CA2145092A1 - Conjugate for the detection and/or assay of a biological compound - Google Patents
Conjugate for the detection and/or assay of a biological compoundInfo
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- CA2145092A1 CA2145092A1 CA002145092A CA2145092A CA2145092A1 CA 2145092 A1 CA2145092 A1 CA 2145092A1 CA 002145092 A CA002145092 A CA 002145092A CA 2145092 A CA2145092 A CA 2145092A CA 2145092 A1 CA2145092 A1 CA 2145092A1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
- C12Q1/708—Specific hybridization probes for papilloma
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- Hematology (AREA)
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- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
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- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Novel conjugate for the detection and/or assay of a biological compound, consisting of a kinase or a dehydrogenase, the sulphur groups being preferably re-duced beforehand, and capable of producing an inter-mediate compound for use in a bioluminescence sys-tem, preferably by means of a luciferase. Said enzyme is coupled to a ligand that was previously activated by a coupling agent and is capable of specifically bonding to said biological compound. The invention also con-cerns a process for the production of said conjugate, the application of said conjugate in detection and/or assay processes and a diagnostic kit containing same.
Description
_ WO 94/06933 - 1 - PCT/BE93/00063 CONJUGATE FOR TEE DETECTION AND/OR ASSAY OF A
BIOLOGICAL COh~O~
Subject-matter of the invention The invention relates to a novel conjugate for the detection and/or the assay of a biological compound.
The invention also extends to a process for producing the said conjugate, to the use of the said conjugate in detectioh and/or assay methods, and to a!diagnostic kit cont~in;ng the said conjugate.
Technological background and state of the art on which the invention is based Many assay methods intended for diagnostis tests in the spheres of biology and medicine are based in principle on the specific recognition by antibodies of their antigens and, for some years now, on the pairing of nucleic acid bases (DNA or RNA). These methods have taken various forms depending on the complexity of the interac-tions involved and on the means for immobilization which are used to fix the assay ligand (antigen/antibody or nucleotide sequence).
The techniques for assaying molecules labeled with a radioactive isotope are precise and very sensitive. The disadvantage of these methods clearly resides in the problems associated with the fitting out of special rooms and with the storage of wastes, with health and safety, due to the installation of radioactive isotopes, and with the legislative constraints which ensue from this.
The cold-probe technique is based on the use of enzymic conjugates composed of a probe and of an enzyme which are linked in a covalent manner. These conjugates can be detected by the conversion of a substrate of the enzyme into a colored product which is then measured by spectrophotometry. This is the case with peroxidase or with alkaline phosphatase. Although this assay permits the detection of ligands which are present in substantial quantities, it has been found to be too insensitive in the case of immunochemical assays - when detecting antigens present in small quantities, such as hormones -21~S092 and - in the case of genetic hybridizations which use DNA
probes -where the quantities to be detected are extremely low.
Chemiluminescence is based on the emission of light from a chemical molecule which is excited. This excitation can be produced from a chemical reaction or by the transfer of energy from another molecule. In the first case, the reaction is generally c!atalyzed by an enzyme. The systems which are used to the greatest extent are either peroxidase in the presence of luminol or of hydrogen peroxide, or alkaline phosphatase in the presence of adamantyl 1,2-dioxyethanephenyl phosphate (L. Kricka, Chim. Chem., 37, 1472-1481, 1991).
These assay systems are sensitive, but it is very difficult to quantify the signal since it is very brief.
One solution would be to use a lanthanide, europium, which, once excited, produces a fluorescence having an excitation time which is particularly long and therefore detectable. It has been used for detecting adenovirus with a detection threshold of 0.3 pg of DNA (Syvanen et al., Nuc. Acids Res. 14, 1017, 1986).
It is also possible to take advantage of the transfer of energy between two molecules: fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC). The first (FITC) is attached to the probe and the second to the sample DNA. When the probe becomes attached to the sample, the two molecules can transfer energy between themselves: thus, the excitation of the TRITC at a specific wavelength comes to be transferred to the FITC which is present in its vicinity - if the pairing has taken place - and which then comes to emit a characteristic fluorescence. The method is sensitive, but numerous parameters still have to be optimized for every day usage (Davins et al., Analyt. Letters 20, 1897, 1987).
A novel chemiluminescence assay system, marketed by Boehringer Mannheim, is based on the detection of DNA
probes which are labeled with digoxigenin and which are detected by labeled antibodies directed against the digoxigenin (Holtke H. and Kessler C. Nucleic Acids Research, 18 (19), 5843-51, 1990).
This molecule is recognized by an antibody which is coupled to alkaline phosphatase, which latter, reac-ting with a substrate, causes a detectable photon to beemitted.
While this method is sufficiently sensitive, it nevertheless suffers from the drawback !of involving a toxic molecule and of being difficult to implement.
The principle of bioluminescence is the natural emission of photons which is caused by the enzymic activity of the luciferases in-the presence of their substrates. Two types of luciferase exist; luceriferase which uses ATP as its substrate and luciferase which uses the oxidation of NADH or of NADPH as the energy source.
In the latter case, the luciferase is linked to an NAD(P)H-FMN oxidoreductase.
These enzymes enable very low quantities (10-l2 mol) of substrate to be measured. The ATP luciferase has a quantum efficiency which is 10 times greater than that of the NAD(P)H luciferase, permitting a substrate detec-tion threshold which is 10 times lower. Depending on the assay conditions, the signal can be stable for very long periods.
Different methods using coupling agents such as agarosebeads,N-succinimidyl-3-(2-pyridylthio)propionate (SDPD) and glutaraldehyde (US.4.604.364) have been proposed for directly linking these enzymes to ligands such as antibodies, antigens or nucleic acid probes.
Patent Application EP 254172 describes an antibody/enzyme (such as beta-galactosidase) conjugate which is coupled using a homobifunctional reagent (bis-maleimidopolyalkylene glycol).
Patent Application EP 137515 describes the use of an immoglobulin G/enzyme (aequorin) conjugate in a bioluminescence test.
In these documents, these two conjugates are obtained by two consecutive activation steps, firstly of the antibody with the coupling agent and then of the 21~09~
enzyme with the coupling agent.
This double activation then makes it possible to react the two activated constitutents with each other, thereby producing a conjugate.
However, these diagnostic methods are based on the direct use of a luciferase, which does not allow a sensitivity to be obtained which is sufficiently high for the detection and/or assay of cert~in biological compounds.
Other methods based on the use of kinases, or of dehydrogenases conjugated to ligands, have also been proposed.
Patent Application EP 304934 describes conjugates comprising enzymes, in particular kinases, which are coupled to ligands such as antibodies, nucleotide sequences, avidin or streptavidin in a customary manner using glutaraldehyde following the procedure described by Avrameas et al. (Bull. of Soc. Chim. Biol. (1968) 50, p. 1169).
However, the use of kinases and dehydrogenases as labels has been limited as a result of their being inactivated by the agents used to effect the coupling of these enzymes to the different ligands.
Patent Application EP 431882 describes a reagent consisting of a protein such as an enzyme (for example bacterial luciferase or a beta-galactosidase) which contains a preactivated thiol group which is capable of attaching a nucleotide sequence, for example.
However, this document does not describe the attachment of this preactivated conjugate to a kinase or a dehydrogenase in order that it can be used in a detection and/or assay method involving bioluminescence.
It is also known from Patent Applications EP 161138 and EP 362042 to use a glucose-6-phosphate dehydrogenase which is attached, in a covalent manner, to an antigen or antibody, to a nucleotide probe or even to avidin for detecting biotinylated probes. This complex permits the detection of NADH produced by glucose-6-phosphate dehydrogenase, luciferase or FMN-oxidoreductase 2l4sns~
which is covalently attached to Sepharose 4B.
However, the sensitivity achieved by this method is too low (doubtless due in part to the previously described inactivation phenomena when the enzymes are coupled), for optimal detection of certain nucleotide sequences from pathogenic or non-pathogenic organisms.
Ob;ects of the invention The invention consists in prov!iding a novel conjugate and a novel process for preparing this conju-gate, which conjugate is intended for the detectionand/or assay of a biological compound; the said conjugate consisting of a ligand which is capable of binding, in a specific manner, to a biological compound, and of a kinase or a dehydrogenase which, after having been coupled to the ligand, retains its enzymic activity.
Another object of the invention consists in providing a conjugate the enzyme of which possesses a specific acitivity, an elevated turnover rate and a stability which is elevated at ambient temperature, is of low cost, can be easily purified, and reacts in a manner which is essentially irreversible.
An additional object of the present invention consists in providing novel nucleic acid sequences which possess the property of pairing with complementary sequences present in the genome of human papilloma viruses (HPV) and which may be used as the ligand of a conjugate according to the invention or be attached to a molecule which recognizes a ligand of a conjugate according to the invention.
A final object of the present invention consists in providing a diagnostic kit which includes the conjugate according to the invention and which displays great sensitivity for detecting and/or assaying a biological compound; this sensitivity being similar to, or more elevated than, that of the diagnostic kits of the state of the art.
Characteristic elements of the invention The invention relates to an active conjugate which is intended for detecting and/or assaying a 214509~
~_ WO 94/06933 - 6 - PCT/B~93/00063 biological compound and which is composed of a kinase or a dehydrogenase, the sulfur groupings of which are preferably reduced in advance and which is capable of producing an intermediate compound which can be used by a bioluminescence system, preferably by a luciferase; the said kinase or dehydrogenase being coupled directly to a -ligand, which ligand is activated in advance by a coupling agent and is capable of bindi~g, directly or indirectly (i.e. by way of a cascade of ligands which ensures recognition of the antibody/antibody, antibody/
antigen, streptavidin/biotin, etc., type), in a specific manner, to the said biological compound.
According to the invention, the said kinase or dehydrogenase, of the conjugate, is not activated in advance and is coupled directly to a ligand which, itself, is activated in advance by a coupling agent.
A biological compound is understood to mean any pathogenic or non-pathogenic microorganism, such as procaryotes, eucaryotes, mycoplasmas, viruses, or vegetable or animal, etc, cell lines; constituents of the said microorganisms or constituents produced by the said microorganisms, in particular antigens, antibodies, nucleotide sequences, where appropriate previously amplified by a genetic amplification (PCR or LCR), lipids or saccharides, etc, and/or a mixture thereof; preferably attached to a solid support or a gel or present in solution.
Advantageously, the kinase is a pyruvate kinase.
Preferably, the coupling agent is a heterobi functional coupling agent such as N-succinimidyl-3-(2-pyridyldithio)propionate, succinimidyl-4-(N-maleimido-methyl)cyclohexane-1-carboxylate or are [sic] sulfo derivative.
According to one embodiment of the invention, the ligand is selected from among the group comprising antigens, hormone receptors, antibodies, nucleotide sequences and compounds such as avidin or streptavidin which are capable of binding to biotinylated nucleotide sequences, and/or a mixture thereof.
,~
A further aspect of the present invention relates to novel nucleotide sequences, or fragments of these sequences, of single-stranded DNA or of RNA, which make it possible to detect and/or assay a variety of viruses which are responsible for human papillomas, such as HPV6B, HPV11, HPV16, HPV18 and HPV33.
The present invention relates, in particular, to:
- all or part of a DNA sequence of 25 nucleotides:
the SEQ ID NO. 1: TGTGCAACAA TGTGCAGACA TTATA.
- all or part if its complementary sequence:
the SEQ ID NO. 2: ACACGTTGTT ACACGTCTGT AATAT
- all or part of the RNA sequences corresponding to the abovementioned sequences:
which permit the simultaneous detection of the 5 types of HPV mentioned above.
These sequences hybridize with a region corresponding to nucleotides 2047-2071 of the HPV6b E1 gene, with two mispairings in positions 9 and 15, and with a region corresponding to nucleotides 2047-2071 of the HPVll E1 gene, with two mispairings at positions 8 and 9, with a region corresponding to nucleotides 2076-2100 of the HPV16 E1 gene, with one mispairing at position 15, with a region corresponding to nucleotides 2157-2171 of the HPV18 E1 gene, with one mispairing at position 6, and with a region corresponding to nucleotides 2070-2094 of the HPV33 E1 gene, with three mispairings at positions 5, 8 and 15.
- all or part of two DNA sequences of 25 nucleotides:
the SEQ ID NO. 3: CCAAGGTTT AGATGCCTGC CAGGA
the SEQ ID NO. 4: TACTACATAC ACCCCCGCAC AGACC
- all or part of their complementary sequences:
the SEQ ID NO. 5: GGTTCGCAAA TCTACGCACG GTCCT
the SEQ ID NO. 6: ATGATGTATG TGGGGGCGTG TGTGG
which permit the simultaneous detection of HPV types 6b and 11.
Sequences 3, 3a, 5 and 5a hybridize with a region corresponding to nucleotides 2736-2760 of the E2 gene of HPV6b and 11, which region is identical in the two viruses.
- 21~50~
Sequences 4, 4a, 6 and 6a hybridize with a region corresponding to nucleotides 3355-3379 of the E2 gene of HPV6b and 11, which region is identical in the two viruses.
- all or part of a DNA sequence of the 21 nucleotides:
the SEQ ID NO. 7: ACCTTAACTG CAGATGTTAT G
- all or part of its complementary sequence:
the SEQ ID Np. 8: TGGAATTGAC GTC~A~TA C
which permit the simultaneous detection of HPV types 16, 18 and 33.
These sequences hybridize with a region corre-sponding to nucleotides 6780-6800 of the HPV16 L1 gene, with a mispairing at position 15, with a region corre-sponding to nucleotides 6759-6779 of the HPV18 L1 gene, with a mispairing at position 3, and with a region corresponding to nucleotides 6734-6754 of the HPV33 L1 gene, with a mispairing at position 15.
- all or part of a DNA sequence of 25 nucleotides:
the SEQ ID NO. 9: ATATCAGATG ACGAGAACGA AAATG
- all or part of its complementary sequence:
the SEQ ID NO. 10: TATAGTCTAC TGCTCTTGCT TTTAC
which permit the simultaneous detection of HPV types 16 and 18.
These sequences hybridize with a region corresponding to nucleotides 931-985 of the HPV16 E1 gene and with a region corresponding to nucleotides 1007-1031 of the HPV18 E1 gene, with one mispairing in the case of this virus.
- all or part of the RNA sequences corresponding to the abovementioned sequences:
the SEQ ID NO. 11: UGUGCAACAA UGUGCAGACA UUAUA
the SEQ ID NO. 12: ACACGUUGUU ACACGUCUGU AAUAU
the SEQ ID NO. 13: CCAAGCGUUU AGAUGCGUGC CAGGA
the SEQ ID NO. 14: UACUACAUAC ACCCCCGCAC AGACC
the SEQ ID NO. 15: GGUUCGCAAA UCUACGCACG GUCCU
the SEQ ID NO. 16: AUGAUGUAUG UGGGGGCGUG UGUGG
the SEQ ID NO. 17: ACCUUAACUG CAGAUGUUAU G
the SEQ ID NO. 18: UGGAAUUGAC GUCUACAAUA C
the SEQ ID NO. 19: AUAUCAGAUG ACGAGAACGA AAAUG
214SO~
- - WO 94/06933 - 9 - ~ PCT/BE93/00063 the SEQ ID NO. 20: UAUAGUCUAC UGCUCUUGCU UUUAC
These sequences possess the property of pairing with complementary sequences which are present in the genome of various types of human papilloma virus (HPV) and which are absent in the human genome.
The hybrids which may thus be formed can be detected by various methods of the state of the art such as in situ hybridization, hybridization ~on a filter, on plastic, in a "sandwich", on a solid support or in solution, dot blot, Northern blot or Southern blot hybridizations, using an isotopic or non-isotopic label which is attached to the fragment, using a cold-probe technique, using genetic amplification, especially using PCR or LCR, and/or using bioluminescence.
These sequences which have been mentioned permit the specific identification of the HPVs which are most frequently encountered in the genital tract (types 6b, 11, 16, 18 and 33). It is known that certain types of HPV
(types 16, 18 and 33) are more frequently associated with the appearance of a malignant tumor, in this case cancer of the uterine cervix in women, while the other types (6b and 11) are characteristic of benign lesions.
Determination of the presence of one or more HPVs, as well as of the HPV type encountered, is thus very impor-tant for establishing a prognosis and, consequently, forinstituting any possible therapeutic intervention.
Preferably, all or part of the abovementioned nucleotide sequences, where appropriate biotinylated, are included in the ligand of the conjugate according to the invention or are capable of binding to a conjugate according to the invention, for the detection and/or assay of the viruses responsible for human papillomas.
Another aspect of the invention relates to the process for preparing the conjugate according to the invention, in which process a coupling agent, which is preferably heterobifunctional, is reacted, in solution, with a ligand, and in which process this activated ligand is coupled to a kinase or to a dehydrogenase, one or more sulfide bridges of which have, where appropriate been 21450~
- WO 94/06933 - 10 - ~ PCT/BE93/00063 reduced.
Advantageously, the kinase or the dehydrogenase of the conjugate according to the invention is i~obi-lized at its active site on an affinity column containing a gel, such as Blue Sepharose, having an affinity for the kinase or the dehydrogenase, and is coupled to the ligand which was previously activated by the coupling agent, and the column is then washed and the conjugate is eluted through the column by modifying the composition of the washing solution, for example by increasing the ionic strength.
Another aspect of the invention also relates to the use of the conjugate according to the invention for detecting and/or assaying a biological compound; in particular to its application for detecting and/or assaying nucleotide sequences by hybridization; these nucleotide sequences preferably being detected by a method selected from among the group comprising in situ hybridization, hybridization on a filter, on plastic, on a solid support, in solution, in a "sandwich" or on a gel, Dot blot, Northern blot or Southern blot hybridiza-tion, the use of an isotopic or a non-isotopic label attached to the fragment, the use of a cold probe technique, the use of genetic amplification, especially using PCR or LCR, and/or the use of a mixture thereof.
A final aspect of the invention relates to the kit for detecting and/or assaying a biological compound, which kit comprises the conjugate according to the invention and, where appropriate, the reagents intended for detecting and/or assaying nucleotide sequences, by a process selected from among the group comprising in situ hybridization, hybridization on a filter, on plastic, on a solid support, in solution, in a sandwich or on a gel, dot blot, Northern blot or Southern blot hybridization, the use of an isotopic or a non-isotopic label attached to the fragment, the use of a cold probe technique, the use of genetic amplification, especially using PCR or LCR, and/or the use of a mixture thereof.
214509~
- - WO 94/06933 - 11 - ~ PCT/BE93/00063 Brief description of the figures Figure 1 is a diagramatic representation of the application of the conjugate according to the invention in a hybridization assay for oligonucleotide sequences.
Figures 2 to 4 show variants of the implementa-tion of the assay depicted in Figure 1.
Figures 5 and 6 show the direct and indirect quantification, respectively, of the DNA 4f an HPV virus using the conjugate according to the invention.
Figure 7 shows the effect of the concentration of SPDP on the activity of the pyruvate kinase and on the number of arms bound per molecule of enzyme.
Figure 8 shows a quantification, by bioluminescence and using the conjugate according to the invention, of human IgG.
Figure 9 shows a concentration curve for a target HIV sequence using the conjugate according to the invention.
DescriPtion of a preferred embodiment of the invention In the present invention, ATP luciferase rather than NADH luciferase is used as the indicating agent, owing to its greater sensitivity for assaying the substrates. In order to increase sensitivity still further, and to obtain a sizeable signal over a long period of time, the nucleotide sequence, the antibody, the antigen, the avidin or the streptavidin is labeled with a kinase which can produce a constant quantity of ATP under the experimental conditions. This ATP is then broken down by the luciferase and the activity of the latter is quantified by detecting the photons which are emitted.
The choice of a kinase The marker enzyme has to be selected in accor-dance with different criteria which are similar to those used for ELISAs (Avrameas, Scand. J. Immunol., 8, 7-22, 1978), namely:
- elevated specific activity and turnover rate;
- relatively good stability at ambient temperature;
- a loss of activity, following the coupling, which has -~ WO 94/06933 - 12 - ~ PCT/BE93/00063 to be as low as possible, and, finally, - the enzyme has to be available in a form which is highly purified; this can be from a commercial source or else the preparation of the enzyme has to be relatively simple, - furthermore, the enzyme has to be selected in accor-dance with the stability of its substrates.
According to Kayne (Kayne F.S., The Enzyme, ed Boyer part. A, 8, 1973, Academic Press, pp. 353-382), while ~yluvate kinase is commercially available in very pure form, it can also be very simply purified in large quantities from rabbit muscle. Moreover, the reaction catalyzed by the kinase is essentially irreversible in favor of the formation of pyruvate and ATP since the st~n~Ard free energy change of the reaction is -7.5 kcal/mol (Muirhead, M., Biochemical Society Transactions, 15 (5), 996-999, 1987).
Phosphoenolpyruvate + ADP ---------> pyruvate + ADP
- A 5 mg/ml solution of the enzyme retains its activity for at least 48 h at ambient temperature. The commercially available solution (contA;n;ng 50% glycerol) is stable at 4c [sic] for at least 6 months.
In addition to this, pyruvate kinase has other advantages, such as its commercial availability, its good thermal stability and the displacement of the reaction in the direction of the formation of ATP.
Furthermore, novel kinases having specific properties can be produced by means of genetic manipula-tion. Thus, it is possible to produce a thermostable pyruvate kinase by cleaving off a part of its sequence.
The latter can then be used at high temperature in hybridization reactions (Walker et al. Journal of Molecular Biology (1992) 228, p. 265-276).
Likewise, other kinases, such as acetate kinase, can be used.
Coupling the ~yruvate kinase The use of customary coupling agents (glutaralde-hyde, SPDP (N-succinimidyl-3-(2-pyridyldithio) propionate), periodate, etc) in fact results in the 214509~
- ~- WO 94/06933 - 13 - PCT/BE93/00063 formation of conjugates in which the loss of activity is very great and exceeds 95%. This confirms the sensitivity of the kinases and the dehydrogenases to the coupling agents and the fact that it is not possible to obtain conjugates which are sufficiently active for use in diagnostic tests using customary coupling methods.
In control experiments, the inventors observed, to their surprise, that it was possible to bind the non-activated native kinase covalently to ligands which were activated with SPDP (N-succinimidyl 3-(2-pyridyldithio) propionate). The explanation for this unexpected observa-tion is probably that the kina-se carries a free -SH
grouping which can produce a disulfide bridge with the 2-pyridyl disulfide grouping which is bound to the activated probe. The conjugate obtained using this approach is completely active.
The inventors have also observed that it is possible advantageously to increase the yield from the coupling, without loss of activity, by prior reduction of one or more natural disulfide bridges in the kinase using low concentrations of dithiothreitol.
The optimized conditions for coupling in solution are described below for antibodies. The inventors observed that activating the antibodies with a 5 times molar excess of SPDP resulted in the attachment of 1 to 2 SPDP arms per molecule of antibody. This concentration of SPDP yields a m~; mllm of active conjugates when equivalent quantities (in mol) of activated antibodies and kinase are brouqht together.
Another method of coupling on a column (solid phase), which makes it possible to improve the yield, to facilitate separation of the antibodies conjugated to the kinase from the unconjugated antibodies and to be able to prepare a large quantity of conjugates, consists in binding the kinase, by means of weak, non-covalent bonds, at its active site, to a column contA; n ing Blue Sepharose (Pharmacia, Uppsala, Sweden) and then allowing the anti-bodies, previously activated with SPDP, to circulate through the column in a closed circuit and for a period .
of time which is adequate (usually 16 h).
The activated antibodies will thus have adequate opportunity to produce the conjugates with the kinase which is immobilized on the Blue Sepharose.
After the reaction, the column is washed and the antibodies which have not reacted are removed, and the conjugate is then eluted by increasing the ionic strength of the washing solution. This method iS described in detail below for the antibodies and for avidin.
The availability of active conjugates makes it possible to use them for diagnostic tests based either on immunological reactions, such -as the enzyme linked immunosorbent assay or ELISA methods, or on the hybridi-zation of nucleotide sequences.
It has been possible to extend the principle of the method for coupling the kinase to the antibodies to other proteins and even to oligonucleotides. Thus, it has been possible to activate avidin and streptavidin with SPDP and then, after elimination of excess reagent, bring them into contact with the pyruvate kinase. The conjugate, formed in this way in solution, was purified on a column of Blue Sepharose which retains the kinase and which proves to be extremely efficacious.
In order to bind the oligonucleotide probes to the kinase, the DNA probes can be terminated by an amine at the 3' and/or 5' terminal position which amine(s~ can be incorporated during the course of synthesizing the nucleotide chain which is produced or else added subsequently.
This terminal amine is then activated by SMCC and then, after the excess reagents have been removed, brought into contact with the pyruvate kinase for the coupling. The conjugate is then easily purified by molecular sieve chromatography or by affinity chromato-graphy.
Immunoenzymic assays (EL~SA) The kinase can be coupled to an antibody in order to form an antibodytkinase conjugate which can be used in various ELISA tests.
- 214~092 For a direct conjugate assay, use is made of a multiwell dish or of tubes or some other support on which the antigens are immobilized.
The antibody/kinase conjugates are then added in decreasing quantity. After washing, the activity of the bound kinase is measured in the presence of its substrates, allowing the production of ATP which, in the presence of luciferin is broken down by the luciferase with an emission of light. The latter can be measured using a bioluminometer or using a sensitive film such as a Polaroid film or else using a negative paper which is sensitive to X-rays. Using this-test, the titre of the antibodies can be determined.
Under the ~luvate kinase assay conditions, the excess of substrates in relation to the quantity of enzymes and the quantity of luciferases is such that the enzymes carry out their activity for several days. In a normal ELISA test, the emission of light can be followed for three days.
The direct test makes it possible to quantify antigen by using the so-called competitive method. In this case, the antibody is immobilized on the assay dish or support. The antigen to be assayed is then incubated in the presence of a fixed quantity of antigen conjugated to the kinase. These two antigens compete for the anti-body attached to the dish: the larger the quantity of antigen to be assayed, the less antigen/kinase will be able to bind to the antibody. It is the latter which is detected by the activity of the kinase. The antigen to be assayed is then estimated by referring to a calibration curve.
Antigen is more readily assayed by the so-called sandwich method. The dish or the solid support contains immobilized antibodies. The antigen to be assayed is then added in increasing dilutions and then, after washing, a fixed quantity of a second antibody, which is labeled with the kinase, is added and the activity of the kinase is measured. A variant of this method consists in using a second antibody which is not labeled and subsequently 214~0~
_ WO 94/06933 - 16 - PCT/BB93/00063 adding an anti-antibody which is labeled with the kinase (double sandwich).
Antibody can also be quantified by the sandwich assay and, in this case, use is made of a support on which the antigen is immobilized. The antibody is added in increasing concentrations and then, after washing, anti-antibodies labeled with the kinase are incubated and the activity of the kinase is measured. Here too, double sandwich assays can be carried out using an anti-antibody which is not labeled and then a second anti-antibody which is conjugated to the kinase.
~ybridization assay of oligonucleotide sequences The possibility of using avidin (or streptavidin), antibodies or nucleotides as probes which can be coupled to the kinase makes it possible to develop tests for recognizing nucleotide sequences. This technique can be applied in a large number of spheres such as diagnostic tests for detecting bacteria, fungi, yeasts, viruses, cancerous cells or any biological organism which possesses a specific nucleic acid sequence.
The principle of oligonucleotide hybridization is well known and we shall review it briefly. It consists in the recognition of a nucleotide sequence (DNA or RNA) by a complementary strand of nucleotides and in their pairing to form a double chain under appropriate temperature, pH and salt conditions. This recognition is permitted by the formation of hydrogen bridges between adenine (A) and thymine (T) (or uracil (U)) and between guanine (g) and cytosine (C). If the DNA (or RNA) sequence to be detected is known, it is possible, using chemical, enzymic or biological methods, to construct the complementary sequence, which will then hybridize specifically with the DNA (or the RNA) to be detected.
Preferably, this hybridization is effected under stringent conditions, in a manner which ensures that the hybridization is specific and thus avoids false posltives .
The advantage of using the kinase conjugates according to the invention for implementing bioluminescence tests i8 a very high degree of sensitivity and a detection time which can extend over several days. In the simplest method, the nucleotide sequence used for the test is labeled with biotin. This labeling can be carried out in various ways:
- either chemically, by using a biotinylated oligonucleo-tide in the synthesis or by reacting the ~equence with an NHS-biotin, biotinamidocaproate N-hydroxysuccinimide ester or a reagent which carries the biotin and which can bind to the nucleotide sequence, - or enzymically, using terrinAl transferase which incorporates the biotinylated nucleotides at the end of a specific nucleotide sequence. Biotin can be incorpo-rated into the nucleic acids using a photoactivatablecompound termed photobiotin.
The`biotinylated sequence can then be recognized by avidin or streptavidin which is itself coupled to the enzyme, or after reaction with an antibody which is labeled with the kinase and which is directed against the biotinylated sequence. After hybridization with the sequence to be detected, an avidin/kinase conjugate is used which binds to the biotin. The activity of the kinase is then measured by its production of ATP which, in the presence of luciferase, emits photons.
The detection of a DNA or RNA sequence which is present in solution is more complex since it requires an immobilization step in order to effect a washing or a competitive reaction. The sequences can be immobilized directly on nitrocellulose or nylon filters in tests termed "dot blot" tests or following separation by gel electrophoresis (Southern blot and Northern blot).
When the DNA 1 (or RNA) to be measured is present in very low quantity, it is also possible to immobilize this DNA 1 by means of a complementary sequence 2 which is itself immobilized on a support 3 (filter, gel, plastic, glass, beads, etc.). The DNA 1 (or RNA) hybridized to the immobilized probe can be measured using another biotinylated sequence 4 and an avidin/kinase - WO 94/06933 - 18 - ~ PCT/BE93/00063 conjugate 5 or an antibody/kinase anti-probe. This method using two nucleotide sequences (2 and 4), - one immobi-lized and termed captor 2 and the other biotinylated and termed detector 4 - is also called a sandwich method.
From the point of view of the methodology, the two, captor and detector, sequences can be added simultaneously to the DNA (or RNA) to be detected, or sequentially, according to the conditions of the test and according to the result to be obtained: greater sensitivity, speed of operation or ease of manipulations (Figure 1).
These methods can be made more complex by emplo-ying not only recognition between avidin and biotin but also between antigens (or haptens) and antibodies (Figures 2, 3 and 4).
For example, it is possible to use a biotinylated nucleic acid sequence 4 and then a reaction with avidin 6 (which is or is not coupled to a kinase) which avidin will, itself, be recognized by anti-avidin antibodies 7 which are coupled to the kinase (Figures 2 and 3).
It is also possible to carry out a direct reac-tion (Figure 4) with antibodies 8 which either are or are not coupled to the kinase and which are directed against the biotin which is bound to the probe. A molecule other than biotin, for example fluorescein or digoxigenin, which can be recognized by specific antibodies, can also be used to label the probe.
Several variants of this technique exist, such as the use of a second antibody conjugated with the kinase or the use of an antibody conjugated with the biotin with which avidin/kinase conjugates may be reacted. Whatever the combination employed, at least one avidin/kinase conjugate 5 or antibody/kinase conjugate 7 according to the invention will be used in one of the steps of the reaction.
These specific avidin/biotin or antibody/antigen recognitions can also be exploited for immobilizing on the solid support. This makes it possible to carry out the hybridization reaction in solution before effecting 214~09~
BIOLOGICAL COh~O~
Subject-matter of the invention The invention relates to a novel conjugate for the detection and/or the assay of a biological compound.
The invention also extends to a process for producing the said conjugate, to the use of the said conjugate in detectioh and/or assay methods, and to a!diagnostic kit cont~in;ng the said conjugate.
Technological background and state of the art on which the invention is based Many assay methods intended for diagnostis tests in the spheres of biology and medicine are based in principle on the specific recognition by antibodies of their antigens and, for some years now, on the pairing of nucleic acid bases (DNA or RNA). These methods have taken various forms depending on the complexity of the interac-tions involved and on the means for immobilization which are used to fix the assay ligand (antigen/antibody or nucleotide sequence).
The techniques for assaying molecules labeled with a radioactive isotope are precise and very sensitive. The disadvantage of these methods clearly resides in the problems associated with the fitting out of special rooms and with the storage of wastes, with health and safety, due to the installation of radioactive isotopes, and with the legislative constraints which ensue from this.
The cold-probe technique is based on the use of enzymic conjugates composed of a probe and of an enzyme which are linked in a covalent manner. These conjugates can be detected by the conversion of a substrate of the enzyme into a colored product which is then measured by spectrophotometry. This is the case with peroxidase or with alkaline phosphatase. Although this assay permits the detection of ligands which are present in substantial quantities, it has been found to be too insensitive in the case of immunochemical assays - when detecting antigens present in small quantities, such as hormones -21~S092 and - in the case of genetic hybridizations which use DNA
probes -where the quantities to be detected are extremely low.
Chemiluminescence is based on the emission of light from a chemical molecule which is excited. This excitation can be produced from a chemical reaction or by the transfer of energy from another molecule. In the first case, the reaction is generally c!atalyzed by an enzyme. The systems which are used to the greatest extent are either peroxidase in the presence of luminol or of hydrogen peroxide, or alkaline phosphatase in the presence of adamantyl 1,2-dioxyethanephenyl phosphate (L. Kricka, Chim. Chem., 37, 1472-1481, 1991).
These assay systems are sensitive, but it is very difficult to quantify the signal since it is very brief.
One solution would be to use a lanthanide, europium, which, once excited, produces a fluorescence having an excitation time which is particularly long and therefore detectable. It has been used for detecting adenovirus with a detection threshold of 0.3 pg of DNA (Syvanen et al., Nuc. Acids Res. 14, 1017, 1986).
It is also possible to take advantage of the transfer of energy between two molecules: fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC). The first (FITC) is attached to the probe and the second to the sample DNA. When the probe becomes attached to the sample, the two molecules can transfer energy between themselves: thus, the excitation of the TRITC at a specific wavelength comes to be transferred to the FITC which is present in its vicinity - if the pairing has taken place - and which then comes to emit a characteristic fluorescence. The method is sensitive, but numerous parameters still have to be optimized for every day usage (Davins et al., Analyt. Letters 20, 1897, 1987).
A novel chemiluminescence assay system, marketed by Boehringer Mannheim, is based on the detection of DNA
probes which are labeled with digoxigenin and which are detected by labeled antibodies directed against the digoxigenin (Holtke H. and Kessler C. Nucleic Acids Research, 18 (19), 5843-51, 1990).
This molecule is recognized by an antibody which is coupled to alkaline phosphatase, which latter, reac-ting with a substrate, causes a detectable photon to beemitted.
While this method is sufficiently sensitive, it nevertheless suffers from the drawback !of involving a toxic molecule and of being difficult to implement.
The principle of bioluminescence is the natural emission of photons which is caused by the enzymic activity of the luciferases in-the presence of their substrates. Two types of luciferase exist; luceriferase which uses ATP as its substrate and luciferase which uses the oxidation of NADH or of NADPH as the energy source.
In the latter case, the luciferase is linked to an NAD(P)H-FMN oxidoreductase.
These enzymes enable very low quantities (10-l2 mol) of substrate to be measured. The ATP luciferase has a quantum efficiency which is 10 times greater than that of the NAD(P)H luciferase, permitting a substrate detec-tion threshold which is 10 times lower. Depending on the assay conditions, the signal can be stable for very long periods.
Different methods using coupling agents such as agarosebeads,N-succinimidyl-3-(2-pyridylthio)propionate (SDPD) and glutaraldehyde (US.4.604.364) have been proposed for directly linking these enzymes to ligands such as antibodies, antigens or nucleic acid probes.
Patent Application EP 254172 describes an antibody/enzyme (such as beta-galactosidase) conjugate which is coupled using a homobifunctional reagent (bis-maleimidopolyalkylene glycol).
Patent Application EP 137515 describes the use of an immoglobulin G/enzyme (aequorin) conjugate in a bioluminescence test.
In these documents, these two conjugates are obtained by two consecutive activation steps, firstly of the antibody with the coupling agent and then of the 21~09~
enzyme with the coupling agent.
This double activation then makes it possible to react the two activated constitutents with each other, thereby producing a conjugate.
However, these diagnostic methods are based on the direct use of a luciferase, which does not allow a sensitivity to be obtained which is sufficiently high for the detection and/or assay of cert~in biological compounds.
Other methods based on the use of kinases, or of dehydrogenases conjugated to ligands, have also been proposed.
Patent Application EP 304934 describes conjugates comprising enzymes, in particular kinases, which are coupled to ligands such as antibodies, nucleotide sequences, avidin or streptavidin in a customary manner using glutaraldehyde following the procedure described by Avrameas et al. (Bull. of Soc. Chim. Biol. (1968) 50, p. 1169).
However, the use of kinases and dehydrogenases as labels has been limited as a result of their being inactivated by the agents used to effect the coupling of these enzymes to the different ligands.
Patent Application EP 431882 describes a reagent consisting of a protein such as an enzyme (for example bacterial luciferase or a beta-galactosidase) which contains a preactivated thiol group which is capable of attaching a nucleotide sequence, for example.
However, this document does not describe the attachment of this preactivated conjugate to a kinase or a dehydrogenase in order that it can be used in a detection and/or assay method involving bioluminescence.
It is also known from Patent Applications EP 161138 and EP 362042 to use a glucose-6-phosphate dehydrogenase which is attached, in a covalent manner, to an antigen or antibody, to a nucleotide probe or even to avidin for detecting biotinylated probes. This complex permits the detection of NADH produced by glucose-6-phosphate dehydrogenase, luciferase or FMN-oxidoreductase 2l4sns~
which is covalently attached to Sepharose 4B.
However, the sensitivity achieved by this method is too low (doubtless due in part to the previously described inactivation phenomena when the enzymes are coupled), for optimal detection of certain nucleotide sequences from pathogenic or non-pathogenic organisms.
Ob;ects of the invention The invention consists in prov!iding a novel conjugate and a novel process for preparing this conju-gate, which conjugate is intended for the detectionand/or assay of a biological compound; the said conjugate consisting of a ligand which is capable of binding, in a specific manner, to a biological compound, and of a kinase or a dehydrogenase which, after having been coupled to the ligand, retains its enzymic activity.
Another object of the invention consists in providing a conjugate the enzyme of which possesses a specific acitivity, an elevated turnover rate and a stability which is elevated at ambient temperature, is of low cost, can be easily purified, and reacts in a manner which is essentially irreversible.
An additional object of the present invention consists in providing novel nucleic acid sequences which possess the property of pairing with complementary sequences present in the genome of human papilloma viruses (HPV) and which may be used as the ligand of a conjugate according to the invention or be attached to a molecule which recognizes a ligand of a conjugate according to the invention.
A final object of the present invention consists in providing a diagnostic kit which includes the conjugate according to the invention and which displays great sensitivity for detecting and/or assaying a biological compound; this sensitivity being similar to, or more elevated than, that of the diagnostic kits of the state of the art.
Characteristic elements of the invention The invention relates to an active conjugate which is intended for detecting and/or assaying a 214509~
~_ WO 94/06933 - 6 - PCT/B~93/00063 biological compound and which is composed of a kinase or a dehydrogenase, the sulfur groupings of which are preferably reduced in advance and which is capable of producing an intermediate compound which can be used by a bioluminescence system, preferably by a luciferase; the said kinase or dehydrogenase being coupled directly to a -ligand, which ligand is activated in advance by a coupling agent and is capable of bindi~g, directly or indirectly (i.e. by way of a cascade of ligands which ensures recognition of the antibody/antibody, antibody/
antigen, streptavidin/biotin, etc., type), in a specific manner, to the said biological compound.
According to the invention, the said kinase or dehydrogenase, of the conjugate, is not activated in advance and is coupled directly to a ligand which, itself, is activated in advance by a coupling agent.
A biological compound is understood to mean any pathogenic or non-pathogenic microorganism, such as procaryotes, eucaryotes, mycoplasmas, viruses, or vegetable or animal, etc, cell lines; constituents of the said microorganisms or constituents produced by the said microorganisms, in particular antigens, antibodies, nucleotide sequences, where appropriate previously amplified by a genetic amplification (PCR or LCR), lipids or saccharides, etc, and/or a mixture thereof; preferably attached to a solid support or a gel or present in solution.
Advantageously, the kinase is a pyruvate kinase.
Preferably, the coupling agent is a heterobi functional coupling agent such as N-succinimidyl-3-(2-pyridyldithio)propionate, succinimidyl-4-(N-maleimido-methyl)cyclohexane-1-carboxylate or are [sic] sulfo derivative.
According to one embodiment of the invention, the ligand is selected from among the group comprising antigens, hormone receptors, antibodies, nucleotide sequences and compounds such as avidin or streptavidin which are capable of binding to biotinylated nucleotide sequences, and/or a mixture thereof.
,~
A further aspect of the present invention relates to novel nucleotide sequences, or fragments of these sequences, of single-stranded DNA or of RNA, which make it possible to detect and/or assay a variety of viruses which are responsible for human papillomas, such as HPV6B, HPV11, HPV16, HPV18 and HPV33.
The present invention relates, in particular, to:
- all or part of a DNA sequence of 25 nucleotides:
the SEQ ID NO. 1: TGTGCAACAA TGTGCAGACA TTATA.
- all or part if its complementary sequence:
the SEQ ID NO. 2: ACACGTTGTT ACACGTCTGT AATAT
- all or part of the RNA sequences corresponding to the abovementioned sequences:
which permit the simultaneous detection of the 5 types of HPV mentioned above.
These sequences hybridize with a region corresponding to nucleotides 2047-2071 of the HPV6b E1 gene, with two mispairings in positions 9 and 15, and with a region corresponding to nucleotides 2047-2071 of the HPVll E1 gene, with two mispairings at positions 8 and 9, with a region corresponding to nucleotides 2076-2100 of the HPV16 E1 gene, with one mispairing at position 15, with a region corresponding to nucleotides 2157-2171 of the HPV18 E1 gene, with one mispairing at position 6, and with a region corresponding to nucleotides 2070-2094 of the HPV33 E1 gene, with three mispairings at positions 5, 8 and 15.
- all or part of two DNA sequences of 25 nucleotides:
the SEQ ID NO. 3: CCAAGGTTT AGATGCCTGC CAGGA
the SEQ ID NO. 4: TACTACATAC ACCCCCGCAC AGACC
- all or part of their complementary sequences:
the SEQ ID NO. 5: GGTTCGCAAA TCTACGCACG GTCCT
the SEQ ID NO. 6: ATGATGTATG TGGGGGCGTG TGTGG
which permit the simultaneous detection of HPV types 6b and 11.
Sequences 3, 3a, 5 and 5a hybridize with a region corresponding to nucleotides 2736-2760 of the E2 gene of HPV6b and 11, which region is identical in the two viruses.
- 21~50~
Sequences 4, 4a, 6 and 6a hybridize with a region corresponding to nucleotides 3355-3379 of the E2 gene of HPV6b and 11, which region is identical in the two viruses.
- all or part of a DNA sequence of the 21 nucleotides:
the SEQ ID NO. 7: ACCTTAACTG CAGATGTTAT G
- all or part of its complementary sequence:
the SEQ ID Np. 8: TGGAATTGAC GTC~A~TA C
which permit the simultaneous detection of HPV types 16, 18 and 33.
These sequences hybridize with a region corre-sponding to nucleotides 6780-6800 of the HPV16 L1 gene, with a mispairing at position 15, with a region corre-sponding to nucleotides 6759-6779 of the HPV18 L1 gene, with a mispairing at position 3, and with a region corresponding to nucleotides 6734-6754 of the HPV33 L1 gene, with a mispairing at position 15.
- all or part of a DNA sequence of 25 nucleotides:
the SEQ ID NO. 9: ATATCAGATG ACGAGAACGA AAATG
- all or part of its complementary sequence:
the SEQ ID NO. 10: TATAGTCTAC TGCTCTTGCT TTTAC
which permit the simultaneous detection of HPV types 16 and 18.
These sequences hybridize with a region corresponding to nucleotides 931-985 of the HPV16 E1 gene and with a region corresponding to nucleotides 1007-1031 of the HPV18 E1 gene, with one mispairing in the case of this virus.
- all or part of the RNA sequences corresponding to the abovementioned sequences:
the SEQ ID NO. 11: UGUGCAACAA UGUGCAGACA UUAUA
the SEQ ID NO. 12: ACACGUUGUU ACACGUCUGU AAUAU
the SEQ ID NO. 13: CCAAGCGUUU AGAUGCGUGC CAGGA
the SEQ ID NO. 14: UACUACAUAC ACCCCCGCAC AGACC
the SEQ ID NO. 15: GGUUCGCAAA UCUACGCACG GUCCU
the SEQ ID NO. 16: AUGAUGUAUG UGGGGGCGUG UGUGG
the SEQ ID NO. 17: ACCUUAACUG CAGAUGUUAU G
the SEQ ID NO. 18: UGGAAUUGAC GUCUACAAUA C
the SEQ ID NO. 19: AUAUCAGAUG ACGAGAACGA AAAUG
214SO~
- - WO 94/06933 - 9 - ~ PCT/BE93/00063 the SEQ ID NO. 20: UAUAGUCUAC UGCUCUUGCU UUUAC
These sequences possess the property of pairing with complementary sequences which are present in the genome of various types of human papilloma virus (HPV) and which are absent in the human genome.
The hybrids which may thus be formed can be detected by various methods of the state of the art such as in situ hybridization, hybridization ~on a filter, on plastic, in a "sandwich", on a solid support or in solution, dot blot, Northern blot or Southern blot hybridizations, using an isotopic or non-isotopic label which is attached to the fragment, using a cold-probe technique, using genetic amplification, especially using PCR or LCR, and/or using bioluminescence.
These sequences which have been mentioned permit the specific identification of the HPVs which are most frequently encountered in the genital tract (types 6b, 11, 16, 18 and 33). It is known that certain types of HPV
(types 16, 18 and 33) are more frequently associated with the appearance of a malignant tumor, in this case cancer of the uterine cervix in women, while the other types (6b and 11) are characteristic of benign lesions.
Determination of the presence of one or more HPVs, as well as of the HPV type encountered, is thus very impor-tant for establishing a prognosis and, consequently, forinstituting any possible therapeutic intervention.
Preferably, all or part of the abovementioned nucleotide sequences, where appropriate biotinylated, are included in the ligand of the conjugate according to the invention or are capable of binding to a conjugate according to the invention, for the detection and/or assay of the viruses responsible for human papillomas.
Another aspect of the invention relates to the process for preparing the conjugate according to the invention, in which process a coupling agent, which is preferably heterobifunctional, is reacted, in solution, with a ligand, and in which process this activated ligand is coupled to a kinase or to a dehydrogenase, one or more sulfide bridges of which have, where appropriate been 21450~
- WO 94/06933 - 10 - ~ PCT/BE93/00063 reduced.
Advantageously, the kinase or the dehydrogenase of the conjugate according to the invention is i~obi-lized at its active site on an affinity column containing a gel, such as Blue Sepharose, having an affinity for the kinase or the dehydrogenase, and is coupled to the ligand which was previously activated by the coupling agent, and the column is then washed and the conjugate is eluted through the column by modifying the composition of the washing solution, for example by increasing the ionic strength.
Another aspect of the invention also relates to the use of the conjugate according to the invention for detecting and/or assaying a biological compound; in particular to its application for detecting and/or assaying nucleotide sequences by hybridization; these nucleotide sequences preferably being detected by a method selected from among the group comprising in situ hybridization, hybridization on a filter, on plastic, on a solid support, in solution, in a "sandwich" or on a gel, Dot blot, Northern blot or Southern blot hybridiza-tion, the use of an isotopic or a non-isotopic label attached to the fragment, the use of a cold probe technique, the use of genetic amplification, especially using PCR or LCR, and/or the use of a mixture thereof.
A final aspect of the invention relates to the kit for detecting and/or assaying a biological compound, which kit comprises the conjugate according to the invention and, where appropriate, the reagents intended for detecting and/or assaying nucleotide sequences, by a process selected from among the group comprising in situ hybridization, hybridization on a filter, on plastic, on a solid support, in solution, in a sandwich or on a gel, dot blot, Northern blot or Southern blot hybridization, the use of an isotopic or a non-isotopic label attached to the fragment, the use of a cold probe technique, the use of genetic amplification, especially using PCR or LCR, and/or the use of a mixture thereof.
214509~
- - WO 94/06933 - 11 - ~ PCT/BE93/00063 Brief description of the figures Figure 1 is a diagramatic representation of the application of the conjugate according to the invention in a hybridization assay for oligonucleotide sequences.
Figures 2 to 4 show variants of the implementa-tion of the assay depicted in Figure 1.
Figures 5 and 6 show the direct and indirect quantification, respectively, of the DNA 4f an HPV virus using the conjugate according to the invention.
Figure 7 shows the effect of the concentration of SPDP on the activity of the pyruvate kinase and on the number of arms bound per molecule of enzyme.
Figure 8 shows a quantification, by bioluminescence and using the conjugate according to the invention, of human IgG.
Figure 9 shows a concentration curve for a target HIV sequence using the conjugate according to the invention.
DescriPtion of a preferred embodiment of the invention In the present invention, ATP luciferase rather than NADH luciferase is used as the indicating agent, owing to its greater sensitivity for assaying the substrates. In order to increase sensitivity still further, and to obtain a sizeable signal over a long period of time, the nucleotide sequence, the antibody, the antigen, the avidin or the streptavidin is labeled with a kinase which can produce a constant quantity of ATP under the experimental conditions. This ATP is then broken down by the luciferase and the activity of the latter is quantified by detecting the photons which are emitted.
The choice of a kinase The marker enzyme has to be selected in accor-dance with different criteria which are similar to those used for ELISAs (Avrameas, Scand. J. Immunol., 8, 7-22, 1978), namely:
- elevated specific activity and turnover rate;
- relatively good stability at ambient temperature;
- a loss of activity, following the coupling, which has -~ WO 94/06933 - 12 - ~ PCT/BE93/00063 to be as low as possible, and, finally, - the enzyme has to be available in a form which is highly purified; this can be from a commercial source or else the preparation of the enzyme has to be relatively simple, - furthermore, the enzyme has to be selected in accor-dance with the stability of its substrates.
According to Kayne (Kayne F.S., The Enzyme, ed Boyer part. A, 8, 1973, Academic Press, pp. 353-382), while ~yluvate kinase is commercially available in very pure form, it can also be very simply purified in large quantities from rabbit muscle. Moreover, the reaction catalyzed by the kinase is essentially irreversible in favor of the formation of pyruvate and ATP since the st~n~Ard free energy change of the reaction is -7.5 kcal/mol (Muirhead, M., Biochemical Society Transactions, 15 (5), 996-999, 1987).
Phosphoenolpyruvate + ADP ---------> pyruvate + ADP
- A 5 mg/ml solution of the enzyme retains its activity for at least 48 h at ambient temperature. The commercially available solution (contA;n;ng 50% glycerol) is stable at 4c [sic] for at least 6 months.
In addition to this, pyruvate kinase has other advantages, such as its commercial availability, its good thermal stability and the displacement of the reaction in the direction of the formation of ATP.
Furthermore, novel kinases having specific properties can be produced by means of genetic manipula-tion. Thus, it is possible to produce a thermostable pyruvate kinase by cleaving off a part of its sequence.
The latter can then be used at high temperature in hybridization reactions (Walker et al. Journal of Molecular Biology (1992) 228, p. 265-276).
Likewise, other kinases, such as acetate kinase, can be used.
Coupling the ~yruvate kinase The use of customary coupling agents (glutaralde-hyde, SPDP (N-succinimidyl-3-(2-pyridyldithio) propionate), periodate, etc) in fact results in the 214509~
- ~- WO 94/06933 - 13 - PCT/BE93/00063 formation of conjugates in which the loss of activity is very great and exceeds 95%. This confirms the sensitivity of the kinases and the dehydrogenases to the coupling agents and the fact that it is not possible to obtain conjugates which are sufficiently active for use in diagnostic tests using customary coupling methods.
In control experiments, the inventors observed, to their surprise, that it was possible to bind the non-activated native kinase covalently to ligands which were activated with SPDP (N-succinimidyl 3-(2-pyridyldithio) propionate). The explanation for this unexpected observa-tion is probably that the kina-se carries a free -SH
grouping which can produce a disulfide bridge with the 2-pyridyl disulfide grouping which is bound to the activated probe. The conjugate obtained using this approach is completely active.
The inventors have also observed that it is possible advantageously to increase the yield from the coupling, without loss of activity, by prior reduction of one or more natural disulfide bridges in the kinase using low concentrations of dithiothreitol.
The optimized conditions for coupling in solution are described below for antibodies. The inventors observed that activating the antibodies with a 5 times molar excess of SPDP resulted in the attachment of 1 to 2 SPDP arms per molecule of antibody. This concentration of SPDP yields a m~; mllm of active conjugates when equivalent quantities (in mol) of activated antibodies and kinase are brouqht together.
Another method of coupling on a column (solid phase), which makes it possible to improve the yield, to facilitate separation of the antibodies conjugated to the kinase from the unconjugated antibodies and to be able to prepare a large quantity of conjugates, consists in binding the kinase, by means of weak, non-covalent bonds, at its active site, to a column contA; n ing Blue Sepharose (Pharmacia, Uppsala, Sweden) and then allowing the anti-bodies, previously activated with SPDP, to circulate through the column in a closed circuit and for a period .
of time which is adequate (usually 16 h).
The activated antibodies will thus have adequate opportunity to produce the conjugates with the kinase which is immobilized on the Blue Sepharose.
After the reaction, the column is washed and the antibodies which have not reacted are removed, and the conjugate is then eluted by increasing the ionic strength of the washing solution. This method iS described in detail below for the antibodies and for avidin.
The availability of active conjugates makes it possible to use them for diagnostic tests based either on immunological reactions, such -as the enzyme linked immunosorbent assay or ELISA methods, or on the hybridi-zation of nucleotide sequences.
It has been possible to extend the principle of the method for coupling the kinase to the antibodies to other proteins and even to oligonucleotides. Thus, it has been possible to activate avidin and streptavidin with SPDP and then, after elimination of excess reagent, bring them into contact with the pyruvate kinase. The conjugate, formed in this way in solution, was purified on a column of Blue Sepharose which retains the kinase and which proves to be extremely efficacious.
In order to bind the oligonucleotide probes to the kinase, the DNA probes can be terminated by an amine at the 3' and/or 5' terminal position which amine(s~ can be incorporated during the course of synthesizing the nucleotide chain which is produced or else added subsequently.
This terminal amine is then activated by SMCC and then, after the excess reagents have been removed, brought into contact with the pyruvate kinase for the coupling. The conjugate is then easily purified by molecular sieve chromatography or by affinity chromato-graphy.
Immunoenzymic assays (EL~SA) The kinase can be coupled to an antibody in order to form an antibodytkinase conjugate which can be used in various ELISA tests.
- 214~092 For a direct conjugate assay, use is made of a multiwell dish or of tubes or some other support on which the antigens are immobilized.
The antibody/kinase conjugates are then added in decreasing quantity. After washing, the activity of the bound kinase is measured in the presence of its substrates, allowing the production of ATP which, in the presence of luciferin is broken down by the luciferase with an emission of light. The latter can be measured using a bioluminometer or using a sensitive film such as a Polaroid film or else using a negative paper which is sensitive to X-rays. Using this-test, the titre of the antibodies can be determined.
Under the ~luvate kinase assay conditions, the excess of substrates in relation to the quantity of enzymes and the quantity of luciferases is such that the enzymes carry out their activity for several days. In a normal ELISA test, the emission of light can be followed for three days.
The direct test makes it possible to quantify antigen by using the so-called competitive method. In this case, the antibody is immobilized on the assay dish or support. The antigen to be assayed is then incubated in the presence of a fixed quantity of antigen conjugated to the kinase. These two antigens compete for the anti-body attached to the dish: the larger the quantity of antigen to be assayed, the less antigen/kinase will be able to bind to the antibody. It is the latter which is detected by the activity of the kinase. The antigen to be assayed is then estimated by referring to a calibration curve.
Antigen is more readily assayed by the so-called sandwich method. The dish or the solid support contains immobilized antibodies. The antigen to be assayed is then added in increasing dilutions and then, after washing, a fixed quantity of a second antibody, which is labeled with the kinase, is added and the activity of the kinase is measured. A variant of this method consists in using a second antibody which is not labeled and subsequently 214~0~
_ WO 94/06933 - 16 - PCT/BB93/00063 adding an anti-antibody which is labeled with the kinase (double sandwich).
Antibody can also be quantified by the sandwich assay and, in this case, use is made of a support on which the antigen is immobilized. The antibody is added in increasing concentrations and then, after washing, anti-antibodies labeled with the kinase are incubated and the activity of the kinase is measured. Here too, double sandwich assays can be carried out using an anti-antibody which is not labeled and then a second anti-antibody which is conjugated to the kinase.
~ybridization assay of oligonucleotide sequences The possibility of using avidin (or streptavidin), antibodies or nucleotides as probes which can be coupled to the kinase makes it possible to develop tests for recognizing nucleotide sequences. This technique can be applied in a large number of spheres such as diagnostic tests for detecting bacteria, fungi, yeasts, viruses, cancerous cells or any biological organism which possesses a specific nucleic acid sequence.
The principle of oligonucleotide hybridization is well known and we shall review it briefly. It consists in the recognition of a nucleotide sequence (DNA or RNA) by a complementary strand of nucleotides and in their pairing to form a double chain under appropriate temperature, pH and salt conditions. This recognition is permitted by the formation of hydrogen bridges between adenine (A) and thymine (T) (or uracil (U)) and between guanine (g) and cytosine (C). If the DNA (or RNA) sequence to be detected is known, it is possible, using chemical, enzymic or biological methods, to construct the complementary sequence, which will then hybridize specifically with the DNA (or the RNA) to be detected.
Preferably, this hybridization is effected under stringent conditions, in a manner which ensures that the hybridization is specific and thus avoids false posltives .
The advantage of using the kinase conjugates according to the invention for implementing bioluminescence tests i8 a very high degree of sensitivity and a detection time which can extend over several days. In the simplest method, the nucleotide sequence used for the test is labeled with biotin. This labeling can be carried out in various ways:
- either chemically, by using a biotinylated oligonucleo-tide in the synthesis or by reacting the ~equence with an NHS-biotin, biotinamidocaproate N-hydroxysuccinimide ester or a reagent which carries the biotin and which can bind to the nucleotide sequence, - or enzymically, using terrinAl transferase which incorporates the biotinylated nucleotides at the end of a specific nucleotide sequence. Biotin can be incorpo-rated into the nucleic acids using a photoactivatablecompound termed photobiotin.
The`biotinylated sequence can then be recognized by avidin or streptavidin which is itself coupled to the enzyme, or after reaction with an antibody which is labeled with the kinase and which is directed against the biotinylated sequence. After hybridization with the sequence to be detected, an avidin/kinase conjugate is used which binds to the biotin. The activity of the kinase is then measured by its production of ATP which, in the presence of luciferase, emits photons.
The detection of a DNA or RNA sequence which is present in solution is more complex since it requires an immobilization step in order to effect a washing or a competitive reaction. The sequences can be immobilized directly on nitrocellulose or nylon filters in tests termed "dot blot" tests or following separation by gel electrophoresis (Southern blot and Northern blot).
When the DNA 1 (or RNA) to be measured is present in very low quantity, it is also possible to immobilize this DNA 1 by means of a complementary sequence 2 which is itself immobilized on a support 3 (filter, gel, plastic, glass, beads, etc.). The DNA 1 (or RNA) hybridized to the immobilized probe can be measured using another biotinylated sequence 4 and an avidin/kinase - WO 94/06933 - 18 - ~ PCT/BE93/00063 conjugate 5 or an antibody/kinase anti-probe. This method using two nucleotide sequences (2 and 4), - one immobi-lized and termed captor 2 and the other biotinylated and termed detector 4 - is also called a sandwich method.
From the point of view of the methodology, the two, captor and detector, sequences can be added simultaneously to the DNA (or RNA) to be detected, or sequentially, according to the conditions of the test and according to the result to be obtained: greater sensitivity, speed of operation or ease of manipulations (Figure 1).
These methods can be made more complex by emplo-ying not only recognition between avidin and biotin but also between antigens (or haptens) and antibodies (Figures 2, 3 and 4).
For example, it is possible to use a biotinylated nucleic acid sequence 4 and then a reaction with avidin 6 (which is or is not coupled to a kinase) which avidin will, itself, be recognized by anti-avidin antibodies 7 which are coupled to the kinase (Figures 2 and 3).
It is also possible to carry out a direct reac-tion (Figure 4) with antibodies 8 which either are or are not coupled to the kinase and which are directed against the biotin which is bound to the probe. A molecule other than biotin, for example fluorescein or digoxigenin, which can be recognized by specific antibodies, can also be used to label the probe.
Several variants of this technique exist, such as the use of a second antibody conjugated with the kinase or the use of an antibody conjugated with the biotin with which avidin/kinase conjugates may be reacted. Whatever the combination employed, at least one avidin/kinase conjugate 5 or antibody/kinase conjugate 7 according to the invention will be used in one of the steps of the reaction.
These specific avidin/biotin or antibody/antigen recognitions can also be exploited for immobilizing on the solid support. This makes it possible to carry out the hybridization reaction in solution before effecting 214~09~
3 - 19 - ~ PCT/BE93/00063 immobilization. The advantage of hybridization in solution lies in its much greater speed as compared with that of hybridization on a solid support, where steric factors and diffusion factors retard the reaction.
A particularly advantageous method consists in hybridizing the DNA to be measured with two nucleotide sequences which are complementary to part of the DNA to be measured. These two sequences are labeled with diffe-rent molecules, for example one with biotin and the other with kinase. After hybridization, the mixture is brought into contact with a support carrying avidin, which will bind the biotinylated sequences,-some of which will form part of the hybrid, which will carry a second kinase sequence which can be measured in the presence of luciferase and the appropriate substrates.
Immobilization can be effected by means of using complementary primer sequences such as poly C or poly A
which respectively recognize poly G or poly T (poly U) sequences.
The probe carrying the biotin, or a second specific probe [sic] DNA or RNA to be assayed, can carry a poly A (or poly T or poly G) sequence which, during the hybridization in solution, will bind to the complementary sequence which is bound to a support.
The two sequences can also carry a biotin or an antigen (or hapten) such as dinitrophenol or fluorescein.
After hybridization, the hybrid can either [sic]
be immobilized on a support carrying avidin and be detected by an antibody which is directed against the antigen or the hapten (in this case, anti-dinitrophenol or anti-digoxigenin) and which is labeled with the kinase. If the antibody is not labeled with the kinase, a second anti-antibody which is labeled with the kinase can be used.
The immobilization can also be effected using antibodies which are bound to the support and which will recognize and bind the antigen or the hapten (in this case, dinitrophenol). The hybrid is then detected by reacting avidin/kinase with the biotinylated sequence of the hybrid. The other methods for detecting the biotin/avidin which have been explained above may also be used, but, in every case, at least one conjugate/kinase - will be employed.
It is also possible to couple the kinase to antibodies which recognize the double strands of DNA.
In these hybrid detection situations described above, recourse was had, in one of the steps at least, to the recognition existing between biotin and avidin or between antibodies and their antigens or haptens.
Especially in the case of pyruvate kinase, it is possible to avoid taking this reaction route (it has been observed that this enzyme is stable at 45c [sic] for 2 h at least, which represents hybridization conditions which can, in particular, be obtained with oligonucleotides).
Based on these observations, a conjugate is produced which is composed of kinase which is coupled directly to the oligonucleotide sequence which will be used for the hybridization; the activity of the kinase being measured in the presence of its substrates and of luciferase.
This technique can be applied after amplifying a sequence by PCR, either in solution or even in a fixed cell. In this case, the method using a thermostable pyruvate kinase is particularly appropriate.
EXAMPLES
IMMnNOLOGICAL TESTS
I - Detection of anti-IqG/kinase coniuqates.
Mouse IgGs are immobilized on multiwell plates.
The wells are then saturated with bovine albumin for 4 h.
After that, the wells are washed 3 times with a solution of Tween. If appropriate, the wells can be dried. The conjugated antibodies are then incubated in the presence of albumin at 20C for 2 h. After washing, the activity of the kinase is assayed by adding a mixed solution contA;n;ng these substrates, namely: phosphenolpyruvate, ADP, luciferin and luciferase, these latter two substances being intended for detecting ATP produced by the kinase from the ADP and the phosphoenolpyruvate. The photons are measured in a bioluminometer during the 21450~2 _ WO 94/06933 - 21 - PCT/BE93/00063 following 5 minutes and the result is expressed in terms of a plateau value or of an integration value over a period of 1 minute. Exposure can also be carried out using a sensitive polaroid film, for example 3,000 or 20,000 ASA for 5 minutes followed by a 45 sec. develop-ment. In a test carried out in this manner, the anti-IgG/kinase was diluted successively and it was possible to detect it down to a dilution of 2,560 times, this representing 2.6 x 10-13 mol of antibody per test.
II - Measurement of IgG, considered as an antigen (sandwich method).
The same experiment was carried out as in the preceding paragraph except that rabbit anti-human IgG
antibodies were immobilized on the multiwell plates.
These plates were incubated with human IgG at increasing dilutions, after which they were washed and mouse ant-human IgG antibodies, which were conjugated to the kinase, were added to all the wells. sy means of measu-ring the kinase by plate photography, it was possible to detect a concentration of 8,310-14 mol/test of human IgG.
III - Determination of the titer of anti-beta-HCG (human chorionic gonadotropin) anti h~ ies.
Two dilutions of 20x and lOOx are prepared, using negative serum (originating from a female human subject), of an anti-beta-HCG [sic] positive serum cont~;n;ng 98,000 mUI/ml. These two dilutions of which been [sic]
incubated at 37C, and under a humid atmosphere for 60 minutes in 2 x 3 series of microwells which have previously been coated with the trapping monoclonal antibody (anti-beta HCG). After rinsing the wells three times, incubation takes place of a commercial preparation of rabbit anti-HCG polyclonal IgG which is dialyzed and diluted lOOx, l,OOOx and lO,OOOx. Rinsing takes place three times in phosphate buffer. Finally, for each series of microwells, we prepared a concentration curve for conjugates which are composed of a sheep anti-rabbit IgG
antibody conjugated to pyruvate kinase. After three rinsings, the activity is assayed using a plate reader for bioluminescence. If the rabbit anti-HCG antibody is 21~5092 - - WO 94/06933 - 22 - ~ PCT/B~93/00063 diluted 100x and if a dilution of 50-fold is used for the conjugate, an activity of 30 RLU is observed for the 20x dilution of the HCG serum containing 98,000 mUI/ml and an activity of 7 RLU (test value minus the blank value) is observed for the 100x dilution. Concentrations of 4,900 mUI/ml of beta-HCG in the serum can therefore be assayed with a signal/noise ratio of 7, and a concentration of 980 mUI/l can be assayed with a signal/noise ratio of 2. This value would, in reality, appear to constitute the detection l;mit of the assay. A
serum is considered as positive when it contains more than 2,000 mUI/l, which agrees well with the requirements stipulated for a clinical biology laboratory.
Optimization of the conditions would permit further improvement in the performance of this assay.
IV - Assay of anti-histone antibodies.
An assay of anti-histone antibodies in the serum of a positive patient was effected by carrying out doubling dilutions of the serum using a control serum in order to obtain dilutions ranging from 800 to 102,400 times. The multiwell dishes were coated with histone proteins. The human anti-histone serum was incubated in these wells as was, after washing, a rabbit anti-human IgG antibody which was labeled with the kinase. This anti-histone serum gave a signal which was linearly related to the dilution up to a dilution of 25,600 times while the same test, carried out using a conjugate labeled with peroxidase and read by spectophotometry following the customary conditions of the supplier (Biolal, Belgium), using ABTS (azinobenzothiazoline sulfonate) as substrate, only detected up to a dilution of 1,600 times.
Genetic tests I. Direct measurement of the DNA of the HPV18 virus A. Covalent attachment of the DNA to the plastic dishes The DNA of the HPV18 virus was immobilized covalently on multiwell plastic dishes which were modi-fied so that they exhibited aminated groupings on their surface. The bonding is effected between the 5' terminal 21~092 ~ - WO 94/06933 - 23 - PCT/BE93/00063 phosphate of the DNA and the ~;ne function which is grafted to the plastic, via the coupling agents which are 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and methylimidazole.
The plates are incubated at 50c [sic] for 5 h and the wells are then washed, at 50c ~sic], with a 5 x SSC solution cont~in;ng 0.25% SDS. These binding conditions are recommended by the firm which markets these Covalink dishes (NUNC, Gibco BRL) B. Detection of the DNA using biotinylated oligos An oligonucleotide of 21 base pairs was selected for its specificity in recognizing a specific sequence of the viral DNA's of HPV 18-33 and 16. This sequence is:
A particularly advantageous method consists in hybridizing the DNA to be measured with two nucleotide sequences which are complementary to part of the DNA to be measured. These two sequences are labeled with diffe-rent molecules, for example one with biotin and the other with kinase. After hybridization, the mixture is brought into contact with a support carrying avidin, which will bind the biotinylated sequences,-some of which will form part of the hybrid, which will carry a second kinase sequence which can be measured in the presence of luciferase and the appropriate substrates.
Immobilization can be effected by means of using complementary primer sequences such as poly C or poly A
which respectively recognize poly G or poly T (poly U) sequences.
The probe carrying the biotin, or a second specific probe [sic] DNA or RNA to be assayed, can carry a poly A (or poly T or poly G) sequence which, during the hybridization in solution, will bind to the complementary sequence which is bound to a support.
The two sequences can also carry a biotin or an antigen (or hapten) such as dinitrophenol or fluorescein.
After hybridization, the hybrid can either [sic]
be immobilized on a support carrying avidin and be detected by an antibody which is directed against the antigen or the hapten (in this case, anti-dinitrophenol or anti-digoxigenin) and which is labeled with the kinase. If the antibody is not labeled with the kinase, a second anti-antibody which is labeled with the kinase can be used.
The immobilization can also be effected using antibodies which are bound to the support and which will recognize and bind the antigen or the hapten (in this case, dinitrophenol). The hybrid is then detected by reacting avidin/kinase with the biotinylated sequence of the hybrid. The other methods for detecting the biotin/avidin which have been explained above may also be used, but, in every case, at least one conjugate/kinase - will be employed.
It is also possible to couple the kinase to antibodies which recognize the double strands of DNA.
In these hybrid detection situations described above, recourse was had, in one of the steps at least, to the recognition existing between biotin and avidin or between antibodies and their antigens or haptens.
Especially in the case of pyruvate kinase, it is possible to avoid taking this reaction route (it has been observed that this enzyme is stable at 45c [sic] for 2 h at least, which represents hybridization conditions which can, in particular, be obtained with oligonucleotides).
Based on these observations, a conjugate is produced which is composed of kinase which is coupled directly to the oligonucleotide sequence which will be used for the hybridization; the activity of the kinase being measured in the presence of its substrates and of luciferase.
This technique can be applied after amplifying a sequence by PCR, either in solution or even in a fixed cell. In this case, the method using a thermostable pyruvate kinase is particularly appropriate.
EXAMPLES
IMMnNOLOGICAL TESTS
I - Detection of anti-IqG/kinase coniuqates.
Mouse IgGs are immobilized on multiwell plates.
The wells are then saturated with bovine albumin for 4 h.
After that, the wells are washed 3 times with a solution of Tween. If appropriate, the wells can be dried. The conjugated antibodies are then incubated in the presence of albumin at 20C for 2 h. After washing, the activity of the kinase is assayed by adding a mixed solution contA;n;ng these substrates, namely: phosphenolpyruvate, ADP, luciferin and luciferase, these latter two substances being intended for detecting ATP produced by the kinase from the ADP and the phosphoenolpyruvate. The photons are measured in a bioluminometer during the 21450~2 _ WO 94/06933 - 21 - PCT/BE93/00063 following 5 minutes and the result is expressed in terms of a plateau value or of an integration value over a period of 1 minute. Exposure can also be carried out using a sensitive polaroid film, for example 3,000 or 20,000 ASA for 5 minutes followed by a 45 sec. develop-ment. In a test carried out in this manner, the anti-IgG/kinase was diluted successively and it was possible to detect it down to a dilution of 2,560 times, this representing 2.6 x 10-13 mol of antibody per test.
II - Measurement of IgG, considered as an antigen (sandwich method).
The same experiment was carried out as in the preceding paragraph except that rabbit anti-human IgG
antibodies were immobilized on the multiwell plates.
These plates were incubated with human IgG at increasing dilutions, after which they were washed and mouse ant-human IgG antibodies, which were conjugated to the kinase, were added to all the wells. sy means of measu-ring the kinase by plate photography, it was possible to detect a concentration of 8,310-14 mol/test of human IgG.
III - Determination of the titer of anti-beta-HCG (human chorionic gonadotropin) anti h~ ies.
Two dilutions of 20x and lOOx are prepared, using negative serum (originating from a female human subject), of an anti-beta-HCG [sic] positive serum cont~;n;ng 98,000 mUI/ml. These two dilutions of which been [sic]
incubated at 37C, and under a humid atmosphere for 60 minutes in 2 x 3 series of microwells which have previously been coated with the trapping monoclonal antibody (anti-beta HCG). After rinsing the wells three times, incubation takes place of a commercial preparation of rabbit anti-HCG polyclonal IgG which is dialyzed and diluted lOOx, l,OOOx and lO,OOOx. Rinsing takes place three times in phosphate buffer. Finally, for each series of microwells, we prepared a concentration curve for conjugates which are composed of a sheep anti-rabbit IgG
antibody conjugated to pyruvate kinase. After three rinsings, the activity is assayed using a plate reader for bioluminescence. If the rabbit anti-HCG antibody is 21~5092 - - WO 94/06933 - 22 - ~ PCT/B~93/00063 diluted 100x and if a dilution of 50-fold is used for the conjugate, an activity of 30 RLU is observed for the 20x dilution of the HCG serum containing 98,000 mUI/ml and an activity of 7 RLU (test value minus the blank value) is observed for the 100x dilution. Concentrations of 4,900 mUI/ml of beta-HCG in the serum can therefore be assayed with a signal/noise ratio of 7, and a concentration of 980 mUI/l can be assayed with a signal/noise ratio of 2. This value would, in reality, appear to constitute the detection l;mit of the assay. A
serum is considered as positive when it contains more than 2,000 mUI/l, which agrees well with the requirements stipulated for a clinical biology laboratory.
Optimization of the conditions would permit further improvement in the performance of this assay.
IV - Assay of anti-histone antibodies.
An assay of anti-histone antibodies in the serum of a positive patient was effected by carrying out doubling dilutions of the serum using a control serum in order to obtain dilutions ranging from 800 to 102,400 times. The multiwell dishes were coated with histone proteins. The human anti-histone serum was incubated in these wells as was, after washing, a rabbit anti-human IgG antibody which was labeled with the kinase. This anti-histone serum gave a signal which was linearly related to the dilution up to a dilution of 25,600 times while the same test, carried out using a conjugate labeled with peroxidase and read by spectophotometry following the customary conditions of the supplier (Biolal, Belgium), using ABTS (azinobenzothiazoline sulfonate) as substrate, only detected up to a dilution of 1,600 times.
Genetic tests I. Direct measurement of the DNA of the HPV18 virus A. Covalent attachment of the DNA to the plastic dishes The DNA of the HPV18 virus was immobilized covalently on multiwell plastic dishes which were modi-fied so that they exhibited aminated groupings on their surface. The bonding is effected between the 5' terminal 21~092 ~ - WO 94/06933 - 23 - PCT/BE93/00063 phosphate of the DNA and the ~;ne function which is grafted to the plastic, via the coupling agents which are 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and methylimidazole.
The plates are incubated at 50c [sic] for 5 h and the wells are then washed, at 50c ~sic], with a 5 x SSC solution cont~in;ng 0.25% SDS. These binding conditions are recommended by the firm which markets these Covalink dishes (NUNC, Gibco BRL) B. Detection of the DNA using biotinylated oligos An oligonucleotide of 21 base pairs was selected for its specificity in recognizing a specific sequence of the viral DNA's of HPV 18-33 and 16. This sequence is:
5' - A C T T T A A C T G C A G A T G T T A T G - 3' This sequence hybridizes with a region corresponding to nucleotides 6780-6800 of the HPV16 Ll gene, with one mispairing at position 15 and one at position 3, with a region corresponding to nucleotides 6759-6779 of the HPV18 L1 gene, and with a region corresponding to nucleo-20 tides 6734-6754 of the HPV33 Ll gene, with one mispairing at position 15 and one mispairing at position 3.
The biotin labeling of this oligonucleotide is carried using terminal transferase. The latter binds some biotinylated dUTPs and some dCTPs (non-biotinylated) to the 3' end.
The hybridization takes place in the wells, where the DNA was attached, in a hybridization solution contai-ning the biotinylated oligo, and incubation is carried out at 50C for 16 h.
After washing, the avidin/kinase conjugate is then added at the rate of 0.01 mg/ml and, after washing, the activity of the kinase is determined in the presence of its substrates and of luciferase, as explained in the examples above. The quantity of HPV 18 viral DNA detected under these conditions corresponds to the solution of 100 ng/well, which is very high.
214509~
~ ~ WO 94/06933 - 24 - PCT/BF93/00063 II - Indirect measurement of the DNA of the HPV18 virus (sandwich methodt usinq two oliqonucleotides A. Genome of 586 bp In order to avoid problems with viscosity, interactions and difficulties associated with manipulation of the complete HPV genome (+/- 8,000 bp), one part of this genome is amplified by PCR, namely a strand of 586 bp from the 6,193rd base to the 6,779th base.
B. Covalent binding of the trappinq oliqo The trapping oligonucleotide of 21 base pairs has a unique specificity for the HPV18 genome and represents the complement of the first 21 bases starting from the 3' end of the fragment selected above.
The sequence is 5' -T T T T G. C A A G A T G G T G A T A
T G G- 3' The trapping oligo is immobilized covalently in the NUNC dishes in the same way as was previously the - case for the DNA of the virusHPV 18.
Incubation is carried out at 50c [sic] for 5 h using 5 ng of oligonucleotides per well, and we bind [sic] approximately 1.2 ng by a covalent bond.
C. Hybridization of the HPV 18 fraqment For the hybridization, the wells contA;n;ng the trapping oligonucleotide are first of all rinsed.
Hybridization solution cont~;n;ng decreasing concentrations of the HPV18 genome fragment, diluted in water and denatured, is then added at the rate of 2C0 micoliters/well. Hybridization takes place at 50c [sic~
over 2 h.
D. Hybridization of the biotinylated oliqonucleotides The detector oligonucleotide of 21 base pairs is specific for the HPV 16, 18 and 33 genomes (cf. indirect measurement of the HPV18 virus - point B) and represents the complement of the last 21 bases starting from the 3' end of the 586 base pair fragment selected in point A.
Its sequence is:
5'-A C T T T A A C T G C A G A T G T T A T G-3' After the wells have been washed, a new WO 94/06933 - 25 - PCT/B~93/00063 hybridization is carried out in the same manner as previously except that biotinylated oligonucleotides are added to the hybridization solution. This hybridization is carried out at 50c [sic] for 1 h.
E.- Determination usinq bioluminescence After hybridization, the plates are washed and then the avidin/kinase conjugate is added at a concentra-tion of 0.01 mg/ml and, after washing, the activity of the kinase is determined in the presence of its substrates and of luciferase, as explained in the examples above.
III. Indirect measurement of the DNA of HPV virus rsandwich method in the Presence of a DNA chain using double recognition of longer probes and detection bY way of antibodies labeled with kinase) A. Genome of 586 bp In order to avoid problems with viscosity, interactions and difficulties associated with manipu-lation of the complete HPV genome, one part of the genome is amplified by PCR, namely a strand of 586 bp from the 6,193rd base to the 6,779th base.
B. Covalent bindinq of a trapping probe A trapping probe, constructed by PCR and 5'-phosphorylated, is covalently bound in the wells 2S
before.
C. Sandwich hybridization A biotinylated detection probe was produced by PCR.
The biotin is incorporated by virtue of the presence of biotinylated dUTP in a ratio of 1/2 relative to dTTP.
The hybridization is carried out in the wells where the trapping probe is immobilized. The hybridiza-tion solution is composed of the HPV target DNA of 586 bp and the biotinylated probe produced by PCR. Hybridization is carried out at 70C for 2 hours.
D. Determination After washing and saturation, avidin is incubated in the wells as is subsequently the anti-avidin antibody, followed by the antibody directed against the latter and labeled with kinase. After washing and equilibration, the 219509~
activity of the kinase is determined in the presence of its substrates and of the luciferase system as explained above, and light production is measured using a luminoscan, or a Polaroid photograph is taken.
E. Results Using a Lumac bioluminometer, the light measure-ment was 10,000 RLU (relative light unit) for 500 pg of HPV target DNA and 2,500 RLU for 50 pg of HPV target DNA.
IV. Indirect measurement of the DNA of HPV virus (sandwich method in the presence of a DNA chain and determination usinq a streptavidin/kinase coniugate) The preparation of the trapping probes bound to the plastic wells and of the detector probes labeled with biotin is identical to that described in Example III
above. The hybridization is carried out under the same conditions. After hybridization, the wells are washed and saturated before incubating with the streptavidin/kinase conjugate, which is diluted 1,OOOx. The wells are washed 4 times before being incubated in the presence of the kinase substrates and luciferase as in the preceding examples. Under these conditions, a concentration curve was constructed for 500 pg to 5 pg of HPV target DNA and the RLU values are greater than the controls obtained in the presence of non-specific DNA.
V. Indirect measurement of the DNA of HIV virus (sandwich method in the presence of a DNA chain and determination usinq streptavidin/kinase conjugate).
A. Tarqet produced by PCR
A segment of the RNA chain of the AIDS virus (HIV
I) was transcribed into DNA and then amplified by PCR.
This fragment comprises 473 bases situated between the 1,214th base and the 1,687th base.
B. Hybridization The preparation of the trapping and detector probes, and also the hybridization and the determination, are carried out in the same manner as in the case of VIII.
C. Results A concentration curve for HIV target DNA
214~092 _ WO 94/06933 - 27 - ~ PCT/BE93/00063 fragments was constructed ranging from 1,000 to 1 pg. At each of these concentrations, the RLU measurement was significantly greater than the controls obtained using non-specific DNA (results presented in Figure 9).
VI. Indirect measurement of the DNA of cytomegalovirus A segment of the RNA chain of the CMV virus was transcribed into DNA and then amplified by PCR. This fragment of 434 bases is situated between the 2,223rd base and the 2,657th base.
The same experimental protocol as for example VIII was carried out and a concentration curve for CMV
target DNA was obtained for the range 1,000 to 10 pg.
VII. Indirect measurement of the DNA of the bacterium Chlamydia trachomatis A segment of Chlamydia trachomatis DNA was amplified by PCR. This fragment of 415 bases is situated downstream of the 5,339th base and represents the target DNA .
The same experimental protocol as for Example VIII was carried out and a concentration curve for Chlamydia trachomatis target DNA was obtained for the range 2,000 to 20 pg.
VIII. Bioluminescence Elisa with polaroid plate readinq New conjugates are synthesized and 96-well plates are coated with rabbit anti-human IgG, human IgG and mouse IgG.
a/ The first operation consists in constructing a saturation curve in order to determine the titer of the conjugates;
b/ The Elisa assay itself was carried out in accordance with two methods which varied slightly as regards the quantity of marker enzyme employed. In a first case, direct measurement, the ATP produced by the pyruvate kinase reaction is measured continually using bioluminescence. This method allows the detection of 2.6 x 10-13 mol/test of human IgG (Figure 5). In a second case, indirect measurement, the ATP produced is accumulated for a period of 60 minutes and is then measured using bioluminescence. In this case, 8.32 x 10-14 mol are detected per test (Fig. 6). It is noted that the sensitivity was not improved as might have been expected.
IX. Effect of the concentration of SPDP on the number of arms which are bonded and on the activitY of the PYruvate kinase, protection by the substrates 100 ml aliquots of 10 mg/ml PK in 100 mM KH2PO4, pH 7, are activated with 200 ml of increasing concentra-tions of SPDP in ethanol. After a reaction of 30 minutes, the excess SPDP is removed on Sephadex G25 and the PK is analyzed with regard to its residual activity and to the number of arms bonded per molecule of enzyme. The experi-ment is also carried out in the presence of substrates at a concentration of 1 mM (ADP, phosphoenolpyruvate and MgCl2). The graph shows that, for a low number of arms bonded to the enzyme (less than 5), the residual activity is similar whether or not the experiment is carried out in the presence of substrates. Over and above an excess of 10 SPDP, which corresponds to +/- 4 arms bonded per enzyme, the residual activity is slightly greater if the experiment tsic] in the presence of substrate (Fig. 7).
A microwell dish is coated, according to a standard method, with immunoglobulins (IgG) of mouse (blank) and human (test) origin. After incubating with conjusates consisting of anti-human IgG IgG coupled to pyruvate kinase, the microwells are washed and is determined by luminescence. The activity of the kinase coupled to the anti-human IgG IgG. [sic] The latter conjugates are bound in the wells contA; n; ng their antigen, namely human IgG
(Fig. 8).
X. Measurement of DNA fraqments produced from the RNA of HIV virus DNA probes specific for the HIV virus were covalently bonded by their 5' ends to a Covalink (NUNC) microwell dish and then incubated in the presence of DNA
fragments derived from a PCR (polymerase chain reaction3 amplification of a 475 bp fragment corresponding to a part of the RNA of the AIDS virus (HIVI). The solutions also containing [sic] a 205 bp biotinylated detector 21450~
_ WO 94/06933 - 29 - PCT/BF93/00063 probe which was also complementary to the DNA sequence of the ~IVI virus.
After hybridization at 70c [sic] and washing, a streptavidin/kinase conjugate is incubated with the hybrids and, after washing, the substrates of the kinase and luciferase are added in order to measure the kinase/streptavidin conjugates which is [sic] present in the wells. Figure 9 shows the slope of the increase in light in RLU (Relative Light Unit) observed per hour of incubation of these reagents. The controls were obtained using a 571 bp target DNA derived from the mouse genome.
The tests were carried out in triplicate and represent the mean. The st~n~rd deviations (+/- s) are indicated in the figure. The figure shows that 1 pg of DNA can be detected with significance by this test.
SEQUENCE LISTING 21~ 5 0 9 ~
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: LAMBDATECH S.A.
(B) STREET: LES BEYOLETTES
(C) CITY: FO~T~
(E) COUNTRY: BELGIQUE
(F) POSTAL CODE (ZIP): 6953 (G) TELEPHONE: 3224263810 (H) TELEFAX: 3224263760 (ii) TITLE OF INVENTION: CONJUGATE FOR THE DETECTION AND/OR OF
A BIOLOGICAL COMPOUND
(iii) NUMBER OF SEQUENCES: 20 (iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (EPO) (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) S~Qu~N~ DESCRIPTION: SEQ ID NO: 1:
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ACAC~Ll~ll ACAC~l~l~l AATAT 25 (2) INFORMATION FOR SEQ ID NO: 3:
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GGTTCGCA~A TCTACGCACG GTCCT 25 (2) INFORMATION FOR SEQ ID NO: 6:
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UACUACAUAC ACCCCCGCAC AGACC 21~ 5 0 9 ~ 25 (2) INFORMATION FOR SEQ ID NO: 15:
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(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: RNA (genomic) (xi) S~Qu~N~ DESCRIPTION: SEQ ID NO: 16:
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(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: RNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
(2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: RNA (genomic) txi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: 21 4 5 0 9 2 (2) INFORMATION FOR SEQ ID NO: 19:
(i) ~U~N-~ CHARACTERISTICS:
(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) sTR~Nn~nNF~s: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: RNA (genomic) (Xi) ~U~N-~ DESCRIPTION: SEQ ID NO: 19:
(2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: RNA (genomic) (Xi ) S~U~N~'~ DESCRIPTION: SEQ ID NO: 20:
The biotin labeling of this oligonucleotide is carried using terminal transferase. The latter binds some biotinylated dUTPs and some dCTPs (non-biotinylated) to the 3' end.
The hybridization takes place in the wells, where the DNA was attached, in a hybridization solution contai-ning the biotinylated oligo, and incubation is carried out at 50C for 16 h.
After washing, the avidin/kinase conjugate is then added at the rate of 0.01 mg/ml and, after washing, the activity of the kinase is determined in the presence of its substrates and of luciferase, as explained in the examples above. The quantity of HPV 18 viral DNA detected under these conditions corresponds to the solution of 100 ng/well, which is very high.
214509~
~ ~ WO 94/06933 - 24 - PCT/BF93/00063 II - Indirect measurement of the DNA of the HPV18 virus (sandwich methodt usinq two oliqonucleotides A. Genome of 586 bp In order to avoid problems with viscosity, interactions and difficulties associated with manipulation of the complete HPV genome (+/- 8,000 bp), one part of this genome is amplified by PCR, namely a strand of 586 bp from the 6,193rd base to the 6,779th base.
B. Covalent binding of the trappinq oliqo The trapping oligonucleotide of 21 base pairs has a unique specificity for the HPV18 genome and represents the complement of the first 21 bases starting from the 3' end of the fragment selected above.
The sequence is 5' -T T T T G. C A A G A T G G T G A T A
T G G- 3' The trapping oligo is immobilized covalently in the NUNC dishes in the same way as was previously the - case for the DNA of the virusHPV 18.
Incubation is carried out at 50c [sic] for 5 h using 5 ng of oligonucleotides per well, and we bind [sic] approximately 1.2 ng by a covalent bond.
C. Hybridization of the HPV 18 fraqment For the hybridization, the wells contA;n;ng the trapping oligonucleotide are first of all rinsed.
Hybridization solution cont~;n;ng decreasing concentrations of the HPV18 genome fragment, diluted in water and denatured, is then added at the rate of 2C0 micoliters/well. Hybridization takes place at 50c [sic~
over 2 h.
D. Hybridization of the biotinylated oliqonucleotides The detector oligonucleotide of 21 base pairs is specific for the HPV 16, 18 and 33 genomes (cf. indirect measurement of the HPV18 virus - point B) and represents the complement of the last 21 bases starting from the 3' end of the 586 base pair fragment selected in point A.
Its sequence is:
5'-A C T T T A A C T G C A G A T G T T A T G-3' After the wells have been washed, a new WO 94/06933 - 25 - PCT/B~93/00063 hybridization is carried out in the same manner as previously except that biotinylated oligonucleotides are added to the hybridization solution. This hybridization is carried out at 50c [sic] for 1 h.
E.- Determination usinq bioluminescence After hybridization, the plates are washed and then the avidin/kinase conjugate is added at a concentra-tion of 0.01 mg/ml and, after washing, the activity of the kinase is determined in the presence of its substrates and of luciferase, as explained in the examples above.
III. Indirect measurement of the DNA of HPV virus rsandwich method in the Presence of a DNA chain using double recognition of longer probes and detection bY way of antibodies labeled with kinase) A. Genome of 586 bp In order to avoid problems with viscosity, interactions and difficulties associated with manipu-lation of the complete HPV genome, one part of the genome is amplified by PCR, namely a strand of 586 bp from the 6,193rd base to the 6,779th base.
B. Covalent bindinq of a trapping probe A trapping probe, constructed by PCR and 5'-phosphorylated, is covalently bound in the wells 2S
before.
C. Sandwich hybridization A biotinylated detection probe was produced by PCR.
The biotin is incorporated by virtue of the presence of biotinylated dUTP in a ratio of 1/2 relative to dTTP.
The hybridization is carried out in the wells where the trapping probe is immobilized. The hybridiza-tion solution is composed of the HPV target DNA of 586 bp and the biotinylated probe produced by PCR. Hybridization is carried out at 70C for 2 hours.
D. Determination After washing and saturation, avidin is incubated in the wells as is subsequently the anti-avidin antibody, followed by the antibody directed against the latter and labeled with kinase. After washing and equilibration, the 219509~
activity of the kinase is determined in the presence of its substrates and of the luciferase system as explained above, and light production is measured using a luminoscan, or a Polaroid photograph is taken.
E. Results Using a Lumac bioluminometer, the light measure-ment was 10,000 RLU (relative light unit) for 500 pg of HPV target DNA and 2,500 RLU for 50 pg of HPV target DNA.
IV. Indirect measurement of the DNA of HPV virus (sandwich method in the presence of a DNA chain and determination usinq a streptavidin/kinase coniugate) The preparation of the trapping probes bound to the plastic wells and of the detector probes labeled with biotin is identical to that described in Example III
above. The hybridization is carried out under the same conditions. After hybridization, the wells are washed and saturated before incubating with the streptavidin/kinase conjugate, which is diluted 1,OOOx. The wells are washed 4 times before being incubated in the presence of the kinase substrates and luciferase as in the preceding examples. Under these conditions, a concentration curve was constructed for 500 pg to 5 pg of HPV target DNA and the RLU values are greater than the controls obtained in the presence of non-specific DNA.
V. Indirect measurement of the DNA of HIV virus (sandwich method in the presence of a DNA chain and determination usinq streptavidin/kinase conjugate).
A. Tarqet produced by PCR
A segment of the RNA chain of the AIDS virus (HIV
I) was transcribed into DNA and then amplified by PCR.
This fragment comprises 473 bases situated between the 1,214th base and the 1,687th base.
B. Hybridization The preparation of the trapping and detector probes, and also the hybridization and the determination, are carried out in the same manner as in the case of VIII.
C. Results A concentration curve for HIV target DNA
214~092 _ WO 94/06933 - 27 - ~ PCT/BE93/00063 fragments was constructed ranging from 1,000 to 1 pg. At each of these concentrations, the RLU measurement was significantly greater than the controls obtained using non-specific DNA (results presented in Figure 9).
VI. Indirect measurement of the DNA of cytomegalovirus A segment of the RNA chain of the CMV virus was transcribed into DNA and then amplified by PCR. This fragment of 434 bases is situated between the 2,223rd base and the 2,657th base.
The same experimental protocol as for example VIII was carried out and a concentration curve for CMV
target DNA was obtained for the range 1,000 to 10 pg.
VII. Indirect measurement of the DNA of the bacterium Chlamydia trachomatis A segment of Chlamydia trachomatis DNA was amplified by PCR. This fragment of 415 bases is situated downstream of the 5,339th base and represents the target DNA .
The same experimental protocol as for Example VIII was carried out and a concentration curve for Chlamydia trachomatis target DNA was obtained for the range 2,000 to 20 pg.
VIII. Bioluminescence Elisa with polaroid plate readinq New conjugates are synthesized and 96-well plates are coated with rabbit anti-human IgG, human IgG and mouse IgG.
a/ The first operation consists in constructing a saturation curve in order to determine the titer of the conjugates;
b/ The Elisa assay itself was carried out in accordance with two methods which varied slightly as regards the quantity of marker enzyme employed. In a first case, direct measurement, the ATP produced by the pyruvate kinase reaction is measured continually using bioluminescence. This method allows the detection of 2.6 x 10-13 mol/test of human IgG (Figure 5). In a second case, indirect measurement, the ATP produced is accumulated for a period of 60 minutes and is then measured using bioluminescence. In this case, 8.32 x 10-14 mol are detected per test (Fig. 6). It is noted that the sensitivity was not improved as might have been expected.
IX. Effect of the concentration of SPDP on the number of arms which are bonded and on the activitY of the PYruvate kinase, protection by the substrates 100 ml aliquots of 10 mg/ml PK in 100 mM KH2PO4, pH 7, are activated with 200 ml of increasing concentra-tions of SPDP in ethanol. After a reaction of 30 minutes, the excess SPDP is removed on Sephadex G25 and the PK is analyzed with regard to its residual activity and to the number of arms bonded per molecule of enzyme. The experi-ment is also carried out in the presence of substrates at a concentration of 1 mM (ADP, phosphoenolpyruvate and MgCl2). The graph shows that, for a low number of arms bonded to the enzyme (less than 5), the residual activity is similar whether or not the experiment is carried out in the presence of substrates. Over and above an excess of 10 SPDP, which corresponds to +/- 4 arms bonded per enzyme, the residual activity is slightly greater if the experiment tsic] in the presence of substrate (Fig. 7).
A microwell dish is coated, according to a standard method, with immunoglobulins (IgG) of mouse (blank) and human (test) origin. After incubating with conjusates consisting of anti-human IgG IgG coupled to pyruvate kinase, the microwells are washed and is determined by luminescence. The activity of the kinase coupled to the anti-human IgG IgG. [sic] The latter conjugates are bound in the wells contA; n; ng their antigen, namely human IgG
(Fig. 8).
X. Measurement of DNA fraqments produced from the RNA of HIV virus DNA probes specific for the HIV virus were covalently bonded by their 5' ends to a Covalink (NUNC) microwell dish and then incubated in the presence of DNA
fragments derived from a PCR (polymerase chain reaction3 amplification of a 475 bp fragment corresponding to a part of the RNA of the AIDS virus (HIVI). The solutions also containing [sic] a 205 bp biotinylated detector 21450~
_ WO 94/06933 - 29 - PCT/BF93/00063 probe which was also complementary to the DNA sequence of the ~IVI virus.
After hybridization at 70c [sic] and washing, a streptavidin/kinase conjugate is incubated with the hybrids and, after washing, the substrates of the kinase and luciferase are added in order to measure the kinase/streptavidin conjugates which is [sic] present in the wells. Figure 9 shows the slope of the increase in light in RLU (Relative Light Unit) observed per hour of incubation of these reagents. The controls were obtained using a 571 bp target DNA derived from the mouse genome.
The tests were carried out in triplicate and represent the mean. The st~n~rd deviations (+/- s) are indicated in the figure. The figure shows that 1 pg of DNA can be detected with significance by this test.
SEQUENCE LISTING 21~ 5 0 9 ~
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: LAMBDATECH S.A.
(B) STREET: LES BEYOLETTES
(C) CITY: FO~T~
(E) COUNTRY: BELGIQUE
(F) POSTAL CODE (ZIP): 6953 (G) TELEPHONE: 3224263810 (H) TELEFAX: 3224263760 (ii) TITLE OF INVENTION: CONJUGATE FOR THE DETECTION AND/OR OF
A BIOLOGICAL COMPOUND
(iii) NUMBER OF SEQUENCES: 20 (iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (EPO) (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) S~Qu~N~ DESCRIPTION: SEQ ID NO: 1:
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) ~Qu~N~ DESCRIPTION: SEQ ID NO: 2:
ACAC~Ll~ll ACAC~l~l~l AATAT 25 (2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs ~ (B) TYPE: nucleic acid 21~ 5 0 9~
_ (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) ~Qu~N~ DESCRIPTION: SEQ ID NO: 3:
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GGTTCGCA~A TCTACGCACG GTCCT 25 (2) INFORMATION FOR SEQ ID NO: 6:
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ATATCAGATG ACGAGAACGA A~ATG 25 (2) INFORMATION FOR SEQ ID NO: 10:
(i) S~U~N~ CHl~RACTERISTICS:
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UACUACAUAC ACCCCCGCAC AGACC 21~ 5 0 9 ~ 25 (2) INFORMATION FOR SEQ ID NO: 15:
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(2) INFORMATION FOR SEQ ID NO: 20:
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Claims (14)
1. Activate conjugate intended for detecting and/or assaying a biological compound, characterized in that it comprises a kinase or a dehydrogenase whose sulfur groups are preferably previously reduced and which is capable of producing an intermediate compound which can be used by a bioluminescence system, preferably by a luciferase, the said enzyme being coupled to a ligand which is previously activated by a coupling agent and which is able specifically to bind, directly or indirectly, to the said biological compound.
2. Conjugate according to Claim 1, characterized in that the kinase is a pyruvate kinase.
3. Conjugate according to Claim 1 or 2, charac-terized in that the coupling agent is heterobifunctional.
4. Conjugate according to Claim 3, characterized in that the coupling agent is N-succinimidyl-3-(2-pyridyldithio) propionate, succinimidyl-4-(N-maleimido-methyl) cyclohexane-l-carboxylate or its sulfo derivative.
5. Conjugate according to any one of the preceding claims, characterized in that the ligand is selected from among the group comprising antigens, hormone receptors, antibodies, nucleotide sequences and compounds capable of binding to biotinylated nucleotide sequences, and/or a mixture thereof.
6. Conjugate according to Claim 5, characterized in that the ligand is avidin or streptavidin, which may, where appropriate, be modified.
7. Conjugate according to one of Claims 5 and 6, characterized in that the nucleotide sequence comprises all or part of a nucleotide sequence selected from among the group comprising the following nucleotide sequences:
the SEQ ID NO. 1 : TGTGCAACAA TGTGCAGACA TTATA
the SEQ ID NO. 2 : ACACGTTGTT ACACGTCTGT AATAT
the SEQ ID NO. 3 : CCAAGCGTTT AGATGCGTGC CAGGA
the SEQ ID NO. 4 : TACTACATAC ACCCCCGCAC AGACC
the SEQ ID NO. 5 : CGTTCGCAAA TCTACGCACG GTCCT
the SEQ ID NO. 6 : ATGATGTATG TGGGGGCGTG TGTGG
the SEQ ID NO. 7 : ACCTTAACTG CAGATGTTAT G
the SEQ ID NO. 8 : TGGAATTGAC GTCTACAATA C
the SEQ ID NO. 9 : ATATCAGATG ACGAACGA AAATG
the SEQ ID.NO, 10 : TATAGTCTAC TGCTCTTGCT TTTAC
the SEQ ID NO. 11 : UGUGCAACAA UGUGCAGACA UUAUA
the SEQ ID NO. 12 : ACACGUUGUU ACACGUCUGU AAUAU
the SEQ ID NO. 13 : CCAAGCGUUU AGAUGCGUGC CAGGA
the SEQ ID NO. 14 : UACUACAUAC ACCCCCGCAC AGACC
the SEQ ID NO. 15 : GGUUCGCAAA UCUACGCACG GUCCU
the SEQ ID NO. 16 : AUGAUGUGUG UGGGGGCGUG UGUGG
the SEQ ID NO. 17 : ACCUUAACUG CAGAUGUUAU G
the SEQ ID NO. 18 : UGGAAUUGAC GUCUACAAUA C
the SEQ ID NO. 19 : AUAUCAGAUG ACGAGAACGA AAAUG
the SEQ ID NO. 20 : UAUAGUCUAC UGCUCUUGCU UUUAC
the SEQ ID NO. 1 : TGTGCAACAA TGTGCAGACA TTATA
the SEQ ID NO. 2 : ACACGTTGTT ACACGTCTGT AATAT
the SEQ ID NO. 3 : CCAAGCGTTT AGATGCGTGC CAGGA
the SEQ ID NO. 4 : TACTACATAC ACCCCCGCAC AGACC
the SEQ ID NO. 5 : CGTTCGCAAA TCTACGCACG GTCCT
the SEQ ID NO. 6 : ATGATGTATG TGGGGGCGTG TGTGG
the SEQ ID NO. 7 : ACCTTAACTG CAGATGTTAT G
the SEQ ID NO. 8 : TGGAATTGAC GTCTACAATA C
the SEQ ID NO. 9 : ATATCAGATG ACGAACGA AAATG
the SEQ ID.NO, 10 : TATAGTCTAC TGCTCTTGCT TTTAC
the SEQ ID NO. 11 : UGUGCAACAA UGUGCAGACA UUAUA
the SEQ ID NO. 12 : ACACGUUGUU ACACGUCUGU AAUAU
the SEQ ID NO. 13 : CCAAGCGUUU AGAUGCGUGC CAGGA
the SEQ ID NO. 14 : UACUACAUAC ACCCCCGCAC AGACC
the SEQ ID NO. 15 : GGUUCGCAAA UCUACGCACG GUCCU
the SEQ ID NO. 16 : AUGAUGUGUG UGGGGGCGUG UGUGG
the SEQ ID NO. 17 : ACCUUAACUG CAGAUGUUAU G
the SEQ ID NO. 18 : UGGAAUUGAC GUCUACAAUA C
the SEQ ID NO. 19 : AUAUCAGAUG ACGAGAACGA AAAUG
the SEQ ID NO. 20 : UAUAGUCUAC UGCUCUUGCU UUUAC
8. Process for preparing the conjugate according to any one of the preceding claims, characterized in that a coupling agent, which is preferably heterobifunctional, is reacted, in solution, with a ligand, and in that this activated ligand is coupled to a kinase or a dehydrogenase, one or more sulfide bridges of which have, where appropriate, been reduced.
9. Process for preparing the conjugate according to Claim 8, characterized in that the kinase or the dehydrogenase of the conjugate is immobilized at its active site on a column containing a gel, preferably Blue Sepharose, possessing an affinity for the kinase or the dehydrogenase, and is coupled to the previously activated ligand by a coupling agent, and in that the column is washed and the conjugate is eluted from the column by changing the composition of the washing solution, prefer-ably by increasing the ionic strength.
10. Application of the conjugate according to any one of Claims 1 to 7 for detecting and/or assaying a biological compound.
11. Application according to Claim 10 for detecting and/or assaying nucleotide sequences by means of hybridization.
12. Application according to Claim 11 for detecting and/or assaying nucleotide sequences which are previously detected by a method selected from among the group comprising in situ hybridization, hybridization on a filter, on plastic, on a solid support, in solution, in a sandwich or on a gel, dot blot, Northern blot or Southern blot hybridization, the use of an isotopic or non-isotopic label attached to the fragment, the use of a cold probe technique or the use of genetic amplifica-tion, especially using PCR or LCR, and/or the use of a mixture thereof.
13. Kit for detecting and/or assaying a biological compound, characterized in that it includes the conjugate according to any one of Claims 1 to 7.
14. Diagnostic kit according to Claim 13, characterized in that it also includes the reagents intended for detecting and/or assaying nucleotide sequences by a method selected from the group comprising in situ hybridization, hybridization on a filter, on a solid support, in solution, in a sandwich or on a gel, dot blot, Northern blot or Southern blot hybridization, the use of an isotopic or non-isotopic label attached to the fragment, the use of a cold probe technique or the use of genetic amplification, especially using PCR or LCR, and/or the use of a mixture thereof.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| BE9200827A BE1006179A3 (en) | 1992-09-22 | 1992-09-22 | Combines for detection and / or biological assay of compound. |
| BE9200827 | 1992-09-22 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2145092A1 true CA2145092A1 (en) | 1994-03-31 |
Family
ID=3886450
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002145092A Abandoned CA2145092A1 (en) | 1992-09-22 | 1993-09-21 | Conjugate for the detection and/or assay of a biological compound |
Country Status (7)
| Country | Link |
|---|---|
| EP (1) | EP0662154B1 (en) |
| JP (1) | JPH08501446A (en) |
| AU (1) | AU4937093A (en) |
| BE (1) | BE1006179A3 (en) |
| CA (1) | CA2145092A1 (en) |
| DE (1) | DE69319127D1 (en) |
| WO (1) | WO1994006933A1 (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE19625137A1 (en) * | 1996-06-24 | 1998-01-08 | Basf Ag | Use of a bioluminescence test for the detection of microorganisms in dispersions which contain polymers and / or pigments |
| BE1011052A3 (en) * | 1997-03-20 | 1999-04-06 | Jose Remacle | Diagnostic and/or assay method and kit using sandwich type hybridisation of nucleic acid sequences on solid substrate |
| GB9902659D0 (en) * | 1999-02-05 | 1999-03-31 | Microbiological Res Authority | Assay with reduced background |
| DE102011077238B4 (en) * | 2011-06-09 | 2016-03-31 | Bayerische Motoren Werke Aktiengesellschaft | Method of detecting microorganisms in a cavity preservative |
| PL235847B1 (en) * | 2014-04-11 | 2020-11-02 | Centrum Onkologii Inst Im Marii Sklodowskiej Curie | Screening examination and method for detection of the presence of oncogenic types of HPV viruses |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DK188184A (en) * | 1983-04-20 | 1984-10-21 | Enzo Biochem Inc | PROCEDURE FOR CREATING A COMPLEX OF BIOLOGICAL ACTIVE OR FUNCTIONAL COMPOUNDS AND USING THE COMPOUNDS |
| ES8607563A1 (en) * | 1983-10-13 | 1986-06-01 | Univ Georgia Res Found | Bioluminescent immunoassays. |
| US4810638A (en) * | 1986-07-24 | 1989-03-07 | Miles Inc. | Enzyme-labeled antibody reagent with polyalkyleneglycol linking group |
| HU204559B (en) * | 1987-08-26 | 1992-01-28 | Akad Wissenschaften Ddr | Process for producing molecular proobes connected with enzymes and detecting biomolecules with them |
| FR2636970B1 (en) * | 1988-09-26 | 1991-11-29 | Inst Nat Sante Rech Med | METHOD FOR DETECTION AND DETERMINATION OF A SPECIFIC SEQUENCE OF DNA OR RNA, REAGENT AND NECESSARY FOR ITS IMPLEMENTATION |
| GB8927365D0 (en) * | 1989-12-04 | 1990-01-31 | Ici Plc | Reagent |
-
1992
- 1992-09-22 BE BE9200827A patent/BE1006179A3/en not_active IP Right Cessation
-
1993
- 1993-09-21 WO PCT/BE1993/000063 patent/WO1994006933A1/en active IP Right Grant
- 1993-09-21 CA CA002145092A patent/CA2145092A1/en not_active Abandoned
- 1993-09-21 EP EP93918817A patent/EP0662154B1/en not_active Expired - Lifetime
- 1993-09-21 JP JP6507616A patent/JPH08501446A/en active Pending
- 1993-09-21 AU AU49370/93A patent/AU4937093A/en not_active Abandoned
- 1993-09-21 DE DE69319127T patent/DE69319127D1/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| AU4937093A (en) | 1994-04-12 |
| DE69319127D1 (en) | 1998-07-16 |
| EP0662154A1 (en) | 1995-07-12 |
| JPH08501446A (en) | 1996-02-20 |
| BE1006179A3 (en) | 1994-05-31 |
| WO1994006933A1 (en) | 1994-03-31 |
| EP0662154B1 (en) | 1998-06-10 |
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