WO1995015497A1 - Dosage immunologique pour la detection du cancer du col de l'uterus - Google Patents

Dosage immunologique pour la detection du cancer du col de l'uterus Download PDF

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WO1995015497A1
WO1995015497A1 PCT/AU1994/000741 AU9400741W WO9515497A1 WO 1995015497 A1 WO1995015497 A1 WO 1995015497A1 AU 9400741 W AU9400741 W AU 9400741W WO 9515497 A1 WO9515497 A1 WO 9515497A1
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sample
protein
hpv
antibody
sds
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PCT/AU1994/000741
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English (en)
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Ian Hector Frazer
Linda Ann Dunn
Robert William Tindle
Germain Jithendra Fernando
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The University Of Queensland
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Priority to AU11035/95A priority Critical patent/AU1103595A/en
Publication of WO1995015497A1 publication Critical patent/WO1995015497A1/fr

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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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57411—Specifically defined cancers of cervix
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3069—Reproductive system, e.g. ovaria, uterus, testes, prostate
    • 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
    • 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

Definitions

  • THIS INVENTION relates to detection of cervical cancer in humans associated with human papilloma virus.
  • the invention relates to the determination of the presence or absence of specific viral antigens in a fluid sample from a patient suspected of suffering from cervical cancer which may be used as the basis for detection of human cervical cancer.
  • papilloma viruses can be classified into several distinct groups based on the host in which they infect.
  • Human papilloma viruses HPV
  • HPV Human papilloma viruses
  • Types 16, 18 and 42 are associated with the majority of in si tu and invasive carcinomas which may occur in the anogenital tract and in particular the cervix.
  • a number of cervical intra epithelial neoplasias and carcinomas of the cervix have been associated with HPV 16 and HPV 18. (Lancaster et al .
  • papilloma viruses are small DNA viruses encoding up to 8 early and 2 late genes.
  • the protein from the expression of the early gene E7 varies between 93 to 127 amino acid residues.
  • the E7 protein is the most abundant viral protein in HPV 16 containing * CaSki and SiHa squamous carcinoma cell lines and in HPV-18 containing HeLa and C4-1 lines. (Seedorf e t al . 1987. EMBO J. 6 , 139).
  • the E7 protein may be recognised by the immune system, since anti E7 antibodies can be detected in the serum of approximately 20% of patients with HPV 16 associated cervical lesions (Jenison e t al . 1988. J. Virol. 62,, 2115; Jochmus-Kudielka e t al . 1989. J. Natl. Cancer Institute 8_, 1698; S illie et al . 1990. Immunol. Infect. Dis. 1, 13).
  • HPV infection is confirmed by histological and cytological criteria or by detection of HPV DNA or RNA (McDougall et al . 1986. In Papillomaviruses, CIBA Foundation Symposium 120. Evered and Clark eds . John Wiley and Sons. Chichester. p86). Tests for detecting antibody against papillomaviruses are difficult and not well standardised. Many have been developed as experimental techniques but none is in routine laboratory use. The basic problem is the lack of standard antigen. Only small amounts of HPV proteins have been isolated from tissues infected with HPV 16 and 18. (Androphy et al . 1987. EMBO J. 6 , 1989; BANKS et al . 1987. J. Gen.
  • Tests for the detection of viral antigens in cells are also typical and suffer from similar problems as tests which detect antibodies.
  • the common tests are indirect immuno fluorescents on frozen sections or the peroxidase-antiperoxidase (i.e. PAP) test on sections from formalin-fixed tissues.
  • Antigens for these tests may be derived from tissue from different HPV lesions or tumours or particles from HPV1 , cotton tail rabbit papillomavirus (CRPV) or bovine papillomavirus (BPV) disrupted by treatment with a detergent.
  • the PAP test uses rabbit anti-HPV antibodies to complex with an antigen in the section followed by the co plexing with a second labelled antibody that is specific for rabbit antibody, that is, for example, goat anti-rabbit antibody linked to an enzyme label such as peroxidase.
  • a second labelled antibody that is specific for rabbit antibody, that is, for example, goat anti-rabbit antibody linked to an enzyme label such as peroxidase.
  • Both groups by ELISA detected reactive monoclonal antibodies which were bound to an antigen comprising the above fusion proteins which antigen was bound to an insert support.
  • the ELISA system used horseradish peroxidase linked sheep anti-mouse Ig as a second antibody wherein the reaction between the peroxidase enzyme and the substrate hydrogen peroxidase was assayed using 0- phenylene diamine as indicator.
  • horseradish peroxidase linked sheep anti-mouse Ig horseradish peroxidase linked sheep anti-mouse Ig as a second antibody wherein the reaction between the peroxidase enzyme and the substrate hydrogen peroxidase was assayed using 0- phenylene diamine as indicator.
  • In the Tindle 1990 reference use was also made of 12:> I conjugated anti- mouse Ig as a second antibody. Both references demonstrated that the majority of raised monoclonal antibodies recognise linear epitopes of the corresponding viral protein.
  • Cervical cancer cells and the epithelial cells of its precursor lesion, cervical intraepithelial neoplasia, generally contain HPV DNA.
  • HPV 16 or closely related genotypes is found in >95% of squamous cervical cancers if a sufficiently sensitive test is employed, whereas adenocarcinomas contain HPV 18 or HPV 16.
  • Epidemiologic evidence supports the hypothesis that HPV 16 and 18 commonly act as initiators of cervical cancer, but measurement of HPV DNA in the cervix has not proved useful as a screening test for risk of cervical cancer, because HPV 16 infection is common in the cervix, and the risk of progression of HPV infection to significant pre alignant disease is less than 1%.
  • HPV E2 protein As the cells mature, trans acting regulatory mechanisms involving the HPV E2 protein normally down regulate E6 and E7 expression, such that little or no message for these proteins is seen in superficial keratinising epithelium.
  • HPV DNA integrates occasionally into the host genome, and integration of HPV DNA with disruption of the E2 open reading frame has been found in all cervical cancers and cancer cell lines examined.
  • ORF HPV E2 open reading frame
  • Premalignant lesions associated with HPV 16 or HPV 18 should therefore have significant levels of E7 protein in superficial cells accessible by cervical scrape, but E7 should not be found in benign lesions associated with episomal HPV 16 or 18 infection, nor in non-HPV related cervical abnormalities.
  • Screening for premalignant cervical lesions is generally carried out by pap smear. While this screening programme is effective at detecting cervical lesions with malignant potential, the overall prevalence of abnormal pap smears is high and there is a significant 'false positive' rate, whether this is defined as the number of women with an abnormal pap smear for whom a repeat smear is negative, or the number for whom a repeat smear is abnormal, but for whom a cervical biopsy shows a lesion without preneoplastic change and for whom no therapy is therefore indicated.
  • the ELISA screening assay for E7 protein of HPV 18 gave encouraging preliminary results, in that cervical cancer cells tested positive, as did a small proportion of samples from women with abnormal pap smears . Reference may also be made to Selvey et al .
  • HPV16 E7 protein at concentrations from 0.10 to 0.69 ng iE7/mg cell protein was detected in 5 of 13 smears from women with abnormal cervical cytology. It was therefore considered that assay of E7 protein may play a role in the detection of HPV-mduced cervical lesions with malignant potential.
  • Tindle et al . 1990 references were nevertheless ineffective as reagents for the capture ELISA assays. The main reason for this result was that presumably many of the linear epitopes for E7 are internal, and antibodies directed against these epitopes do not see E7 in solution in the ELISA.
  • the Selvey et al . reference also established that E7-dependent cell lines contain about 10 "M immunoreactive E7. this level is at the threshold of sensitivity of ELISA assays, and this and the relative instability of ⁇ E7 at room temperature and 4°C presents problems for the measurement of this protein in clinical samples. Attempts to stabilise the E7 by a number of conventional techniques including addition of a wide range of protease inhibitors and manipulation of divalent cations showed no improvement in E7 stability .
  • the present invention therefore provides an assay for determination of E7 protein which includes the following steps:
  • step d) it has been found that in relation to the HPV16 E7 antigen boiling may increase the level of this antigen by a mean of about 100% and has little effect on the half life of the E7 protein.
  • SDS treatment above has little effect on the amount of detectable antigen, but stabilises antigen, prolonging the T** ? of E7 at room temperature; the kinetics of the decay of E7 are first order and the T-J is approximately 10 fold increased at any given E7 concentration and temperature.
  • the invention therefore in another aspect refers to a method of stabilizing HPV E7 protein by the use of step (i) above.
  • the HPV E7 samples may be collected into an SDS vehicle of 0.01-0.1% concentration and boiled for an appropriate time at the point of collection in a simple manner, such that the stablized antigen may then be transported to the laboratory in such a way that transport conditions are not critical.
  • an assay for HPV E7 may be carried out on the same day and at room temperature as opposed to the antigen being snap frozen at -70°C as was the case previously.
  • the sample is preferably boiled for 10-15 mins and also treated with a final concentration of 0.01-0.1% SDS.
  • the final concentration of SDS is preferably from 0.01 to 0.1%. However, boiling is preferably avoided.
  • the sample from the patient is subjected to a mild lysis process whereby any infected cells may be lysed so that minimal disruption occurs.
  • a mild lysis buffer such as a non-ionic detergent.
  • Suitable non-ionic detergents include BRIJ56, BRIJ58, BRIJ35, Triton X-45, Triton X-114, Nonidet P-40, Triton X-100, Triton N-101, Tween 20, Tween 40, Tween 80, and oxtyl-3-glucoside.
  • Nonidet P40 is the preferred non-ionic detergent.
  • the concentration of the non-ionic detergent may vary between 0.01% to 1% in buffered solution. Preferably a 0.1% solution in phosphate buffered saline or other suitable buffer is used.
  • the cells may be subsequently disrupted m a variety of ways but preferably by means of external pressure whereby the sample may be forced under pressure through a small orifice or tubular passage of small cross section such as a hollow bore or cannula of a needle.
  • a French press may be used where large volumes of sample are being processed.
  • the disruption of the cells may occur in the presence of a protease inhibitor such as phenylmethylfluorane sulphonate.
  • the sample may be incubated in boiling water for a suitable time span (e.g. 5 mm-30 nun) and suitably a denaturing agent such as SDS is added which may have the effect of stabilising any E7 protein located in the sample.
  • a denaturing agent such as SDS
  • Any appropriate amount of SDS may be utilised such as 1-20 ⁇ l of 10% SDS which may be added to 500 ml samples for example.
  • the lysed sample may be purified by centrifugation of filtration and especially ultraflltration wherein insoluble matter may be discarded.
  • a first antibody that is specific for the E7 protein and demonstrates high reactivity to the E7 protein.
  • the first antibody is preferably onoclonally derived and is suitably raised against MS2 replicase/E7 fusion protein described above in the Tindle 1990 et al . and Selvey et al . 1990 references.
  • the level of reactivity is determined by the ability with which the antibody may bind to the E7 protein under assay conditions. High reactivity is when significant binding occurs.
  • Selvey et al The abovementioned Selvey et al .
  • Suitable first antibodies of HPV16E7, that is anti-E7 HPV16MAb include 1 OF and 6D referred to in the Tindle et al . 1990 reference above.
  • Suitable first antibodies for HPV18E7, that is anti-E7 HPVI ⁇ MAb include 7E10, 15H4, and monoclonal antibodies that recognise epitopes A, B, C, C0NF1 and CONF2 referred to in the Selvey et al . 1990 reference above.
  • anti-E7 HPV18MAb7E10 is used for the capture of HPV18E7 protein and anti-E7 HPV16MAb1 OF is used for the capture of HPV16E7 protein.
  • the first antibody is preferably bound to a solid support surface.
  • the solid support surface may be of any suitable material including hydrocarbon polymers such as polystyrene, polyethylene and polybutylene; polyesters and polyamides; cellulose and cellulosic derivatives; and vinyl polymers.
  • a preferred form of the solid support surface is a test tube or a microtitre plate well.
  • binding sites on the solid support surface may be blocked by any suitable material but preferably by skim milk powder.
  • the sample from the patient may then be applied to the first antibody in any suitable manner.
  • a second antibody may then be applied in the assay specific for the E7 protein.
  • the second antibody may be onoclonally derived or from polyvalent antiserum.
  • Suitable monoclonal antibodies that can serve as a second antibody are the monoclonal antibodies that recognise the epitopes in epitope C and C0NF2.
  • Suitable polyvalent antiserum may be raised in rabbits against purified or partially purified E7 protein.
  • a source of E7 protein may be obtained from the expression of a recombinant vector which encodes the E7 gene.
  • a suitable E7 protein is the MS2-replicase fusion protein expressed from the MS2/E718 or MS2/E716 vectors discussed previously.
  • rabbit polyvalent antiserum of appropriate specificity is used as the source of the second antibody .
  • the first antibody and the second antibody may be added together in the one reaction mix in order to achieve a simultaneous immunoassay although preferably the antibodies are added in a step-wise addition in order to- achieve a forward immuno assay reaction.
  • a labelled third antibody may then be utilised which may bind to the second antibody.
  • the second antibody may be labelled but the former procedure is preferred for convenience.
  • a suitable label may be selected from a radioactive isotope, an enzyme, or a fluoro etric molecule. Enzyme labels may include catalase, peroxidase, urease, glucose oxidase and alkaline phosphatase.
  • the third antibody is preferably labelled with a peroxidase enzyme label. Standard techniques known to a person expert in the art may be used to determine the concentration of the labelled antibody in the third complex or the unreacted labelled antibody.
  • the first antibody may be selected from any of those mentioned above but preferably anti-E7 HPV18 MAb 7E10 anti-E7 HPV16 MAb 10F.
  • the first antibody is provided in the form of a coating on a solid support such as on the surface of the wells of a microtitre tray.
  • a blocking solution may be optionally provided.
  • the preferred block solution is a 5% skim milk powder in 0.15 molar phosphate buffer saline PH 7.4.
  • An ELISA wash buffer may be optionally provided.
  • the preferred ELISA wash buffer consists of 0.1% skim milk powder and 0.1% tween 20 in phosphate buffer saline.
  • the second antibody may be selected from those mentioned above.
  • HPV 16E7 antigen antibodies of appropriate specificity from polyvalent rabbit anti-E7 sera are preferably used.
  • HPV 18E7 antigen antibodies of appropriate specificity from rabbit polyvalent anti-E7 18 sera are preferably used.
  • the third antibody may consist of any appropriate label but preferably a peroxidase enzyme label.
  • the third antibody is preferably a goat anti- rabbit immunoglobulin coupled to horseradish peroxidase.
  • the second antibody is derived from a mice or mice cell line, then the third antibody may be rabbit anti-mice immunoglobulin coupled to horseradish peroxidase.
  • a OPD substrate buffer may be optionally provided.
  • the preferred OPD substrate buffer comprises of 0.012% hydrogen peroxide, 0.2M o-phenylenediamine, 25 mM citric acid, and 50 mM disodium hydrogen phosphate PH 5.0.
  • a stop solution may be optionally provided.
  • the preferred stop solution is 2.5 M HC1.
  • the scope of the present invention includes the detection and determination of E7 antigens from other papilloma viruses and that types HPV 16 and HPV 18 are used here by way of example.
  • the assay procedure of the invention may be utilised in regard to not only a forward type reaction in which the antibody bound to the solid phase is first contacted with the sample so as to form a solid phase antibody :antigen complex and then subsequently detected with a second (or third) labelled antibody; but also a simultaneous type reaction which involves a single incubation step where the antibody bound to the solid support and the labelled antibody are both added to the sample at the same time.
  • the present invention may be further described in the following preferred embodiment which relates to an appropriate experimental procedure. Having regard to the foregoing, the assay of the present invention reduces the cost of screening for cervical cancer by allowing simple further evaluation by a medical officer who first performs the test of the malignant potential of an abnormal pap smear.
  • the assay of the present invention allows for evaluation of abnormal pap smears for malignant potential by a further cervical scrape at the time of the repeat pap smear, and only those women with a positive screening test for malignant potential would require further specialist evaluation.
  • This may reduce the cost and increase the acceptability of pap smear programmes and thus may also indirectly increase uptake of the programme.
  • the assay of the invention may also involve collection of one extra cervical scrape from women with known abnormal pap smears who are undergoing routine evaluation of their lesion by a gynaecologist. It is clear that no extra discomfort is envisaged.
  • the assay of the invention may be utilised as a confirmatory test for the diagnosis of cervical disease of significant malignant potential.
  • the assays of the invention are reproducible, sensitive and HPV genotype specific and give positive results with cervix cancer cells but negative results with non-cervical tissues.
  • the assays of the invention may also be used as confirmatory assays in the field, using patients with abnormal pap smears who are to proceed to biopsy for confirmation of the nature of their disease.
  • Tissue samples collected are immediately snap frozen in a dry ice/ethanol bath or in liquid nitrogen after which they are crushed with a mortar and pestel or tissue homogeniser, resuspended in 500 ⁇ l of lysis buffer and snap frozen again.
  • Samples are thawed on ice and if possible passed through a 27 gauge needle five times. Samples are then spun down (top speed microfuge for 5 min) and 30 ⁇ l removed from the supernatant into a separate tube for total protein assay.
  • the monoclonal 6D is preferably used whereas for the detection of HPV18E7 the monoclonal 7E10 is preferably used.
  • 16 and 18E7MS2 fusion protein (alternatively 16E7GST fusion protein or the cleaved E7 derived therefrom) as positive and negative controls and for the production of a standard curve.
  • 16E7MS2 fusion protein (alternatively 16E7GST fusion protein or the cleaved E7 derived therefrom) as positive and negative controls and for the production of a standard curve.
  • 16E7MS2 fusion protein as the cross fusion protein and zero value for 16E7, diluted 1 :1000 in PBS.
  • the 16E7 fusion protein curve should cross the y axis at an OD point approximately equal to the mean of the negative control values (distilled water treated with
  • the ng E7/mg total protein of these positive samples can then be calculated by dividing the value obtained for 16E7 mg/ml by total protein mg/ml.
  • Example 1 A sample of Caski cells grown m vi tro under conventional conditions was taken and subjected to the assay outlined in Example 1. Caski cells have been known to contain HPV16E7. The results of the experiment are given in Tables 1 and 2 and FIGS. 1 and 2. Bovine serum albumin (BSA) was used as the total protein standard.
  • BSA Bovine serum albumin
  • the fusion protein 16E7MS2 was used as the standard for E7 ng/ml determinations. When using 16E7MS2 (or 18E7MS2) as an E7 standard only half the total protein concentration is taken into account as approximately only half of the fusion protein is E7.
  • the total protein concentration of 16E7MS2 is 600 ⁇ g/ml, only a figure of 300 ⁇ g/ml is used with respect to E7 ng/ml.
  • 18E7MS2 is used as a control for the E7 ng/ml determinations and is the zero or base line value as there is no cross reactivity with the anti-16E7 monoclonal antibody. (Alternatively any protein that does not cross react with the anti-16E7 monoclonal antibody, may be used as the control . )
  • the OD reading at 540 nm of the Caski cell sample prepared for total protein determination was 0.429 which from the total protein standard curve (FIG. 1 ) gave a total protein concentration of 1.95 mg/ml.
  • the OD reading at 492 nm of the Caski cell sample prepared for 16E7 ng/ml determination was 0.688 which from the 16E7 standard curve (FIG. 2) gave a 16E7 concentration of 41.75 ng 16E7/ml.
  • the amount of 16E7 per total protein in the Caski cell sample was 21.41 mgE7 per mg total protein.
  • the mean OD reading at 492 nm of the negative controls plus two standard deviations was 0.023.
  • a cervical scrape sample was obtained from a human subject and assayed in accordance with the assay in Example 1. Standard total protein and 16E7 values were determined and appropriate standard curves were prepared. The values of the standards and the standard curves were substantially identical to those given in Example 2 (Tables 1 and 2; FIGS. 1 and 2).
  • the OD reading at 540 nm of the cervical scrape sample prepared for total protein determination was 0.042 which from the total protein standard curve gave a total protein concentration in the sample of 0.107 mg/ml.
  • the OD reading at 492 nm of the cervical scrape sample prepared for 16E7 concentration determination was 0.034 which from the 16E7 standard curve gave an 16E7 concentration of 0.252 ng/ml.
  • the amount of 16E7 per total protein in the cervical scrape sample was 2.36 16E7 ng/total protein mg.
  • the mean OD at 492 nm of the 18E7 MS2 plus two standard deviations was 0.037. As this value was close to the OD 492 value for the cervical scrape sample (i.e. 0.034), it was considered worthwhile to re-examine the subject for the presence of 16E7.
  • EXAMPLE 4 Determina tion of E7 assay condi tions
  • Caski cell samples were used.
  • the monoclonal antibody 6D specific for 16E7 was used.
  • Caski cell lysates were syringed and boiled prior to assay . (i) kept at room temp for 3 hrs then stored at
  • Hela and STO cell lysates with combination of SDS and boiling (final concentration of SDS is given as a percentage).
  • Hela cells have been shown to contain 18E7 protein.
  • the monoclonal antibody 7E10, specific for 18E7 was used.
  • the fusion protein (FP) used was 18E7MS2. Boiling was for approximately 10 mins. STO cells do not contain E7 protein.
  • FIG. 2 16E7 concentration standard curve

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Abstract

L'invention concerne une méthode pour stabiliser la protéine codée par le gène E7 du virus du papillome humain, consistant à faire bouillir l'échantillon et/ou à traiter l'échantillon avec un agent dénaturant qui peut être le dodécylsulfate de sodium, lequel peut également être utilisé à une concentration finale entre 0,01 et 0,1 %. L'échantillon peut également être bouilli pendant 10-15 minutes avant le traitement par le dodécylsulfate de sodium.
PCT/AU1994/000741 1993-11-30 1994-11-30 Dosage immunologique pour la detection du cancer du col de l'uterus WO1995015497A1 (fr)

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AU11035/95A AU1103595A (en) 1993-11-30 1994-11-30 Immunoassay for cervical cancer

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AUPM2706 1993-11-30

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997002491A1 (fr) * 1995-07-04 1997-01-23 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Procede pour deceler des anticorps specifiques diriges contre des proteines hpv
WO2005008246A1 (fr) * 2003-07-16 2005-01-27 Joint-Stock Company Ltd. 'melofarma' Procede de diagnostic in vitro pour detecter, a un stade precoce, des dysplasies cervicales et des cancers du col de l'uterus associes a des hpv

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS55136233A (en) * 1979-04-11 1980-10-23 Green Cross Corp:The Hb peptide vaccine
WO1982004396A1 (fr) * 1981-06-19 1982-12-23 Russell Roy Robert Baird Protection contre la carie dentaire
EP0495401A1 (fr) * 1991-01-17 1992-07-22 Kao Corporation Protéinase alkaline et son procédé de production

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS55136233A (en) * 1979-04-11 1980-10-23 Green Cross Corp:The Hb peptide vaccine
WO1982004396A1 (fr) * 1981-06-19 1982-12-23 Russell Roy Robert Baird Protection contre la carie dentaire
EP0495401A1 (fr) * 1991-01-17 1992-07-22 Kao Corporation Protéinase alkaline et son procédé de production

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997002491A1 (fr) * 1995-07-04 1997-01-23 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Procede pour deceler des anticorps specifiques diriges contre des proteines hpv
US6214541B1 (en) 1995-07-04 2001-04-10 Deutsches Krebsforschungszentrum Stiftung Des Offentlichen Rechts Process for detecting specific antibodies acting against HPV proteins
WO2005008246A1 (fr) * 2003-07-16 2005-01-27 Joint-Stock Company Ltd. 'melofarma' Procede de diagnostic in vitro pour detecter, a un stade precoce, des dysplasies cervicales et des cancers du col de l'uterus associes a des hpv

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