EP0848822A1 - Procede pour deceler des anticorps specifiques diriges contre des proteines hpv - Google Patents

Procede pour deceler des anticorps specifiques diriges contre des proteines hpv

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Publication number
EP0848822A1
EP0848822A1 EP96921890A EP96921890A EP0848822A1 EP 0848822 A1 EP0848822 A1 EP 0848822A1 EP 96921890 A EP96921890 A EP 96921890A EP 96921890 A EP96921890 A EP 96921890A EP 0848822 A1 EP0848822 A1 EP 0848822A1
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EP
European Patent Office
Prior art keywords
hpv
antibodies
protein
buffer
proteins
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP96921890A
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German (de)
English (en)
Inventor
Hanswalter Zentgraf
Manfred Frey
Iris Velhagen
Regina Martens
Wolfgang Meschede
Michael Pawlita
Joris Braspenning
Massimo Tommasino
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Deutsches Krebsforschungszentrum DKFZ
Original Assignee
Deutsches Krebsforschungszentrum DKFZ
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Publication of EP0848822A1 publication Critical patent/EP0848822A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/01—DNA viruses
    • G01N2333/025—Papovaviridae, e.g. papillomavirus, polyomavirus, SV40, BK virus, JC virus
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/815—Test for named compound or class of compounds
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
    • Y10S530/82—Proteins from microorganisms
    • Y10S530/826—Viruses

Definitions

  • the invention relates to a method for the detection of specific antibodies directed against HPV proteins in body fluids.
  • the invention also relates to a kit that can be used for this purpose.
  • the invention relates to native HPV proteins which are suitable for carrying out the method according to the invention.
  • HPVs human papilioma virus infections
  • the detection of such an expression could therefore be a way to detect HPV-associated carcinomas at an early stage.
  • the object of the present invention is therefore to provide a method with which the uncontrolled expression of HPV genes, in particular genes E6 and / or E7, can be detected.
  • the invention thus relates to a method for the detection of specific antibodies directed against HPV proteins, which comprises the following method steps:
  • the method according to the invention is based on the knowledge of the applicant that antibodies which are directed against the expression products of these genes often exist in people in whom there is uncontrolled expression of HPV genes, in particular genes E6 and / or E7.
  • HPV protein encompasses any protein of any HPV type.
  • the expression relates to an HPV-E6 and HPV-E7 protein, very particularly of HPV 1 6 and HPV 1 8.
  • the HPV protein can have a wild-type sequence. It can also have a sequence that deviates therefrom, wherein the deviations can take the form of additions, deletions and / or substitutions of one or more amino acids.
  • the HPV protein can be part of a fusion protein.
  • HPV protein Conventional methods can be used to produce an HPV protein. It is favorable to use a nucleic acid coding for an HPV protein, in particular a DNA to insert into an expression vector and to use this for the transfection or transformation of host cells.
  • HPV-E6 and HPV-E7 protein in particular of HPV 1 6 and HPV 1 8, he also knows the following: EMBL database, AC K0271 8 for HPV 1 6; Swiss protein database P031 26 for HPV 1 6-E6; Swiss protein database P031 29 and Durst, M. et al., Proc. Natl. Acad. Be.
  • Expression vectors are also known to the person skilled in the art.
  • these are e.g. pGEMEX, pUC derivatives, pGEX-2T, pET3b and pQE-8, the latter being preferred.
  • yeast these are e.g. pY100, Ycpad I and pREP-L20, the latter being preferred.
  • pAcSGHisNT-A is particularly suitable for expression in insect cells.
  • host cells examples include the E. coli strains HB101, DH 1, x1 776, JM 101, JM 109, BL21 and SG 1 3009, the latter being preferred, the yeast strains Saccharomyces cerevisiae and Saccharomyces Pombe, the latter strain is preferred, and the animal cells L, NIH 3T3, FM3A, CHO, COS, Vero and HeLa and the insect cells sf9.
  • HPV proteins in particular an HPV-E6 and / or HPV-E7 protein, very particularly of HPV 16 and HPV 18, can be used simultaneously or in succession. They are in native form. All natural epitopes of specific antibodies lying on the HPV proteins are thus provided. This optimizes the detection of specific antibodies against HPV proteins in denatured form.
  • an HPV protein in its native form can be refolded by conventional methods if it is in denatured form. It is beneficial to the denatured protein in a urea buffer, e.g. 10mM Na phosphate, pH 7.4, 10% glycerol, 2mM DTT, 8M urea, on a standard hydroxyapatite column, e.g. HA Ultrogel, IBF Biotechnics, with a conventional elution buffer of the above urea molarity, e.g.
  • a urea buffer e.g. 10mM Na phosphate, pH 7.4, 10% glycerol, 2mM DTT, 8M urea
  • a standard hydroxyapatite column e.g. HA Ultrogel, IBF Biotechnics
  • a conventional elution buffer of the above urea molarity e.g.
  • a urea buffer for example 100 mM phosphate buffer, pH 8.0, 8M urea
  • stepwise dialysis against conventional dialysis buffer for example 50 mM pug / NaOH, pH 7.8, 500 mM NaCl, 20% glycerol, 5 mM DTT, 1 00 ⁇ M ZnCl 2 .
  • the dialysis buffers have decreasing urea molarities, for example 8M, 3M, IM or OM. Dialysis is carried out in the usual way, for example within 24 hours.
  • HPV protein After the last dialysis, a refolded HPV protein is obtained, which in the usual way, for example by gel filtration, in particular on Superdex 200 (Pharmacia) is collected.
  • An HPV protein that has been refolded in the above manner is also the subject of the present invention.
  • an HPV protein is bound to a carrier material.
  • Any material suitable for binding proteins in particular microtiter plates, tubes, microspheres and slides, can be used as such.
  • the binding between the HPV protein and the carrier material can be carried out by customary methods. It is favorable if the HPV protein together with a C-terminus, e.g. 1 1 amino acids of SV40t antigen-forming tag polypeptide is present as a fusion protein, as a result of which this can be bound to the carrier material via an antibody which is generally available and which is directed against the tag polypeptide.
  • an HPV protein bound to a carrier material is incubated with body fluids.
  • body fluids can be obtained from an animal body, in particular a mammal and very particularly a human being.
  • the fluids preferably include serum, lymph, saliva, sputum, urine, stool, cerebrospinal fluid, bile and gastrointestinal secretions. They also include liquids made from solid tissues such as lungs, brain and bone marrow, smears and biopsies, and tumors, e.g. Anogenital carcinomas, can be isolated.
  • the HPV protein can be incubated with the body fluids by customary methods.
  • Antibodies which are specific for an HPV protein are bound to this by the above incubation. Such antibodies (hereinafter referred to as (a)) are then labeled with antibodies (b) directed against the antibodies (a), or with unlabelled antibodies (b) and the latter with labeled antibodies directed against the antibodies (b) ( c) implemented.
  • the label can be radioactive or non-radioactive. In the latter case other common markers are used. Fluorescent dyes such as fluorescein isothiocyanate and enzymes such as alkaline phosphatase or peroxidase are particularly suitable. A biotin / streptavidin complex can be used as the enhancer system.
  • the markers are generally available. The conjugation with the antibodies (b) or (c) takes place according to the manufacturer's instructions. Labeled antibodies (b) and (c) are also generally available.
  • suitable antibodies (b) whether labeled or unlabeled, depends on the animal or animal species from which the body fluid used originates. If, for example, it is a liquid from a human, then antibodies (b) which are directed against human immunoglobulin are used. If antibodies (c) are additionally used, these are selected in a corresponding manner with regard to the animal or the animal species from which the antibodies (b) originate.
  • suitable antibodies is known to the person skilled in the art and can be carried out without further ado.
  • Bound antibodies (a) can be reacted with labeled antibodies (b) or with unlabeled antibodies (b) and then with labeled antibodies (c) in the usual way. It is favorable to allow the reaction with the antibodies (b) in both alternatives to take place within 1 h at 37 ° C. After several washes, a substrate solution corresponding to the marker is then added in the first alternative to develop the detection reaction. This is done according to the manufacturer's instructions. In the second alternative, the antibodies (c) are added after the washing processes. Their implementation and the development of the detection reaction take place in a corresponding manner.
  • the method according to the invention has a high specificity. This is particularly high if an HPV-tag fusion protein is used in process step (I). Furthermore, the method according to the invention has a high sensitivity. This is particularly high if, in process step (II), the antibodies (c) be used. Furthermore, it is often advantageous if one or more denatured HPV proteins and / or one or more fragments thereof are additionally used in process step (I). In this way, specific antibodies can be detected which are directed, for example, against degradation products of an HPV protein.
  • kit is also provided which is suitable for carrying out the above method.
  • This kit preferably contains
  • one or more native, carrier-bound HPV proteins optionally one or more denatured, carrier-bound HPV proteins and / or fragments thereof, and labeled antibodies (b) according to claim 1 and conventional washing buffers and optionally one of the brands Appropriate substrate, or one or more native, carrier-bound HPV proteins, optionally one or more denatured, carrier-bound HPV proteins and / or fragments thereof and unlabeled antibodies (b) and labeled antibodies (c) according to claim 1 as well as usual wax buffers and possibly a substrate corresponding to the marking.
  • HPV genes in particular genes E6 and / or E7.
  • the invention is therefore suitable for early detection of HPV-associated carcinomas.
  • Example 1 Production of an HPV 16-E7 protein and an HPV 16-E7 tag fusion protein
  • the original DNA of HPV 16 was assumed (cf. Durst M. et al., Above). This DNA was used as a template for a PCR method.
  • the primer pair used was: 5'-TTTGGATCCATGCATGG-AGATACACCTACATTG-3 'and 5'-TTTGTCGACTTATGGTTTCTGAGA-3'.
  • the PCR approach and conditions were standard.
  • the amplified DNA was digested with BamHI and Sall and inserted into the yeast shuttle vector pREP-L20 opened with BamHI and Sall.
  • This vector corresponds to pREP3 (cf. Maundrell, K., Gene 1 23 (1 993), 1 27-1 30), but contains the following "multiple cloning site":
  • the expression plasmid pREP3-L20 HPV1 6-E7 was obtained. This was used for the transfection of Schizosaccharomyces Pombe LEU 1, 32 (cf. Broker, M., Biotechniques, (1 993), 1 1 9). The transfected yeast cells were cultured overnight in a conventional "pombe min” medium in the presence of thiamine. This suppressed the "no message thiamine" promoter of the above expression plasmid. After washing away the thiamine and culturing in thiamine-free medium, the promoter was switched on and the HPV 16-E7 gene was expressed. The HPV1 6-E7 protein was isolated after mechanical disruption of the yeast cells.
  • the original DNA of HPV 16 was assumed (cf. Durst, M. et al., Above). This DNA was used as a template for a PCR method.
  • the primer pair used was: 5'-TTTTCTAGAA-GATCTATGCATGGAGATACACCT-3 'and 5'-TTTGGATCCTGGTTTCT-GAGAACA-3.
  • the PCR approach and the conditions were standard.
  • the amplified DNA was digested with BglII and BamHI and inserted into the Bluescript vector pL441, which was opened with BglII and BamHI and encodes a tag polypeptide.
  • the DNA molecule obtained was cleaved with Xbal and Sall and the HPV 16-E7 tag DNA was isolated.
  • This DNA was inserted into the expression vector pREP-L20 (see above) opened with Xbal and Sall.
  • the expression plasmid pREP-L20 HPV 1-6-7 days was obtained. This was used for the transfection of Schizosaccharomyces Pombe Leu 1.32 (see above).
  • the cultivation of the yeast cells and the expression of the HPV 16-E7 tag gene and the isolation of the HPV 16-E7 tag fusion protein were carried out as described in FIG. 1 (a).
  • Example 2 Refolding of an HPV 16-E7 protein and an HPV 16-E7 fusion protein
  • HPV 16-E7 protein obtained in Example 1 (a) was in insoluble, denatured form. It was dissolved in a buffer, 25 mM Tris-HCl, pH 8.0, 10% glycerol, 2 mM DTT, 6 M guanidine hydrochloride, 50 mM NaF, 0.1 mM Na-o-yanadate. To refold it, it was diluted 1:12 in a urea buffer, 10 mM Na phosphate, pH 7.4, 10% glycerol, 2 mM DTT, 8 M urea and onto a hydroxylapatite column (HA Ultragel, IBF Biotechnics).
  • HPV protein was treated with a urea elution buffer, 1 50 mM NaPi, pH 7.8, 10% glycerol, 2mM DTT, 8 M urea, eluted and dialyzed against a urea dialysis buffer, 50 mM Tris-HCl, pH 7.5, 0.1 mM ZnAc, 1 mM DTT, 50 mM NaCl, 4 M urea.
  • the dialysate was loaded onto an anion exchanger, Q-Sepharose (Pharmacia), and the HPV protein with a urea dialysis buffer, 50 mM Tris-HCl, pH 7.5, 0.1 mM ZnAc, 1 mM DTT , 1 M NaCl, 4 M urea, eluted.
  • the eluate was subjected to conventional gel filtration and then dialyzed against a dialysis buffer, 5 mM Hepes, 5% glycerol, 1 mM DTT, 20 ⁇ M ZnAc.
  • a native HPV 16-E7 protein was obtained.
  • Example 1 Refolding of the denatured HPV 16-E7 tag fusion protein obtained in Example 1 (b) was carried out as described in Example 2 (a). A native HPV 1 6-E7 tag fusion protein was obtained.
  • the HPV 16-E7 protein solution from Example 2 (a) was diluted in a carbonate buffer, pH 9.6.
  • a carbonate buffer pH 9.6.
  • 100 ng of the HPV protein and once carbonate buffer were added as an empty control per well.
  • 6 short washing steps with PBS, 0.05% Tween 20 followed.
  • the free binding sites of the polymeric carrier were then blocked by incubation for one hour with an irrelevant protein, for example pig skin gelatin, BSA or casein, the latter being preferred, in PBS at 37 ° C.
  • Sera from patients with cervical carcinoma and from controls from Mexico were incubated in PBS (1:50 dilution) for 1 hour at 37 ° C on the plate.
  • TMB developing solution 50 mM sodium acetate, 0.4 mM 3,3 ', 5,5'-tetramethyl-benzidine-dihydrochloride, 4, 4 mM H 2 0 2
  • HPV 16-E7 protein can detect HPV-specific antibodies in sera from cervical carcinoma patients.
  • the plate was incubated with the antibody dissolved in the above carbonate buffer overnight at 4 ° C. After six short washing steps with PBS, 0.05% Tween 20, the HPV 16-E7 tag protein dissolved in the above carbonate buffer was added in an amount of 100 ng per hole. The further process steps were carried out as described in Example 3 (a).
  • HPV-specific antibodies can be detected in sera from cervical carcinoma patients by means of an HPV 16-E7 tag protein.
  • Example 3 (a) and (b) Both detections in Example 3 (a) and (b) were more specific than a comparative ELISA in which an HPV 1 6-E7 fragment, ie no native protein, was used. The specificity of the detection of 3 (b) was even greater than that of the detection of 3 (a). In 3 (a), 64 positive values were obtained in 64 control values, while in 3 (b) all of these control values were negative. Furthermore, in 3 (b) 28 of 73 sera from the cervical carcinoma patients could be recognized as HPV-specific, while in 3 (a) there were only 20.
  • the plasmid pGem-2/1 6 E6 was started, which contains a DNA coding for the E6 protein of HPV 16 (cf. Werness, BA et al., Science, Volume 248, (1 990), 76-79) .
  • This DNA was used as a template for a PCR method.
  • the following was used as a primer pair: 5'-CAGGGATCCGATGACGATGACAAAATGTTTCAGGACCCACAGG-3 'and 5'-GGGAAGCTTATTACAGCTGGGTTTCTCTAC-3'.
  • the PCR approach and the PCR conditions were as follows:
  • the amplified DNA was digested with BamHI and HindIII and inserted into the expression vector pQE-8 (Diagen) opened with BamHI and HindIII.
  • the expression plasmid pQ / 16 / E6 was obtained. This codes for a fusion protein composed of 6 histidine residues and an enterokinase interface (N-terminus partner) and the E6 protein of HPV 16 (C-terminus partner).
  • pQ / 16 / E6 was used to transform E.coli SG 13009 (see Gottesman, S. et al., J. Bacteriol. 148, (1 981), 265-273).
  • the bacteria were cultivated in an LB medium with 100 ⁇ g / ml ampicillin and 25 / yg / ml kanamycin and induced for 4 h with 60 // M isopropyl- ⁇ -D-thiogalactopyranoside (IPTG). Lysis of the bacteria was achieved by adding 6 M guanidine hydrochloride, then chromatography (Ni-NTA resin) was carried out with the lysate in the presence of 8 M urea in accordance with the manufacturer's (Diagen) instructions for the chromatography material. The bound fusion protein was eluted in a buffer at pH 3.5.
  • the fusion protein was subjected to 1 8% SDS-polyacrylamide gel electrophoresis and stained with Coomassie blue (cf. Thomas, JO and Kornberg, RD, J. Mol. Biol. 149 (1 975), 709 -733).
  • HPV1 6-E6 fusion protein (MW approx. 18 kD) was obtained. This was in denatured form.
  • HPV 1 6-E7 and HPV 1 8-E6 (E7) proteins were produced and purified as described in FIG. 4 (a). The following deviations were guided:
  • the plasmid pWV 2916 which contains a DNA coding for HPV 16, was used to produce HPV 16-E7 (cf. Durst, M. et al., Proc. Natl. Acad. Sei. USA, Volume 60, (1983), 3812-3815).
  • the primer pair used for the PCR method 5'-CAGGGATCCATGC-ATGGAGATACACCTAC-3 'and 5'-GGGAAGCTTATTATGGTTTCTGA-GAACAGATG-3'.
  • HPV 18-E7 the plasmid pGEM 3/91 was used, which contains a DNA coding for HPV 18 (cf. Roggenbuck, B. et al., J. Virol., Volume 65, (1991), 5068-5072).
  • the primer pair used for the PCR method 5'-CAGGGATCCATGCATGG-ACCTAAGGCAAC-3 'and 5'-GGGAAGCTTATTACTGCTGGGATGCA-CACC-3'
  • pQE-8 pQ / 1 8 / E7
  • SG 1 3009 a pure HPV 1 8-E7 fusion protein (MW approx. 1 3 kD) received. This was in denatured form.
  • the HPV 16-E6 protein obtained in Example 4 (a) was dissolved in a urea buffer (100 mM phosphate buffer, 8M urea, pH 8.0) and subjected to gradual dialysis.
  • the dialysis buffers contained 50 mM pug / NaOH, pH 7.8, 500 mM NaCl, 20% glycerol, 5 mM DTT, 100 ⁇ M ZnCl 2 . They also had 8M, 3M, 1M and 0M urea, respectively.
  • the individual dialyses were carried out within 24 hours. Gel filtration was then carried out on Superdex 200 (Pharmacia).
  • HPV 16 - E6 protein was obtained in native form.
  • HPV 16-E7, HPV 18-E6 (E7) proteins obtained in Example 4 (b) were refolded as described in FIG. 5 (a). Corresponding HPV proteins were obtained in native form.
  • Example 6 Detection of HPV-E6 and HPV-E7 specific antibodies in the serum of patients
  • HPV 1 6-E6 and HPV 1 6-E7 proteins from Example 5 were taken up in buffer (0.1 M NaH 2 PO 4 , pH 8.0).
  • buffer 0.1 M NaH 2 PO 4 , pH 8.0.
  • 100 ⁇ l of 20 ng or 8 ng of the HPV proteins and 1% of BSA as an empty control were pipetted into each hole. After incubation Overnight at 4 ° C followed by 4 short washing steps with PBS, 0.05% Tween 20 every 5 min. Free binding sites of the polymeric support were then blocked by overnight incubation with 1% BSA in PBS, 0.05% Tween 20 at 4 ° C.
  • a serum to be tested from a patient with cervical cancer was incubated in a 1: 100 dilution (PBS, 1% BSA, 0.05% Tween 20) on the plate for 1 hour at 37 ° C. ("checkerboard titration"). After 4 short washing steps with PBS, 0.05% Tween 20 at intervals of 5 min, a generally available peroxidase-coupled goat anti-human antibody (dilution according to the manufacturer) was added.
  • HPV-E6 and HPV-E7 specific antibodies can be detected in the serum of a patient with cervical cancer.

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Abstract

L'invention concerne un procédé pour déceler des anticorps spécifiques dirigés contre les protéines HPV et contenus dans le volume humoral. Ce procédé comporte les étapes suivantes: (I) incubation d'une protéine HPV native, liée à un matériau support, dans le volume humoral, et (II) réaction d'anticorps (a) spécifiques liés à la protéine HPV: - avec les anticorps (b) marqués, dirigés contre les anticorps (a) ou - avec les anticorps (b) non marqués et de ces derniers avec les anticorps (c) marqués, dirigés contre les anticorps (b). L'invention concerne également un kit pour mettre en oeuvre ce procédé.
EP96921890A 1995-07-04 1996-07-04 Procede pour deceler des anticorps specifiques diriges contre des proteines hpv Withdrawn EP0848822A1 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
DE19524346 1995-07-04
DE19524347 1995-07-04
DE19524347 1995-07-04
DE19524346 1995-07-04
PCT/DE1996/001195 WO1997002491A1 (fr) 1995-07-04 1996-07-04 Procede pour deceler des anticorps specifiques diriges contre des proteines hpv

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EP0848822A1 true EP0848822A1 (fr) 1998-06-24

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EP96921890A Withdrawn EP0848822A1 (fr) 1995-07-04 1996-07-04 Procede pour deceler des anticorps specifiques diriges contre des proteines hpv

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US (1) US6214541B1 (fr)
EP (1) EP0848822A1 (fr)
JP (1) JPH11508685A (fr)
DE (1) DE19627031C2 (fr)
WO (1) WO1997002491A1 (fr)

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DE19649604C1 (de) * 1996-11-29 1997-09-25 Deutsches Krebsforsch Verfahren zur Reinigung und Rückfaltung von HPV-Proteinen
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JPH11508685A (ja) 1999-07-27
US6214541B1 (en) 2001-04-10
DE19627031A1 (de) 1997-01-09
WO1997002491A1 (fr) 1997-01-23
DE19627031C2 (de) 1998-07-02

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