. nucleotides into a nucleic acid sequence complementa- [0016] One approach to overcome the problem of ry to each strand of the target nucleic acid. After disso- cross-reactivity is disclosed in U.S. Pat. No. 5612,458 10 ciation of the extension products from the target nucleic co Hyldig-Nielson and Pluzek They use antibodies to acid strands, the extension products become target se- complexes between peptide nucleic acid (PNA) and nu- quences for the next cycle. In order to obtain satisfactory cleic acids, particularly antibodies to nucleic acid probe- amounts of the amplified DNA, repeated cycles must be DNA or nucleic acid probe-RNA hybrids. carried out:, between which cycles, the complementary [0017] Another approach is to attempt to improve the 15 DNA strands must be denatured under elevated tem- affinity or selectivity of the antibody used. Fliss et al. (Ap- peratures. plied and Environmental Microbiology (1993) 59:2698 - [0022] A method of detecting a specific nucleic acid 2705) disclose murine monoclonal antibodies specific sequence present in low copy in a mixture of nucleic ac- for DNA-RNA hybrids which are used to detect Lysteria ids, called ligase chain reaction (LCR), has also been DNA-RNA hybrids formed in solution from a biotinylated 20 described. WO 89/09835 describes this method. Target gene probe and rRNA extracted from Lysteria. They al- nucleic acid in a sample is annealed to probes contain- so teach that the endonuclease digestion approach ing contiguous sequences. Upon hybridisation, the used by Atlas and Stefan (see above) does not efficient- probes are ligated to form detectable fused probes com- ly separate hybridised from unhybridised molecules. plementary to the original target nucleic acid. The fused Significantly, they do not teach that treatment with a nu- 25 probes are disassociated from the nucleic acid and clease to remove any single-stranded nucleic acids prior serve as a template for further hybridisation's and fu- to capture with the murine monoclonal antibodies spe- sions of the probes, thus amplifying geometrically the cific for DNA-RNA hybrids would offer an improvement nucleic acid to be detected. The method does not use to their assay. DNA polymerase. [0018] In summary, Carrico discloses a simple meth- 30 [0023] Other known nucleic acid amplification proce- od for capturing hybrids formed in solution between a dures include transcription-based amplification systems target nucleic acid and a nucleic acid probe which uti- (Kwoh et al., Proc. Natl. Acad. Sci. (U.S.A.) (1989) 86: lises antibodies specific for double-stranded nucleic ac- 1173; Gingeras et al., WO 88/10315; Davey et al., Ep id. A disadvantage of this approach is the cross-talk be- 329,822; Miller et al., WO 89/06700), RACE (Frohman, tween antibody and any single-stranded nucleic acid 35 In: PCR Protocols: A Guide to Methods and Applica- which may be present. This limits the sensitivity of the tions, Academic Press, NY (1990)) and one-sided PCR assay. Atlas and Steffan disclose another solution (Ohara, et al., Proc. Natl. Acad. Sci. (U.S.A.) (1989) 86: phase hybridisation assay in which hybrids are separat- 5673-5677). Particularly suitable amplification proce- ed by column chromatography following an endonucle- dures include Nucleic Acid Sequence-Based Amplifica- ase digestion step. A similar approach is utilised in nu- 40 tion, Strand Displacement Amplification, and Cycling clease protection assays. This is a sensitive technique Probe Amplification. for the detection, quantitation, and characterisation of [0024] Methods based on ligation of two (or more) ol- RNA. The hybridisation reaction occurs in solution al- igonucleotides in the presence of nucleic acid having the lowing complete hybridisation of the probe to the target sequence of the resulting di-oligonucleotide, thereby mRNA. After hybridisation, remaining single-stranded 45 amplifying the di-oligonucleotide, are also known (Wu probe RNA and unhybridised sample RNA are removed et al., Genomics (1989) 4:560). by digestion with a mixture of ribonucleases A and T1, [0025] An isothermal amplification method has been or S1 nuclease. Then, in a single step, the nucleases described in which restriction endonucleases and ligas- are inactivated and the remaining hybrids precipitated. es are used to achieve the amplification of target mole- Nuclease protection assays are not used for the detec- 50 cules that contain nucleotide 5'-[a-thio]triphosphates in tion of DNA. A disadvantage of these latter two methods one strand of a restriction site (Walker et al., Proc. Natl. is that they are cumbersome and do not accurately Acad. Sci, (U.S.A.) (1992) 89:392-396). quantify the amount of target nucleic acid present. [0026] It is important that enzymes employed as la- [0019] There remains a need to combine the advan- bels catalyse a reaction which has an easily detectable tageous features of Carrico's invention with one having 55 product, and have a high turnover number to allow sen- reduced cross-talk, lower background and improved sitive detection: horseradish peroxidase and alkaline sensitivity. phosphatase are most common. Although sensitive [0020] In U.S. Pat. Nos. 4,683,195 and 4,683,202, chemiluminometric assays for horseradish peroxidase

4 7 EP 0 975 805 B1 8 have been described which allow small amounts of en- biotin and avidin, streptavidin or neutravidin, or a nucleic zyme to be detected, problems associated with its use acid binding protein specific for a sequence present in include lack of enzyme and substrate stability and the the nucleic acid probe. Any of these agents may be la- presence of endogenous peroxidases in samples. belled with a detectable label which may an enzyme, a [0027] For alkaline phosphatase, enzyme amplifica- 5 fluorescent moiety, a chemiluminescent moiety, an elec- tion cycles have been described which further reduce tro-chemiluminescent moiety or a coloured moiety. the amount of enzyme which can be detected, thereby [0031] In a further aspect the invention discloses a extending the detection limit. In one method, the ampli- method for detecting DNA-RNA hybrids, DNA-DNA, fication system comprises an apoenzyme which is con- RNA-RNA, DNA-RNA or DNA-PNA hybrids between a vertible into a holoenzyme by interaction with an acces- 10 target nucleic acid and a nucleic acid probe having a sory subunit and a masked form of the subunit which is sequence complementary to part of the target nucleic convertible into its active unmasked form by the action acid. of the enzyme to be detected. For example, in US Patent [0032] In a another further aspect the invention dis- No. 5,445,942 to Rabin et al., a method is disclosed for closes a method for detecting hybrids between nucleic detecting a hydroiase enzyme able to hydrolyse a syn- 15 acid amplification products and a nucleic acid probe thetic derivative of FAD substituted in such a way that it having a sequence complementary to part of the ampli- yields FAD when hydrolysed, and is incorporated herein fied nucleic acid. by reference in its entirety. Here the subunit is FAD and [0033] In a further aspect the invention discloses a the masked form is 3'FADP, and the apoenzyme is ap- method for detecting hybrids between target nucleic ac- oglucose oxidase or apo-D-aminoacid oxidase. 20 id extracted from a clinical specimen, a veterinary spec- [0028] The FAD produced forms an active holoen- imen, a food specimen or an environmental sample and zyme from the corresponding apoenzyme. This ap- a nucleic acid probe having a sequence complementary proach allows the detection of small amounts of alkaline to part of the target nucleic acid. phosphatase in short periods of time. For example, us- [0034] In a another further aspect the invention dis- ing such an amplification system in which the apoen- 25 closes a method for the detection of multiple nucleic acid zyme is apo-D-amino acid oxidase has permitted the de- targets in a sample. tection of 0.1 amol of alkaline phosphatase in less than [0035] In further aspects the invention provides a kit 30 minutes (Harbron et al., Anal. Biochem. (1992) 206: for carrying out the method. 119 - 124). In GB9622524.8 this approach is further ex- [0036] Preferred embodiments of the invention may tended to an amplification assay for nuclease P. 30 enable one to achieve one or more of the following ob- jects and advantages: Disclosure of Invention (a) to provide a method for detecting hybrids be- [0029] Broadly, the present invention combines ad- tween a target nucleic acid and a nucleic acid probe vantageous aspects of the above techniques and dis- 35 having a sequence complementary to part of the closes a new and improved method for detecting single- target nucleic acid by means of a specific binding stranded target nucleic acid comprising the steps of: member, in which any single-stranded nucleic acid is removed by treatment with an enzyme reagent (a) forming a hybrid between said target nucleic ac- attached to said probe and which is specific for sin- id and a nucleic acid probe, said nucleic acid probe 40 gle-stranded nucleic acids. Advantages of the labelled with an enzyme reagent which hydrolyses present invention are: only a single probe is re- single-stranded nucleic acid but is substantially quired; highly sensitive detection systems, such as without effect on double-stranded nucleic acid, said chemiluminescence or enzyme amplification cas- hybrid formed under conditions of pH which are out- cades may be used to detect the hybrids; and the side the activity range of said enzyme reagent, 45 sensitive detection of target nucleic acid may be (b) adjusting said pH to a value within the activity achieved without using target amplification tech- range of said enzyme reagent, niques, such as PCR or LCR. (c) allowing said enzyme reagent substantially to (b) to provide a method for detecting multiple nucle- hydrolyse any single-stranded nucleic acid present, ic acid targets. An advantage of the present inven- and 50 tion is that a single sample may be screened for a (d) detecting said hybrid. number of targets, thereby increasing the speed of assay and reducing the number of sample which are [0030] In a further aspect, the invention provides a va- required. riety of means for detecting the hybrid by means of a (c) to provide a universal method for detecting tar- hybrid-binding reagent such as an antibody or DNA- 55 get nucleic acid. An advantage of the present inven- binding protein specific for double-stranded nucleic action is that it may be used with existing nucleic acid id, or by means of a pair or pairs of sbm's. These may probes and their corresponding detection systems. be a antigen or hapten and the corresponding antibody, (d) to provide a method for detecting hybrids be-

5 9 EP 0 975 805 B1 10

tween DNA-RNA, RNA-DNA, RNA-RNA, RNA- physical/chemical disruption (detergents such as Tri- PNA and DNA-PNA hybrids by appropriate selec- ton™, Tween, or sodium dodecylsulfate, alkali treat- tion of the hybrid binding reagent and enzyme rea- ment, osmotic shock, or heat), or enzymatic lysis (lys- gent used. ozyme, proteinase K, pepsin). The resulting test medi- 5 um will contain the target nucleic acid in single-stranded Brief Description of Drawings form. [0041] The nucleic acid probe may be a DNA probe [0037] an RNA probe, or a PNA probe. The nucleic acid probe will comprise at least one single-stranded base se- Figure 1 is a diagrammatic representation of three 10 quence substantially complementary to at least part of preferred embodiments of the present invention for the target nucleic acid sequence. However, such base the detection of single-stranded nucleic acids. sequence need not be a single continuous polynucleotide segment, but can be comprised of two or more in- Figure 2 shows a standard curve for the 3'FADP- dividual segments interrupted by non-complementary 15 based enzyme amplification assay of nuclease P1 sequences. These non-hybridisable sequences are lin- (filled triangles) and nuclease S1 (filled squares). ear. In addition, the complementary region of the nucleic The abscissa represents the amount of each en- acid probe can be flanked at the 3'- and 5'-termini by zyme present in amol (10-8 mol), and the ordinate non-hybridisable sequences, such as those comprising represents the absorbance obtained after 15 mins the DNA or RNA of a vector into which the complemen- incubation at 25° C after subtraction of the blank 20 tary sequence had been inserted for propagation. In ei- reading. Both scales are logarithmic. The dotted ther instance, the nucleic acid probe as presented as an line represents the detection limit. analytical reagent will exhibit detectable hybridisation at one or more points with target nucleic acids of interest. Reference Numerals Used in the Drawings The nucleic acid probe sequence can be of any conven- 25 ient or desired length, ranging from as few as a dozen [0038] to as many as 10,000 bases, and including oligonucleotides having less than about 50 bases. The nucleic acid 2 - single-stranded target nucleic acid probe may be an oligonucleotide produced by solid- 4 - nucleic acid probe phase chemistry by a nucleic acid synthesiser. The RNA 6 - enzyme reagent 30 or DNA probe can be obtained in a variety of other con- 8 - first member of a specific binding pair ventional manners. It should be understood that in using 10 - specific binding member the expressions RNA probe and DNA probe herein, it is 12 - solid surface not implied that all nucleotides comprised in the probe 14 - second member of a specific binding pair be ribonucleotides or 2'-deoxyribonucleotides. There- 16 - product 35 fore, one or more of the 2'-positions on the nucleotides comprised in the probe can be chemically modified pro- Best Modes for Carrying Out the Invention vided the antibody binding characteristics necessary for performance of the present assay are maintained to a [0039] The present invention provides a method for substantial degree. Likewise, in addition or alternatively detecting hybrids between a target nucleic acid and a 40 to such limited 2'-deoxy modification, the nucleic acid nucleic acid probe having a sequence complementary probe can have in general any other modification along to part of the target nucleic acid by treating the sample its ribose phosphate backbone provided there is no sub- with an enzyme reagent to remove single-stranded nu- stantial interference with the specificity of the antibody cleic acids, and detecting the hybrid. to the double stranded hybridisation product compared [0040] The target nucleic acid may be DNA or RNA, 45 to its individual single strands. In preferred embodi- and is obtained from any medium of interest, for exam- ments, in addition to the enzyme label, the nucleic acid ple, a liquid sample of medical, veterinary, environmen- probe is labelled with either a detectable moiety or an tal, or industrial significance. The target nucleic acid immobilisable moiety. For example, the nucleic acid may also be the product of a nucleic acid amplification probe is prepared by solid-phase chemistry using a nu- assay, such as PCR or LCR. If the target nucleic acid is 50 cleic acid synthesiser and has a trityl-hexyl thiol deriva- principally double stranded, it will be treated to denature tised 5'-end. The covalent attachment of the label to this it prior to the formation of the hybrid. Denaturation of moiety may be achieved by a number of well-known nucleic acids is preferably accomplished by heating in methods using a wide range of heterobifunctional rea- boiling water or alkali treatment (e.g., 0.1 N sodium hy- gents. For example, the method of Carlsson et al. :Bio- droxide), which if desired, can simultaneously be used 55 chem J (1978) 173: 723 - 737) may be used: the label to lyse cells. Also, release of nucleic acids from cellular is reacted with 3-[(2)-pyridyldithio]propionic acid N-hy- or viral sources can, for example, be obtained by me- droxysuccinimide ester (SPDP). to give a 2-pyridyl di- chanical disruption (freeze/thaw, abrasion, sonication), sulphide-activated label. This allows disulphide ex-

6 11 EP 0 975 805 B1 12 change with trityl-hexyl thiol derivatised described nucleic acid may also be obtained from commercial above to yield a labelled nucleic acid probe. Other ap- sources. In preferred embodiments the antibody is la- proaches for labelling the nucleic acid probe will be ap- belled with either a detectable moiety or an immobilisa- parent to one skilled in the art. Additionally, a wide range ble moiety. The covalent attachment of the label may be of labelled nucleic acids is available from commercial 5 achieved by a number of well-known methods using a sources. Preferred labels include the enzymes alkaline wide range of heterobifunctional reagents. For example, phosphatase, peroxidase, β-galactosidase, nuclease the method of Carlsson et al. (Biochem J (1978) 173: P1 and nuclease S1; the haptens digoxin, digoxygenin, 723 - 737) may be used: the label is reacted with 3-[(2)- fluorescein, fluorescein isothiocyanate, and biotin or bi- pyridyldithio]propionic acid N-hydroxysuccinimide ester otin analogues. 10 (SPDP) to give a 2-pyridyl disulphide-activated label. [0042] A preferred embodiment of the present inven- This is mixed with an IgG antibody, and a disulphide ex- tion employs a nuclease as the enzyme reagent. A change reaction yields a labelled antibody conjugate. number of nucleases are known which are specific for Other approaches for labelling the antibody will be ap- single-stranded nucleic acids. For example, ribonucle- parent to one skilled in the art. Preferred labels include 15 ase A and ribonuclease T1 may be used in combination the enzymes alkaline phosphatase, peroxidase, b-ga- to hydrolyse single-stranded RNA. Other preferred nu- lactosidase, and nuclease P1; the haptens digoxin and cleases include exodeoxyribonuclease I (E.C. 3.1.11.1, digoxigenin, and biotin or biotin analogues. In a partic- similar enzymes: mammalian DNase III, exonuclease ularly preferred embodiment the antibody is immobilised IV, T2- and T4-induced exodeoxyribonucleases), exo- directly onto a microtitre plate. This may be achieved by deoxyribonuclease (phage sp3-induced) (E.C. 3.1.11.4, 20 a number of means well known to those skilled in the exodeoxyribonuclease V (E.C. 3.1.11.5, similar en- art. For example, Immulon II microtitre plates may be zyme: Haemophilus influenzae ATP-dependent coated with the antibody by incubating them with the an- DNase), exodeoxyribonuclease VII (E.C. 3.1.11.6, sim- tibody dissolved in 60 mM carbonate buffer pH 9.6. Oth- ilar enzyme: Micrococcus luteus exonuclease), exoribo- er approaches will be apparent to one skilled in the art. nuclease II (E.C. 3.1.13.1, similar enzymes: RNase Q, 25 [0044] Particularly attractive applications, which illus- RNase 3N, RNase PIII, RNase Y), venom exonuclease trate the operation of the present invention, are de- (E.C. 3.1.15.1, similar enzymes: hog kidney phosphodi- scribed below. esterase, Lactobacillus exonuclease), spleen exonucle- [0045] Referring now to Fig 1, which shows three par- ase (E.C. 3.1.16.1, similar enzymes: Lactobacillus aci- ticularly preferred embodiments of the present inven- dophilus nuclease, B subtilis nuclease, salmon testis 30 tion, the first row shows the target nucleic acid (2), de- nuclease, deoxyribonuclease IV (phage T4-induced) (E. natured if necessary to render it single-stranded, being C. 3.1.21.2, similar enzymes: DNase V (mammalian, contacted under hybridisation conditions with a nucleic Aspergillus sojae DNase, 3 subtilis endonuclease, T4 acid probe (4) having a sequence complementary to at endonuclease III, T7 endonuclease I, Aspergillus least part of the target nucleic acid and labelled at its 5'- 35 DNase K2, Vaccinia virus DNase VI, yeast DNase, Chlo- end with an enzyme reagent (6), preferably nuclease P1. rella DNase), Aspergillus deoxyribonuclease K1 (E.C. In two embodiments, shown in the 2nd and 3rd columns, 3.1.22.2, Aspergillus nuclease Si (E.C. 3.1.30.1, similar nucleic acid probe (4) is additionally labelled at its 3'- enzymes: N crassa nuclease, mung bean nuclease, end with a first member of a specific binding pair (8), Penicillium citrinum nuclease P1). Particularly preferred preferably biotin. 40 nucleases are nuclease P1 and nuclease S1 which have [0046] In the second row of Fig. 1, the pH of the mix- a relatively broad specificity against single-stranded ture is adjusted to allow enzyme reagent (6) to remove DNA and RNA. single-stranded nucleic acids. These single-stranded [0043] Where the hybrid binding reagent is an anti- nucleic acids comprise unhybridised probe and unhy- body, this may be obtained in any available manner such bridised target. as conventional antiserum and monoclonal techniques. 45 [0047] In the final row of Fig. 1, an sbm (10) immobi- Antiserum can be obtained by well-established tech- lised on a solid surface (12) recognises and binds to the niques involving immunisation of an animal, such as a hybrids which have formed. Sbm (10) is preferably an mouse, rabbit, guinea pig or goat, with an appropriate antibody specific for double-stranded nucleic acid, as immunogen. The immunoglobulins can also be obtained shown in columns 1 and 2 of Fig. 1, or streptavidin as by somatic cell hybridisation techniques, also involving 50 shown in column 3. In one embodiment, shown in col- the use of an appropriate immunogen. The antibody re- umn 2, a second member of a specific binding pair la- agent may also be a recombinant antibody, a chimeric belled with a detection enzyme (14), preferably bioti- antibody, or a single chain antibody. The antibody may nylated alkaline phosphatase is also introduced. Un- be specific for RNA-DNA hybrids, DNA-DNA hybrids or bound materials are washed off and the amount of RNA-RNA hybrids. An example of the production of anti- 55 bound probe nucleic acid-target nucleic acid-antibody DNA-RNA monoclonal antibodies is given by Fliss et al. complex is determined by measuring the amount of (Applied and Environmental Microbiology (1993) 59: product (16) produced by enzyme label (6 or 14), pref- 2698 - 2705). Antibodies specific for double-stranded erably using an amplification assay for the nuclease P1,

7 13 EP 0 975 805 B1 14 as shown in columns 1 and 3, or alkaline phosphatase, amine, thiol or aldehyde chemistry or biospecifically by as shown in column 2, attached to the probe. e.g. biotin-avidin interaction. An sbm specific for the hy- [0048] In Fig. 1, the nuclease P1 is shown to be joined brid or a moiety present on the nucleic acid probe is cou- directly to the nucleic acid probe. Embodiments are en- pled to the dextran layer of a sensor chip used in the visaged in which the probe is labelled with a moiety, 5 BIAcore™ biosensor-system (or other types of biosen- such as flourescein isothiocyanate, and nuclease P1 is sor systems). A sample containing target nucleic acid, attached thereto by means of an anti-FITC antibody la- denatured if necessary to render it single-stranded, is belled with nuclease P1. contacted under hybridisation conditions with a nucleic [0049] Other embodiments of the invention employing acid probe labelled with an enzyme so that a complex the principles described above will be obvious to one 10 is formed. The pH of the mixture is adjusted to allow the skilled in the arts. Thus the method may be applied to enzyme reagent to hydrolyse single-stranded nucleic hybridisation and detection in solution. The target nu- acids. The sample is passed through the flow system of cleic acid, denatured if necessary to render it single- the BIAcore™ and the sbm coupled to the dextran sur- stranded, is contacted under hybridisation conditions face will bind the nucleic acid probe-target nucleic acid with a nucleic acid probe having a sequence comple- 15 complex if present. Based on the SPR detection em- mentary to at least part of the target nucleic acid and ployed by the BIAcore™ this binding will generate a sig- labelled with an enzyme reagent. The pH of the mixture nal dependent on the amount of target material in the is adjusted to allow the enzyme reagent to remove sin- sample which becomes bound to the surface. gle-stranded nucleic acids, and hybrids which have [0052] In a yet further application, the method may be formed bind to a sbm specific for the hybrid or a moiety 20 applied to the detection of bound nucleic acid probe in present on the nucleic acid probe. These reactions will cells. Under suitable conditions, nucleic acid probe oli- result in a large complex which may be detected, for ex- gomers may be able to penetrate the cell-wall of living ample by a turbidimetric assay. or fixed cells, such as cell-lines, hemopoetic cells, and [0050] In another further application, the method may animal/human tissues. Labelling the nucleic acid probe be applied to the detection of sbm-nucleic acid complex- 25 with haptens or other reporter molecules can inhibit pen- es on a solid phase. Compiexes formed between a nu- etration into the cells. After the pH of the mixture is ad- cleic acid probe and a target nucleic acid in which either justed to allow the enzyme to remove single-stranded the nucleic acid probe or the target nucleic acid is im- nucleic acid, hybrids formed between the nucleic acid mobilised on a solid phase can be detected by the meth- probe and target nucleic acid are detected, either by im- od of the present invention. The pH of the mixture is ad- 30 munohistochemistry (in frozen or fixed tissue biopsies) justed to allow the enzyme reagent to remove single- or by flow-cytometry (e.g. on cells treated with deter- stranded nucleic acids from the immobilised complex, gent, acetone or alcohol), or in an in vivo set up to detect and detection can be performed either directly using a binding and/or tissue distribution of nucleic acid probe's sbm conjugated to an enzyme, a fluorescent marker or added to a cell culture or administered to a living animal. another signal generating system, or indirectly using 35 [0053] In another application, the method may be ap- one of the detection systems commonly used for detect- plied to hybridisation and detection of multiple targets in ing sbm's bound to their target. The solid includes nylon a single sample solution. The target nucleic acids, de- or nitro-cellulose membranes (Southern or Northern natured if necessary to render them single-stranded, are blots), a tissue section (in situ hybridisation), or a plastic contacted under hybridisation conditions with corre- surface (an ELISA format). This approach has the ad- 40 sponding labelled nucleic acid probes having sequenc- vantage that the extensive washing procedures normal- es complementary to at least part of the respective tar- ly used in these assays can be reduced to a minimum get nucleic acids and which are labelled with an enzyme as single-stranded nucleic acid probe will be hydrolysed reagent. The pH of the mixture is adjusted to allow the by the enzyme reagent. enzyme reagent to remove single-stranded nucleic ac- [0051] In a further application, the method may be ap- 45 ids, and hybrids which have formed bind to an immobi- plied to biosensor systems. One example of dynamic lised sbm specific for the hybrid or a moiety present on reaction detection using a biosensor surface is the sur- the nucleic acid probe. Unbound materials are washed face plasmon resonance (SPR) detection system, such away. The captured hybrids may be detected in several as that employed by the BIAcore™ biosensor system ways. In one approach, each set of probes used are la- (Pharmacia). The interaction of biomolecules with an 50 belled with different fluorescent or absorbing moieties. immobilised ligand on a sensor chip is measured at the These may be interrogated at different wavelengths, surface using evanescent light. The system includes a and the amounts of each target present in the original sensor chip to which the ligand can be immobilised in a sample are thereby determined. In another approach, hydrophilic dextran matrix, a miniaturised fluidics car- each set of probes used are labelled with different en- tridge for the transport of analytes and reagents to the 55 zymes. After treatment with the enzyme reagent, aliq- sensor surface, a SPR detector, an autosampler and uots of the solution are dispensed into different wells of system control and evaluation software. Specific ligands a microtitre plate coated with sbm specific for the hybrid are covalently immobilised to the sensor chip through or a moiety present on the nucleic acid probe. After

8 15 EP 0 975 805 B1 16 washing to remove unbound components, different de- manner, and the detection limit was 4 amol (Fig. 2). tection reagents are added to each of the wells, and the amounts of each target present in the original sample Example 3 are thereby determined. [0054] A kit for carrying out the described methods ac- 5 Oligonucleotide Synthesis cording to the present invention contains a sbm specific: for the hybrid or a moiety present on the nucleic acid [0059] Oligonucleotides were synthesised on a Cy- probe in labelled or unlabelled form, a nucleic acid probe clone™ DNA synthesiser using the Expedite™ chemis- that is complementary to the target nucleic acid to be try. 10 detected and which is labelled with an enzyme reagent [0060] The DNA to be labelled with nuclease P1 was specific for single-stranded nucleic acids, and a detec- complementary to a region in the middle of the ribonu- tion system. clease gene. containing the K66E mutation. This probe [0055] In a preferred embodiment, the kit contains a was derivatised at the 5' end with a trityl-hexyl thiol sbm specific for the hybrid or a moiety present on the group to facilitate linkage to nuclease P1. The sequence nucleic acid probe immobilised in the wells of a microti- 15 was: tre plate, a nucleic acid probe that is complementary to the target nucleic acid to be detected and which is labelled with an enzyme reagent specific for single- stranded nucleic acids, and a detection system. [0056] The following examples illustrate various fur- 20 ther aspects of the operation of the invention. These ex- [0061] Another oligonucleotide specific for repeat re- amples are not intended to limit the invention in any way. gions of the genomic DNA of Streptococcus pneumoniae (SEQ ID No 6 of U.S. Pat. No. 5,656,432) and having Example 1 the sequence: 25

Standardisation of Nuclease P1.

[0057] Nuclease P1 (1 mg; obtained from sigma Chemical Company, batch no: 107F0799) was dissolved in 1 ml of water to give a concentration of 22.7 30 and having a biotinylated 5'-end and an FITC-la- µM and stored at 4°C. The activity of this solution was belled 3'-end was obtained from Cruachem Ltd. assayed in the following mixture: 0.16 mM NADH, 1 mM [0062] The oligonucleotides were freeze-dried and ATP, 1 mM PEP, 1 mM MgSO4, 20 mM KCl, 0.5 mM ad- stored at 4°C until required. enosine 3',5'-bisphosphate, 1 U pyruvate kinase, 1 U lactate dehydrogenase and 1 U myokinase in 50 mM 35 Example 4 HEPES buffer, pH 7.2, in a total volume of 1 ml. From the change in absorbance at 340 nm the activity of nu- Derivatisation of Nuclease P1 clease P1 was solution was found to be 320 U/ml, as- suming a molar extinction coefficient of 6220 for NADH. [0063] Nuclease P1 (5 mg) was dissolved in 0.5 ml 0.1 40 M sodium bicarbonate pH 7.5 containing 0.1 M sodium Example 2 chloride and desalted by gel filtration on Sephadex G25 (NAP-5 column, Pharmacia) equilibrated with the same Amplification Assay of Nuclease P1 and Nuclease buffer. This enzyme solution was incubated with a S1 50-fold molar excess of 3-(2)-pyridyldithio)-propionic 45 acid N-hydroxysuccinimide ester (SPDP) at room tem- [0058] A solution of nuclease P1 standardised accord- perature for 30 minutes. Unreacted SPDP was removed ing to Example 1 was serially diluted in 50 mM citrate by gel filtration on Sephadex G25 (NAP 10 column, buffer adjusted to pH 6.5 with NaOH. The assay mixture Pharmacia) equilibrated with the bicarbonate buffer. contained 20 mM 3'FADP, 0.1 mM 4-aminoantipyrine, 2 The 2-pyridyl disulphide-activated nuclease P1 was mM DHSA, 1 µg horseradish peroxidase, 0.1 M glucose 50 stored at 4°C. and 0.1 µM apoglucose oxidase in a total volume of 0.1 ml. The change in absorbance was monitored at 520 nm Example 5 in a Dynatech MR7000 plate reader fitted with a ther- mostatically controlled plate holder set to 25°C. Fig. 2 Conjugation of Nuclease P1 to an Oligonucleotide 55 shows the performance of the nuclease P1 assay. After a 15 minute assay period, the detection limit (defined as [0064] Nuclease P1 was linked to 2-pyridyl disulphide 3 times the standard deviation of the background read- as described in Example 4 and stored in 0.1 M sodium ing) was 0.2 amol. Nuclease S1 was assayed in a similar bicarbonate, pH 7.5, containing 0.1 M sodium chloride

9 17 EP 0 975 805 B1 18 at 4°C. The K66E oligonucleotide of Example 3 was dis- isation with 8 µl of 0.5 M sodium citrate buffer, pH 3.0, solved in 0.5 ml 0.1 M sodium bicarbonate buffer, pH containing 2.21 M sodium chloride and 0.1% Tween 20. 7.5, containing 0.1 M sodium chloride to give a final con- 50 µl (34 fmol) of the S pneumoniae probe described in centration of 0.36 µM. This was incubated with activated Example 3, dissolved in 0.1 M Tris-HCl buffer, pH 7.5, 5 nuclease P1 prepared according to Example 4 at a mole containing 7 mM zinc sulphate, 1% (w/v) PVP, 0.1 % N- ratio of 1:2 at room temperature for 45 minutes, followed lauroylsarkosine and 150 mM sodium chloride, is add- by an incubation at 4°C for 16 h. ed, together with 50 µlof1µg/ml nuclease P1-labelled [0065] The conjugate was transferred to 20 mM bis- anti-FITC antibody, prepared by linking anti-FITC anti- Tris propane buffer, pH 7.5, containing 1 mM CHAPS by body treated with 2-mercaptoethylamine (to yield free chromatography on Sephadex G25, and purified by ion- 10 sulphydryl groups) with the SPDP-activated nuclease exchange chromatography on a Pharmacia Mono Q col- P1 of Example 4 in an analogous way to that described umn. A sodium chloride gradient in the same buffer was in Example 5. After hybridisation at 40°C for 1 hour, the used applied to the column and the conjugate was elut- pH is adjusted to about 6.0 by the addition of citrate buff- ed at a molar concentration of 0.25 M. er, and the temperature maintained at 40°C for 10 min- 15 utes, after which time more than 95% of unhybridised Example 6 reporter probe will be hydrolysed. [0070] The mixture is then added to a commercial mi- Hybridisation and Detection of Plasmid DNA on crotitre plate coated with either anti-double stranded Antibody-Coated Plates DNA antibodies or streptavidin. After incubation at 37°C 20 for 30 minutes, the plates are washed 6 times with 20 [0066] 50 pg of λDNA, dissolved in 95 µl sterile water, mM Tris-HCl buffer, pH 7.5, containing 7 mM zinc sul- which serves as a control for non-complementary bind- phate, 1% (w/v) PVP, 0.1 % N-lauroylsarkosine and 150 ing, is mixed with a further 5 µl of a known amount of mM sodium chloride. the piasmid containing the human RNase mutant and [0071] The amount of hybrid captured on the microti- 10 µl 1 M sodium hydroxide in a microtitre plate well. 25 tre plate is quantified using the amplification assay de- This mixture is incubated at room temperature for 10 scribed in Example 2. minutes to denature the plasmid before neutralisation with 8 µl of 0.5 M sodium citrate buffer, pH 3.0, contain- Industrial Applicability ing 2.21 M sodium chloride and 0.1 % Tween 20. 50 µl 30 (34 fmol; of the nuclease P1-conjugated reporter probe, [0072] Accordingly, it will be seen that the method of prepared according to Example 5, dissolved in 0.1 M the present invention can be used to detect hybrids Tris-HCl buffer, pH 7.5, containing 7 mM zinc sulphate, formed between a target nucleic acid and a nucleic acid 1% (w/v) PVP, 0.1 % N-lauroylsarkosine and 150 mM probe labelled with an enzyme reagent which removes sodium chloride, is added to each well. After hybridisa- single-stranded nucleic acid. This approach eliminates tion at 40°C for 1 hour, the pH is adjusted to about 6.0 35 the possibility of cross-talk arising out of the binding of by the addition of citrate buffer, and the temperature sbm to any single-stranded nucleic acid present. This maintained at 40°C for 10 minutes, after which time means that the complex formed between hybrid and more than 95% of unhybridised reporter probe will be sbm can be detected using highly sensitive approaches, hydrolysed. such as enzyme amplification or chemiluminescence. In [0067] The mixture is then added to a commercial mi- 40 addition, the nucleic acid probe may be labelled with nu- crotitre plate coated with anti-double stranded DNA an- clease P1 at each end, thereby giving an increase in the tibodies. After incubation at 37°C, the plates are washed overall sensitivity of the detection reaction. 6 times with 20 mM Tris-HCl buffer, pH 7.5, containing [0073] In addition to the methods described above, 7 mM zinc sulphate, 1% (w/v) PVP, 0.1 % N-lauroyl- many other techniques for detecting the complex sarkosine and 150 mM sodium chloride. 45 formed between a sbm and the hybrid will be apparent [0068] The amount of hybrid captured on the microti- to one skilled in the art. For example the complex formed tre plate is quantified using the amplification assay de- may be captured. A number of approaches are known scribed in Example 2. for effecting the capture. For example, the complex may be captured by means of an antibody specific for the Example 7 50 sbm and which is immobilised on a solid phase. Alter- natively, the sbm may be labelled, and the complex cap- Detection of S pneumoniae genomic DNA. tured by means of an antibody immobilised on a solid phase, and which is specific: for said label. Another ap- [0069] Genomic DNA from 5 pneumoniae was ex- proach is to label the sbm with an antibody specific for tracted and treated with PstI to break the DNA up into 55 a hapten or antigen immobilised on a solid support. A fragments. 95 µl of the treated DNA is mixed with 10 µl further approach is to label the sbm with one partner of 1 M sodium hydroxide and incubated at room tempera- a pair of sbm's, and capture the complex by means of ture for 10 minutes to denature the DNA before neutral- the second partner immobilised on a solid surface. A yet

10 19 EP 0 975 805 B1 20 further approach involves labelling the nucleic acid b) contacting said probe with said target under probe, and capturing the complex by means of an anti- conditions of pH which are outside the activity body immobilised on a solid phase which is specific for range of said enzyme, so as to form a hybrid, said label. Another approach is to label the nucleic acid c) adjusting said pH to a value within the activity probe with an antibody specific for a hapten or antigen 5 range of said enzyme reagent by means of a immobilised on a solid support. A further approach is to base, an acid, or a buffer, label the nucleic acid probe with one partner of a pair of d) hydrolysing all or part of any single-stranded sbm's, and capture the complex by means of the second nucleic acid present by means of said enzyme partner immobilised on a solid surface. Other approach- reagent, es for the capture of the complex will be apparent to one 10 e) detecting said hybrid. skilled in the art. Unbound materials are washed off and the amount of bound probe nucleic acid-target nucleic 2. A method according to claim 1 wherein said enzyme acid-antibody complex is determined. reagent is detectable, whereby said hybrid is de- [0074] Again a number of approaches are known for tected. detecting such a complex. For example, the sbm or the 15 nucleic acid probe may be labelled with a detectable la- 3. A method according to claim 1 or 2 wherein said bel. Alternatively, a label on the sbm or a label on the enzyme reagent is a nuclease. nucleic acid probe may be detected using an antibody detection system. Other approaches for the detection of 4. A method according to claim 3 wherein said nuclease the complex will be apparent to one skilled in the art. 20 is selected from the group consisting of: ribonucle- [0075] The method has the additional advantage that ase A and ribonuclease T1 in combination, exodeox- it utilises a single probe, which offers cost savings and yribonuclease I (E.C. 3.1.11.1), mammalian DNase simplifies the design of assay protocols. III, exonuclease IV, T2- and T4-induced exodeoxyri- [0076] The method has the further advantage that it bonucleases, exodeoxyribonuclease (phage sp3-in- permits the detection of multiple targets in a sample, 25 duced) (E.C. 3.1.11.4), exodeoxyribonuclease V (E. again offering economic advantage over the detection C. 3.1.11.5), Haemophilus influenzae ATP-depend- of each target singly. ent DNase, exodeoxyribonuclease VII (E.C. [0077] Although the description above contains many 3.1.11.6), Micrococcus luteus exonuclease, exoribo- specificities, these should not be construed as limiting nuclease II (E.C. 3.1.13.1), RNase Q, RNase BN, the scope of the invention but as merely providing illus- 30 RNase PIII, RNase Y, venom exonuclease (E.C. trations of some of the presently preferred embodiments 3.1.15.1), hog kidney phosphodiesterase, Lactoba- of this invention. For example, the nucleic acid probe cillus exonuclease, spleen exonuclease (E.C. may be a peptide nucleic acid probe, or another nucleic 3.1.16.1), Lactobacillus acidophilus nuclease, B sub- acid analogue having modified basis or an altered back- tilis nuclease, deoxyribonuclease IV (phage T4-in- bone. When the nucleic acid probe is a peptide nucleic 35 duced) (E.C. 3.1.21.2), DNase V (mammalian), As- acid probe the enzyme reagent may be a protease spe- pergillus sojae DNase, B subtilis endonuclease, T4 cific for single stranded peptide nucleic acid. endonuclease III, T7 endonuclease I, Aspergillus [0078] Thus the scope of the invention should be de- DNase K2, Vaccinia virus DNase VI, yeast DNase, termined by the appended claims and their legal equiv- Chlorella DNase, Aspergillus deoxyribonuclease K1 alents, rather than by the examples given. 40 (E.C. 3.1.22.2, Aspergillus nuclease S1 (E.C. 3.1.30.1), N crassa nuclease, mung bean nuclease, and Penicillium citrinum nuclease P1. Claims 5. A method according to claim 3 wherein said enzyme 1. A method for detecting a single-stranded target nu- 45 reagent is mung bean nuclease. cleic acid comprising the steps of: 6. A method according to any of claims 1 to 5 addition- a) providing a nucleic acid probe, said probe ally comprising the step of contacting said hybrid comprising: with a hybrid-binding reagent. 50 i) a nucleotide sequence complementary to 7. A method according to claim 6 wherein said hybrid- at least part of said target, and binding reagent is an antibody specific for double- ii) an enzyme reagent joined to said se- stranded nucleic acid or a DNA-binding protein spe- quence, said enzyme reagent able to hy- cific for double-stranded nucleic acid. drolyse single-stranded nucleic acids but 55 substantially not able to hydrolyse double- 8. A method according to claim 7 wherein said anti- stranded nucleic acid, body is selected from the group consisting of monoclonal antibody, polyclonal antibody, recombinant

11 21 EP 0 975 805 B1 22

antibody, chimeric antibody and single-chain anti- is convertible into a holoenzyme by interaction with body. an accessory subunit; and a masked form of said subunit which is convertible into its active un- 9. A method according to claim 6 wherein said hybrid- masked form by the action of the said enzyme. binding reagent is labelled. 5 24. A method according to claim 23 wherein said sub- 10. A method according to claim 6 wherein said hybrid- unit is FAD and said masked form is 3'FADP. binding reagent is immobilised. 25. A method according to claim 23 or 24 wherein said 11. A method according to any of claims 1 to 10 wherein 10 apoenzyme is apoglucose oxidase or apo-D-ami- said nucleic acid probe additionally comprises a first noacid oxidase. member of a specific binding pair. 26. A method according to claim 1 wherein said nucleic 12. A method according to claim 11 wherein said first acid probe is immobilised on a solid surface. member is selected from the group consisting of di- 15 goxin, digoxygenin, fluorescein, fluorescein isothi- 27. A method according to any of the preceding claims ocyanate and biotin. wherein said target nucleic acid is isolated from a test sample. 13. A method according to claim 11 additionally comprising the step of contacting said hybrid with a sec- 20 28. A method according to any of the preceding claims ond member of a specific binding pair. wherein said target nucleic acid is produced by a target amplification means. 14. A method according to claim 13 wherein said second member is selected from the group consisting 29. A method according to any of the preceding claims of anti-digoxin antibody, anti-digoxygenin antibody, 25 wherein said target nucleic acid is selected from the anti-fluorescein antibody, anti-fluorescein isothio- group consisting of DNA, RNA or PNA. cyanate antibody, avidin, streptavidin and neutravidin. 30. A method according to claim 29 wherein said target amplification means is selected from the group 15. A method according to claim 13 wherein said sec- 30 comprising polymerase chain reaction, ligase chain ond member has a label. reaction, nucleic acid sequence-based amplification, cycling probe amplification and strand dis- 16. A method according to claim 13 wherein said sec- placement amplification. ond member is immobilised. 35 31. A method according to any of the preceding claims 17. A method according to claim 9 or 15 wherein said wherein said probe nucleic acid is selected from the label is an immobilisable label. group consisting of DNA, RMA or PNA.

18. A method according to claim 9 or 15 wherein said 32. An assay kit for detecting a single-stranded target label is a detectable label. 40 nucleic acid comprising a nucleic acid probe complementary to the target nucleic acid to be detected 19. A method according to claim 18 wherein said de- which is labelled with an enzyme able to substan- tectable label is selected from the group consisting tially hydrolyse single-stranded nucleic acid but not of enzyme, fluorescent moiety, chemiluminescent double-stranded nucleic acid. moiety, and electrochemiluminescent moiety. 45 33. The assay kit according to claim 32 wherein said 20. A method according to claim 19 wherein said en- nuclease is selected from the group consisting of: zyme is β-galactosidase or horseradish peroxidase. ribonuclease A and ribonuclease T1 in combination, exodeoxyribonuclease I (E.C. 3.1.11.1), mam- 21. A method according to claim 19 wherein said en- 50 malian DNase III, exonuclease IV, T2- and T4-in- zyme is selected from the group consisting of alka- duced exodeoxyribonucleases, exodeoxyribonu- line phosphatase, nuclease P1 and nuclease S1. clease (phage sp3-induced) (E.C. 3.1.11.4), exodeoxyribonuclease V (E.C. 3.1.11.5), Haemophilus in- 22. A method according to claim 5 or 21 wherein said fluenzae ATP-dependent DNase, exodeoxyribonu- hybrid is detected by an amplification system. 55 clease VII (E.C. 3.1.11.6), Micrococcus luteus exonuclease, exoribonuclease II (E.C. 3.1.13.1), 23. A method according to claim 22 wherein said am- RNase Q, RNase BN, RNase PIII, RNase Y, venom plification system comprises an apoenzyme which exonuclease (E.C. 3.1.15.1), hog kidney phos-

12 23 EP 0 975 805 B1 24

phodiesterase, Lactobacilius exonuclease, spleen 43. An assay kit according to claim 42 wherein said sub- exonuclease (E.C. 3.1.16.1), Lactobacillus acido- unit is FAD and said masked form is 3'FADP. philus nuclease, B subtilis nuclease, deoxyribonuclease IV (phage T4-induced) (E.C. 3.1.21.2), 44. An assay kit according to claim 42 or 43 wherein DNase V (mammalian), Aspergillus sojae DNase, 5 said apoenzyme is apoglucose oxidase or apo-D- B subtilis endonuclease, T4 endonuclease III, T7 aminoacid oxidase. endonuclease I, Aspergillus DNase K2. Vaccinia virus DNase VI, yeast DNase, Chlorella DNase, As- pergillus deoxyribonuclease K1 (E.C. 3.1.22.2, As- Patentansprüche pergillus nuclease S1 (E.C. 3.1.30.1), N crassa nu- 10 clease, mung bean nuclease, and Penicillium citri- 1. Verfahren zum Nachweisen einer einzelsträngigen num nuclease ?1. Zielnucleinsäure, umfassend die Stufen von:

34. An assay kit according to claim 32 wherein said en- a) zur Verfügung stellen einer Nucleinsäuren- zyme reagent is nuclease P1 or nuclease S1. 15 sonde, wobei die Sonde umfaßt:

35. An assay kit according to any of claims 32 to 34 ad- i) eine Nucleotidsequenz komplementär zu ditionally comprising a specific binding member mindestens Teil des Ziels und specific either for hybrids formed between said sin- ii) ein Enzymreagens, verbunden mit der gle-stranded target nucleic acid and said nucleic ac- 20 Sequenz, wobei das Enzymreagens fähig id probe or for a moiety present on said nucleic acid ist, einzelsträngige Nucleinsäuren zu hy- probe. drolysieren, aber im wesentlichen nicht fä- hig ist, doppelsträngige Nucleinsäure zu 36. An assay kit according to claim 35 wherein said spe- hydrolysieren, cific binding member is an antibody specific for dou- 25 ble-stranded nucleic acid or a DNA-binding protein b) in Kontakt bringen der Sonde mit dem Ziel specific for double-stranded nucleic acid. unter pH Bedingungen, die außerhalb des Ak- tivitätsbereichs des Enzyms sind, so daß ein 37. An assay kit according to claim 36 wherein said an- Hybrid gebildet wird, tibody is selected from the group consisting of mon- 30 c) Einstellen des pH auf einen Wert innerhalb oclonal antibody, polyclonal antibody, recombinant des Aktivitätsbereichs des Enzymreagenzes antibody, chimeric antibody and single-chain anti- durch eine Base, eine Säure oder einen Puffer, body. d) Hydrolysieren alles oder Teil irgendeiner vorhandenen einzelsträngigen Nucleinsäure 38. An assay kit according to claim 35 wherein said moi- 35 durch das Enzymreagens, ety is selected from the group consisting of digoxin, e) Nachweisen des Hybrids. digoxygenin, fluorescein, fluorescein isothiocyanate and biotin. 2. Verfahren nach Anspruch 1, bei dem das Enzym- reagens nachweisbar ist, wodurch das Hybrid nach- 39. An assay kit according to claim 35 wherein said spe- 40 gewiesen wird. cific binding member is selected from the group consisting of anti-digoxin antibody, anti-digoxygen- 3. Verfahren nach Anspruch 1 oder 2, bei dem das En- in antibody, anti-fluorescein antibody, anti-fluores- zymreagens eine Nuclease ist. cein isothiocyanate antibody, avidin, streptavidin and neutravidin. 45 4. Verfahren nach Anspruch 3, bei dem die Nuclease ausgewählt wird aus der Gruppe, bestehend aus: 40. An assay kit according to any of claims 35 to 39 ad- Ribonuclease A und Ribonuclease T1 in Kombina- ditionally comprising a detection system. tion, Exodeoxyribonuclease I (E.C. 3.1.11.1), Säu- ger DNase III, Exonuclease IV, T2- und T4-induzier- 41. An assay kit according to claim 40 wherein said de- 50 te Exodeoxyribonucleasen, Exodeoxyribonuclease tection system is an amplification system. (Phage sp3-induziert) (E.C. 3.1.11.4), Exodeoxyri- bonuclease V (E.C. 3.1.11.5), Haemophilus influ- 42. An assay kit according to claim 41 wherein said am- enzae ATP-abhängige DNase, Exodeoxyribonu- plification system comprises an apoenzyme which clease VII (E.C. 3.1.11.6), Micrococcus luteus is convertible into a holoenzyme by interaction with 55 Exonuclease, Exoribonuclease II (E.C. 3.1.13.1), an accessory subunit; and a masked form of said RNase Q, RNase BN, RNase PIII, RNase Y, (tieri- subunit which is convertible into its active un- sches) Gift Exonuclease (E.C. 3.1.15.1), Haus- masked form by the action of the said enzyme. schweinnierenphosphodiesterase, Lactobacillus

13 25 EP 0 975 805 B1 26

Exonuclease, Milz Exonuclease (E.C. 3.1.16.1), 15. Verfahren nach Anspruch 13, wobei das zweite Lactobacillus acidophilus Nuclease, B. subtilis Nu- Glied eine Markierung hat. clease, Deoxyribonuclease IV (Phage T4-induziert) (E.C. 3.1.21.2), DNase V (Säuger), Aspergillus so- 16. Verfahren nach Anspruch 13, wobei das zweite jae DNase, B subtilis Endonuclease, T4 Endonu- 5 Glied immobilisiert wird. clease III, T7 Endonuclease I, Aspergillus DNase K2, Vaccinia Virus DNase VI, Hefe DNase, Chlorel- 17. Verfahren nach Anspruch 9 oder 15, wobei die Mar- la DNase, Aspergillus Deoxyribonuclease K1 (E.C. kierung eine immobilisierte Markierung ist. 3.1.22.2, Aspergillus Nuclease SI (E.C. 3.1.30.1) N crassa Nuclease, Mung Bohnen Nuclease und Pe- 10 18. Verfahren nach Anspruch 9 oder 15, wobei die Mar- nicillium citrinum Nuclease Pl. kierung eine nachweisbare Markierung ist.

5. Verfahren nach Anspruch 2, wobei das Enzymrea- 19. Verfahren nach Anspruch 18, wobei die nachweis- gens Nuclease P1 oder Nuclease S1 ist. bare Markierung ausgewählt wird aus der Gruppe, 15 bestehend aus Enzym, Fluoreszenzrest, Chemilu- 6. Verfahren nach einem der Ansprüche 1 bis 5, zu- mineszenzrest und Elektrochemilumineszenzrest. sätzlich umfassend die Stufe von in Kontakt bringen des Hybrids mit einem Hybridbindungsreagens. 20. Verfahren nach Anspruch 19, wobei das Enzym β- Galactosidase oder Meerrettich-Peroxidase ist. 7. Verfahren nach Anspruch 6, bei dem das Hybrid- 20 bindungsreagens ein Antikörper, spezifisch für dop- 21. Verfahren nach Anspruch 19, wobei das Enzym aus pelsträngige Nucleinsäure, oder ein DNA-Bin- der Gruppe, bestehend aus alkalische Phosphata- dungsprotein, spezifisch für doppelsträngige Nu- se, Nuclease P1 und Nuclease S1, ausgewählt cleinsäure, ist. wird. 25 8. Verfahren nach Anspruch 7, wobei der Antikörper 22. Verfahren nach Anspruch 5 oder 21, wobei das Hy- ausgewählt wird aus der Gruppe, bestehend aus brid durch ein Amplifikationssystem nachgewiesen monoklonalem Antikörper, polyklonalem Antikör- wird. per, rekombinantem Antikörper, chimärem Antikör- per und einzelsträngigem Antikörper. 30 23. Verfahren nach Anspruch 2,2, wobei das Amplifika- tionssystem umfaßt ein Apoenzym, das in ein Ho- 9. Verfahren nach Anspruch 6, bei dem das Hybrid- loenzym umwandelbar ist durch Wechselwirkung Bindungsreagens markiert wird. mit einer akzessorischen Untereinheit, und eine maskierte Form der Untereinheit, die in ihre aktive 10. Verfahren nach Anspruch 6, bei dem das Hybrid- 35 unmaskierte Form durch die Wirkung des Enzyms Bindungsreagens immobilisiert wird. umwandelbar ist.

11. Verfahren nach einem der Ansprüche 1 bis 10, wo- 24. Verfahren nach Anspruch 23, wobei die Unterein- bei die Nucleinsäurensonde zusätzlich ein erstes heit FAD ist, und die maskierte Form ist 3'FADP. Glied eines spezifischen Bindungspaares umfaßt. 40 25. Verfahren nach Anspruch 23 oder 24, wobei das 12. Verfahren nach Anspruch 11, wobei das erste Glied Apoenzym Apo-Glucose-Oxidase oder Apo-D-Ami- ausgewählt wird aus der Gruppe, bestehend aus Di- nosäure-Oxidase ist. goxin, Digoxygenin, Fluorescein, Fluoresceinisot- hiocyanat und Biotin. 45 26. Verfahren nach Anspruch 1, wobei die Nucleinsäu- rensonde auf einer festen Oberfläche immobilisiert 13. Verfahren nach Anspruch 11, zusätzlich umfassend wird. die Stufe von in Kontakt bringen des Hybrids mit einem zweiten Glied eines spezifischen Bindungs- 27. Verfahren nach einem der vorhergehenden Ansprü- paares. 50 che, wobei die Zielnucleinsäure aus einer Testpro- be isoliert wird. 14. Verfahren nach Anspruch 13, wobei das zweite Glied ausgewählt wird aus der Gruppe, bestehend 28. Verfahren nach einem der vorhergehenden Ansprü- aus Anti-Digoxin-Antikörper, Anti-Digoxygenin-An- che, wobei die Zielnucleinsäure durch ein Zielam- tikörper, Anti-Fluorescein-Antikörper, Anti-Fluore- 55 plifikationsmittel hergestellt wird. sceinisothiocyanat-Antikörper, Avidin, Streptavidin und Neutravidin. 29. Verfahren nach einem der vorhergehenden Ansprü- che, wobei die Zielnucleinsäure aus der Gruppe,

14 27 EP 0 975 805 B1 28

bestehend aus DNA, RNA oder PNA, ausgewählt 36. Assaykit nach Anspruch 35, wobei das spezifische wird. Bindeglied ein Antikörper, spezifisch für doppelst- rängige Nucleinsäure, oder ein DNA Bindungspro- 30. Verfahren nach Anspruch 29, wobei das Zielampli- tein, spezifisch für doppelsträngige Nucleinsäure, fikationsmittel aus der Gruppe, umfassend Polyme- 5 ist rasekettenreaktion, Ligasekettenreaktion, auf Nu- cleinsäuresequenz basierende Amplifikation, Zy- 37. Assaykit nach Anspruch 36, wobei der Antikörper klussondenamplifikation und Strangverdrängungs- aus der Gruppe, bestehend aus monoklonalem An- amplifikation, ausgewählt wird. tikörper, polyklonalem Antikörper, rekombinantem 10 Antikörper, chimärem Antikörper und einzelsträngi- 31. Verfahren nach einem der vorhergehenden Ansprü- gem Antikörper, ausgewählt ist. che, wobei die Sondennucleinsäure aus der Grup- pe, bestehend aus DNA, RNA oder PNA, ausge- 38. Assaykit nach Anspruch 35, wobei der Rest aus der wählt wird. Gruppe, bestehend aus Digoxin, Digoxygenin, 15 Fluorescein, Fluoresceinisothiocyanat und Biotin 32. Assaykit zum Nachweisen einer einzelsträngigen ausgewählt ist. Zielnucleinsäure, umfassend eine Nucleinsäuren- sonde komplementär zu der nachzuweisenden Zi- 39. Assay nach Anspruch 35, wobei das spezifische elnucleinsäure, die mit einem Enzym markiert ist, Bindeglied aus der Gruppe, bestehend aus Anti-Di- das fähig ist, wesentlich einzelsträngige Nuclein- 20 goxin-Antikörper, Anti-Digoxygenin-Antikörper, An- säure aber nicht doppelsträngige Nucleinsäure zu ti-Fluorescein-Antikörper, Anti-Fluoresceinisothio- hydrolysieren. cyanat-Antikörper, Avidin, Streptavidin und Neutra- vidin ausgewählt ist. 33. Assaykit nach Anspruch 32, wobei die Nuclease aus der Gruppe, bestehend aus: Ribonuclease A 25 40. Assaykit nach einem der Ansprüche 35 bis 39, zu- und Ribonuclease T1 in Kombination, Exodeoxyri- sätzlich ein Nachweissystem umfassend. bonuclease I (E.C. 3.1.11.1), Säuger DNase III, Exonuclease IV, T2- und T4-induzierte Exodeoxyri- 41. Assaykit nach Anspruch 40, wobei das Nachweis- bonucleasen, Exodeoxyribonuclease (Phage system ein Amplifikationssystem ist. sp3-induziert) (E.C. 3.1.11.4), Exodeoxyribonu- 30 clease V (E.C. 3.1.11.5), Haemophilus influenzae 42. Assaykit nach Anspruch 41, wobei das Amplifikati- ATP-abhängige DNase, Exodeoxyribonuclease VII onssystem ein Apoenzym umfaßt, das in ein Ho- (E.C. 3.1.11.6), Micrococcus luteus Exonuclease, loenzym durch Wechselwirkung mit einer akzesso- Exoribonuclease II (E.C. 3.1.13.1), RNase Q, RNa- rischen Untereinheit umwandelbar ist, und eine se BN, RNase PIII, RNase Y, (tierisches) Gift 35 maskierte Form der Untereinheit, die in ihre aktive Exonuclease (E.C. 3.1.15.1), Hausschweinnieren- unmaskierte Form durch die Wirkung des Enzyms phosphodiesterase, Lactobacillus Exonuclease, umwandelbar ist. Milz Exonuclease (E.C. 3.1.16.1), Lactobacillus acidophilus Nuclease, B. subtilis Nuclease, Deoxyri- 43. Assaykit nach Anspruch 42, wobei die Untereinheit bonuclease IV (Phage T4-induziert) (E.C. 3.1.21.2), 40 FAD ist, und die maskierte Form ist 3'FADP. DNase V (Säuger), Aspergillus sojae DNase, B. subtilis Endonuclease, T4 Endonuclease III, T7 En- 44. Assaykit nach Anspruch 42 oder 43, wobei das donuclease I, Aspergillus DNase K2, Vaccinia Virus Apoenzym Apo-Glucose-Oxidase oder Apo-D-Ami- DNase VI, Hefe DNase, Chlorella DNase, Aspergil- nosäure-Oxidase ist. lus Deoxyribonuclease K1 (E.C. 3.1.22.2), Asper- 45 gillus Nuclease S1 (E.C. 3.1.30.1), N crassa Nu- clease, Mung Bohnen Nuclease und Penicillium ci- Revendications trinum Nuclease P1 ausgewählt ist. 1. Procédé de détection d'un acide nucléique cible 34. Assaykit nach Anspruch 32, wobei das Enzymrea- 50 monocaténaire comprenant les étapes de : genz Nuclease P1 oder Nuclease S1 ist. a) fourniture d'une sonde d'acide nucléique, la- 35. Assaykit nach einem der Ansprüche 32 bis 34, zu- dite sonde comprenant : sätzlich umfassend ein spezifisches Bindeglied, spezifisch für entweder Hybride, gebildet zwischen 55 i) une séquence nucléotidique complé- der einzelsträngigen Zielnucleinsäure und der Nu- mentaire d'au moins une partie de ladite ci- cleinsäurensonde oder für einen auf der Nuclein- ble, et säurensonde vorhandenen Rest. ii) un réactif enzymatique réuni à ladite sé-

15 29 EP 0 975 805 B1 30

quence, ledit réactif enzymatique étant ca- 6. Procédé selon l'une quelconque des revendications pable d'hydrolyser un acide nucléique mo- 1 à 5 comprenant en outre l'étape de mise en con- nocaténaire mais pratiquement incapable tact dudit hybride avec un réactif de liaison à l'hy- d'hydrolyser un acide nucléique bicaténai- bride. re, 5 7. Procédé selon la revendication 6 dans lequel ledit b) mise en contact de ladite sonde avec ladite réactif de liaison à l'hybride est un anticorps spéci- cible dans des conditions de pH qui sont en de- fique d'acide nucléique bicaténaire ou une protéine hors de la plage d'activité de ladite enzyme, de se fixant à l'ADN spécifique d'acide nucléique bica- façon à former un hybride, 10 ténaire. c) ajustement dudit pH à une valeur située dans la plage d'activité dudit réactif enzymatique au 8. Procédé selon la revendication 7 dans lequel ledit moyen d'une base, d'un acide, ou d'un tampon, anticorps est choisi dans le groupe constitué d'un d) hydrolyse de la totalité ou d'une partie de tout anticorps monoclonal, d'un anticorps polyclonal, acide nucléique monocaténaire présent au 15 d'un anticorps recombinant, d'un anticorps chimé- moyen dudit réactif enzymatique, rique et d'un anticorps simple chaîne. e) détection dudit hybride. 9. Procédé selon la revendication 6 dans lequel ledit 2. Procédé selon la revendication 1 dans lequel ledit réactif de liaison à l'hybride est marqué. réactif enzymatique est détectable, grâce à quoi le- 20 dit hybride est détecté. 10. Procédé selon la revendication 6 dans lequel ledit réactif de liaison à l'hybride est immobilisé. 3. Procédé selon la revendication 1 ou 2 dans lequel ledit réactif enzymatique est une nucléase. 11. Procédé selon l'une quelconque des revendications 25 1 à 10 dans lequel ladite sonde d'acide nucléique 4. Procédé selon la revendication 3 dans lequel ladite comprend en outre un premier membre d'une paire nucléase est choisie dans le groupe constitué de : liante spécifique. la ribonucléase A et la ribonucléase T1 en combinaison, l'exodésoxyribonucléase I (E.C. 3.1.11.1), 12. Procédé selon la revendication 11 dans lequel ledit la DNase III de mammifère, l'exonucléase IV, les 30 premier membre est choisi dans le groupe constitué exodésoxyribonucléases induites par T2 et T4, de la digoxine, de la digoxygénine, de la fluorescéi- l'exodésoxyribonucléase (induite par le phage sp3) ne, de l'isothiocyanate de fluorescéine et de la bio- (E.C. 3.1.11.4), l'exodésoxyribonucléase V (E.C. tine. 3.1.11.5), la DNase ATP-dépendante de Haemo- philus influenzae, l'exodésoxyribonucléase VII (E. 35 13. Procédé selon la revendication 11 comprenant en C. 3.1.11.6), l'exonucléase de Micrococcus luteus, outre l'étape de mise en contact dudit hybride avec l'exoribonucléase II (E.C. 3.1.13.1), la RNase Q, la un second membre d'une paire liante spécifique. RNase BN, la RNase PIII, la RNase Y, l'exonucléa- se de venin (E.C. 3.1.15.1), la phosphodiestérase 14. Procédé selon la revendication 13 dans lequel ledit de rein de porc, l'exonucléase de Lactobacillus, 40 second membre est choisi dans le groupe constitué l'exonucléase splénique (E.C. 3.1.16.1), la nucléa- d'un anticorps anti-digoxine, d'un anticorps anti-di- se de Lactobacillus acidophilus, la nucléase de B goxygénine, d'un anticorps anti-fluorescéine, d'un subtilis, la désoxyribonucléase IV (induite par le anticorps anti-isothiocyanate de fluorescéine, de phage T4) (E.C. 3.1.21.2), la DNase V (mammifè- l'avidine, de la streptavidine et de la neutravidine. re), la DNase de Aspergillus sojae, l'endonucléase 45 de B subtilis, l'endonucléase III de T4, l'endonuclé- 15. Procédé selon la revendication 13 dans lequel ledit ase I de T7, la DNase K2 de Aspergillus, la DNase second membre a un marqueur. VI du virus de la vaccine, la DNase de levure, la DNase de Chlorella, la désoxyribonucléase K1 de 16. Procédé selon la revendication 13 dans lequel ledit Aspergillus (E.C. 3.1.22.2), la nucléase S1 de As- 50 second membre est immobilisé. pergillus (E.C. 3.1.30.1), la nucléase de N crassa, la nucléase de haricot mungo, et la nucléase P1 de 17. Procédé selon la revendication 9 ou 15 dans lequel Penicillium citrinum. ledit marqueur est un marqueur immobilisable.

5. Procédé selon la revendication 2 dans lequel ledit 55 18. Procédé selon la revendication 9 ou 15 dans lequel réactif enzymatique est la nucléase P1 ou la nuclé- ledit marqueur est un marqueur détectable. ase S1. 19. Procédé selon la revendication 18 dans lequel ledit

16 31 EP 0 975 805 B1 32

marqueur détectable est choisi dans le groupe 31. Procédé selon l'une quelconque des revendications constitué d'une enzyme, d'un groupement fluores- précédentes dans lequel ledit acide nucléique de la cent, d'un groupement chimioluminescent, et d'un sonde est choisi dans le groupe constitué de l'ADN, groupement électrochimioluminescent. de l'ARN et de l'acide nucléique peptidique. 5 20. Procédé selon la revendication 19 dans lequel ladi- 32. Trousse d'analyse pour détecter un acide nucléique te enzyme est la β-galactosidase ou la peroxydase cible monocaténaire comprenant une sonde d'acide raifort. de nucléique complémentaire de l'acide nucléique cible à détecter qui est marquée avec une enzyme 21. Procédé selon la revendication 19 dans lequel ladi- 10 capable d'hydrolyser substantiellement un acide te enzyme est choisie dans le groupe constitué de nucléique monocaténaire mais pas un acide nucléi- la phosphatase alcaline, de la nucléase P1 et de la que bicaténaire. nucléase S1. 33. Trousse d'analyse selon la revendication 32 dans 22. Procédé selon la revendication 5 ou 21 dans lequel 15 laquelle ladite nucléase est choisie dans le groupe ledit hybride est détecté par un système d'amplifi- constitué de : la ribonucléase A et la ribonucléase cation. T1 en combinaison, l'exodésoxyribonucléase I (E. C. 3.1.11.1), la DNase III de mammifère, l'exonuclé- 23. Procédé selon la revendication 22 dans lequel ledit ase IV, les exodésoxyribonucléases induites par T2 système d'amplification comprend une apoenzyme 20 et T4, l'exodésoxyribonucléase (induite par le pha- qui est convertible en holoenzyme par interaction ge sp3) (E.C. 3.1.11.4), l'exodésoxyribonucléase V avec une sous-unité auxiliaire; et une forme mas- (E.C. 3.1.11.5), la DNase ATP-dépendante de Hae- quée de ladite sous-unité qui est convertible en sa mophilus influenzae, l'exodésoxyribonucléase VII forme non masquée active par l'action de ladite en- (E.C. 3.1.11.6), l'exonucléase de Micrococcus lu- zyme. 25 teus, l'exoribonucléase II (E.C. 3.1.13.1), la RNase Q, la RNase BN, la RNase PIII, la RNase Y, l'exo- 24. Procédé selon la revendication 23 dans lequel ladi- nucléase de venin (E.C. 3.1.15.1), la phosphodies- te sous-unité est le FAD et ladite forme masquée térase de rein de porc, l'exonucléase de Lactoba- est le 3'FADP. cillus, l'exonucléase splénique (E.C. 3.1.16.1), la 30 nucléase de Lactobacillus acidophilus, la nucléase 25. Procédé selon la revendication 23 ou 24 dans le- de B subtilis, la désoxyribonucléase IV (induite par quel ladite apoenzyme est l'apo-glucose oxydase le phage T4) (E.C. 3.1.21.2), la DNase V (mammi- ou l'apo-D-aminoacide oxydase. fère), la DNase de Aspergillus sojae, l'endonucléa- se de B subtilis, l'endonucléase III de T4, l'endonu- 26. Procédé selon la revendication 1 dans lequel ladite 35 cléase I de T7, la DNase K2 de Aspergillus, la DNa- sonde d'acide nucléique est immobilisée sur une se VI du virus de la vaccine, la DNase de levure, la surface solide. DNase de Chlorella, la désoxyribonucléase K1 de Aspergillus (E.C. 3.1.22.2), la nucléase S1 de As- 27. Procédé selon l'une quelconque des revendications pergillus (E.C. 3.1.30.1), la nucléase de N crassa, précédentes dans lequel ledit acide nucléique cible 40 la nucléase de haricot mungo, et la nucléase P1 de est isolé à partir d'un échantillon d'essai. Penicillium citrinum.

28. Procédé selon l'une quelconque des revendications 34. Trousse d'analyse selon la revendication 32 dans précédentes dans lequel ledit acide nucléique cible laquelle ledit réactif enzymatique est la nucléase P1 est produit par un moyen d'amplification de cible. 45 ou la nucléase S1.

29. Procédé selon l'une quelconque des revendications 35. Trousse d'analyse selon l'une quelconque des re- précédentes dans lequel ledit acide nucléique cible vendications 32 à 34 comprenant en outre un mem- est choisi dans le groupe constitué de l'ADN, de bre liant spécifique, spécifique soit des hybrides for- l'ARN et de l'acide nucléique peptidique. 50 més entre ledit acide nucléique cible monocaténai- re et ladite sonde d'acide nucléique, soit d'un grou- 30. Procédé selon la revendication 29 dans lequel ledit pement présent sur ladite sonde d'acide nucléique. moyen d'amplification de cible est choisi dans le groupe comprenant la réaction en chaîne de la po- 36. Trousse d'analyse selon la revendication 35 dans lymérase, la réaction en chaîne de la ligase, l'am- 55 laquelle ledit membre liant spécifique est un anti- plification basée sur une séquence d'acide nucléi- corps spécifique d'acide nucléique bicaténaire ou que, l'amplification par sonde en succession de cy- une protéine se fixant à l'ADN spécifique d'acide nu- cles et l'amplification par déplacement de brins. cléique bicaténaire.

17 33 EP 0 975 805 B1 34

37. Trousse d'analyse selon la revendication 36 dans laquelle ledit anticorps est choisi dans le groupe constitué d'un anticorps monoclonal, d'un anticorps polyclonal, d'un anticorps recombinant, d'un anticorps chimérique et d'un anticorps simple chaîne. 5

38. Trousse d'analyse selon la revendication 35 dans laquelle ledit groupement est choisi dans le groupe constitué de la digoxine, de la digoxygénine, de la fluorescéine, de l'isothiocyanate de fluorescéine et 10 de la biotine.

39. Trousse d'analyse selon la revendication 35 dans laquelle ledit membre liant spécifique est choisi dans le groupe constitué d'un anticorps anti-digoxi- 15 ne, d'un anticorps anti-digoxygénine, d'un anticorps anti-fluorescéine, d'un anticorps anti-isothiocyanate de fluorescéine, de l'avidine, de la streptavidine et de la neutravidine. 20 40. Trousse d'analyse selon l'une quelconque des revendications 35 à 39 comprenant en outre un sys- tème de détection.

41. Trousse d'analyse selon la revendication 40 dans 25 laquelle ledit système de détection est un système d'amplification.

42. Trousse d'analyse selon la revendication 41 dans laquelle ledit système d'amplification comprend une 30 apoenzyme qui est convertible en holoenzyme par interaction avec une sous-unité auxiliaire; et une forme masquée de ladite sous-unité qui est convertible en sa forme non masquée active par l'action de ladite enzyme. 35

43. Trousse d'analyse selon la revendication 42 dans laquelle ladite sous-unité est le FAD et ladite forme masquée est le 3'FADP. 40 44. Trousse d'analyse selon la revendication 42 ou 43 dans laquelle ladite apoenzyme est l'apo-glucose oxydase ou l'apo-D-aminoacide oxydase.

18 EP 0 975 805 B1