J Clin Pathol 1990;43:488-492

Production and characterisation of a monoclonalantibody to human papillomavirus type 16 usingrecombinant vaccinia virus

C S McLean, M J Churcher, J Meinke, G L Smith, G Higgins, M Stanley, A C Minson

AbstractA monoclonal antibody was raisedagainst the major capsid protein LI ofhuman papillomavirus type 16, using arecombinant vaccinia virus that ex-presses the LI protein, as a target forscreening. This antibody, designatedCAMVIR-1, reacted with a 56 kilodaltonprotein in cells infected with LI-vacciniavirus, and the protein was present in apredominantly nuclear location. Theantibody also detects the HPV-16 Liantigen in formalin fixed, paraffin waxembedded biopsy specimens and onroutine cervical smears. The antibodyreacts strongly and consistently withbiopsy specimens containing HPV-16 orHPV-33, but very weak reactions wereoccasionally observed with biopsyspecimens or smears containing HPV-6or HPV- 1.The potential advantages of using a

vaccinia recombinant are (i) the targetprotein is synthesised in a eukoryotic cellso that its "processing" and location arenormal; (ii) cells infected with vacciniarecombinants can be subjected to variousfixing procedures similar to those usedfor routine clinical material. This greatlyincreases the probability that an identi-fied antibody will be useful in a clinicalsetting.

subjects. The ubiquity of viral DNA highlightsthe need for reagents that will detect viralproteins. Several groups have recently tried toovercome the problems associated with the lackof a permissive in vitro culture system bycloning and expressing viral genes in prok-aryotic and eukaryotic expression systems."'2In an earlier paper we reported the construc-tion of a vaccinia virus recombinant containingthe HPV 16 Li gene (encoding the majorcapsid antigen), and the preparation of poly-clonal antisera raised against a bacterial fusionprotein containing HPV 16 LI sequences.'3Polyclonal antisera, however, are of limited usein that they cannot give information about thepresence of type specific epitopes on the LIprotein and tend to result in high backgroundin immunohistochemical and immunoprecipi-tation studies. There is a need for type specificreagents for diagnostic as well as for researchpurposes for viruses such as HPV 16, which hasbeen identified in 50-600/o of cases of cervicalcarcinoma subjected to viral typing.5

In this report we describe the production of amonoclonal antibody to HPV 16 L1 using anovel protocol in which viral antigen is gen-erated for immunisation using a bacterialexpression system and hybridoma supernantsare screened for specific antibody using avaccinia recombinant expressing the targetprotein.

Department ofPathology, Divisionsof Cellular Pathologyand Virology,University ofCambridgeC S McLeanM J ChurcherJ MeinkeG L SmithM StanleyA C MinsonDivision of Virology,Institute of Medicaland VeterinarySciences, Adelaide,South AustraliaG HigginsCorrespondence to:Dr C SMcLean,Department of Pathology,University of Cambridge,Cambridge CB2 IQP

Accepted for publication15 February 1990

Human papillomaviruses (HPV) are smallDNA viruses which cause a variety of pro-liferative lesions on epithelial or mucosal sur-

faces.' More than 50 HPV types have now beenidentified, and several of these are associatedwith premalignant and malignant disease of theanogenital region. In particular, HPV 6 and 11

have been identified in benign lesions; HPV 16,18, 31 and 33 are highly significantly associatedwith all grades of cervical intraepithelial neo-plasia (CIN) and cervical cancer.2 3There is no suitable tissue culture system for

the propagation of these viruses and the iden-tification of viral DNA and classification ofviruses is based on DNA homology usinghybridisation techniques.4' Viral DNAsequences have been identified and typed inabnormal cervical lesions and in cervicalsmears using blot hybridisation techniques,and more recently by the polymerase chainreaction.7 The exquisite sensitivity of the latterhas resulted in the detection of low copy viralDNA in histologically and cytologically normal

MethodsThe myeloma cell line NSO and establishedhybridoma cell lines were maintained inGlasgow modified Eagle's medium (GMEM)containing 50o fetal calf serum (FCS). Africangreen monkey kidney (CV- 1) cells were main-tained in the same medium containing 100/0FCS. Baby hamster kidney 21 clone 13 cells(BHK) were maintained in GMEM containing1000 tryptose phosphate broth and 1000/ new-born calf serum. The construction of recom-binant vaccinia virus (VLlRK) expressing theentire HPV 16 LI open reading frame underthe control of a vaccinia late promoter has beendescribed previously.'3 The recombinant virusand the parent WT vaccinia virus (strain WR)were grown in BHK cells and titrated in CV-1cells.

MOUSE IMMUNISATION AND CELL FUSION

Mice were immunised subcutaneously with 20ig of a # galactosidase-LI fusion protein (con-taining amino acids 198-C terminus ofHPV-16L1 13) in complete Freund's adjuvant. After

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four weeks a series ofsubsequent doses of 15 Mgprotein in Freund's incomplete adjuvant weregiven intraperitoneally at two weekly intervals.A final intravenous immunisation of 15 Mg wasgiven three days before the fusion. The spleenwas removed aseptically and fused to the mousehybridoma NSO, using the original proto-col.'41 5 Cells were plated at a density of2 x 106/well in 24-well Linbro plates, and the growthmedium changed every two to three days.Positive hybrids were cloned twice by limitingdilution, and clones secreting antibody to L1were grown in 75 cm2 flasks and stored in liquidnitrogen.

SCREENING OF HYBRIDOMA SUPERNATANTSUSING ELISAThe supernatants of wells containing hybridswere tested in an ELISA as follows: CV-1 cellswere grown in 5 mm wells to confluency andinfected with 30 plaque forming units/cell WTor VL1RK vaccinia virus. After 18 hours at37°C cells were fixed in 5% formaldehyde,10% sucrose, in phosphate buffered saline(PBS), and air dried. Before use cells werepermeabilised with 1% Triton X-100, 10%sucrose in PBS. After blocking with 5% FCS,monolayers were reacted with hybridomasupernatants, followed by incubation withperoxidase-linked protein A, each for 30 min-utes at room temperature. The wells were thenreacted with 0-phenylene diamine and theabsorption of the supernatants at 492 nm wasmeasured in an ELISA reader.

IMMUNOFLUORESCENCECV-1 cells were grown on coverslips andinfected, fixed, and permeabilised as describedfor the ELISA method. Cells were reacted withhybridoma supernatant and subsequentlyincubated with an anti-mouse IgG fluoresceinconjugate.

WESTERN BLOT ANALYSISBHK-cell monolayers, infected with 30 pfu/cell WT or VL1RK vaccinia virus, were har-vested in PBS 20 hours after infection, lysed insample buffer (24 mm TRIS-HCI, pH 6-18;100 mm dithiothreitol; 2% sodium dodceylsulphate (SDS); 20% glycerol; 0-02% bromo-phenol blue), and sonicated. Samples weredenatured at 100°C for three minutes, subjec-ted to SDS-polyacrylamide gel electrophoresis(PAGE) and the proteins transferred to nitro-cellulose by electroblotting as describedpreviously.9 After blocking in 5% dried milkpowder in PBS for 30 minutes at roomtemperature, filters were incubated with 1/2diluted supernatant for one hour at roomtemperature, washed three times in PBS-1%NP40 and reacted with affinity purified 125I1protein A (Amersham International, UK) at aconcentration of 0 I pCi-ml. The blots werewashed again in PBS-i% NP40, dried, andautoradiographed.

IMMUNOCYTOCHEMICAL STUDIESFormalin fixed, paraffin wax embedded sec-tions were obtained from diagnostic biopsyspecimens taken from patients attending a

routine colposcopy clinic. Sections weredewaxed and rehydrated through a xylenealcohol series. Before immunostaining with theVectastain ABC complex (Vector Labora-tories, Peterborough, England) sections wereincubated in 0-10% trypsin at 37°C for fiveminutes and quenched in 0-3% H202 for 15minutes.

Routine cervical Papanicolau smears werefixed in 500 formaldehyde in PBS for fiveminutes, washed in PBS, permeabilised in 1%Triton and subsequently treated with trypsin,quenched, and stained in the same way as thecervical sections.

IN SITU HYBRIDISATIONSections of cervical lesions embedded in paraf-fin wax were dewaxed in xylene and alcohol andhybridised with probes containing DNA ofHPV 16/18, HPV 31/33, or HPV 6/1 1, usingthe Vira Type in situ hybridisation kit (LifeTechnologies Inc, Gaithersburg, Maryland,USA) according to the manufacturer's instruc-tions. In certain cases in situ hybridisation wascarried out using 35S-labelled DNA, accordingto the method described by Gowans et al.'6Briefly, slides were rehydrated, digested withproteinase K and pronase, and treated withacetic anhydride before hybridisation with 35S-labelled HPV-16 DNA. After coating withradiographic emulsion slides were stored foreight days at 4°C and developed, fixed, andstained with haematoxylin.

ResultsMice repeatedly immunised with the f galac-tosidase-HPV 16 Li fusion protein developedstrong responses to the fusion protein, but theresponse to the vaccinia recombinant express-ing HPV-16 Li was variable. Some mice alsogave high "background" responses to cellsinfected with wild type vaccinia virus. Spleencells from mice showing a good differentialresponse in ELISAs to the Li recombinantvaccinia virus relative to the wild type viruswere used in fusions with cell line NSO. Theresults of a series of three fusions were dis-appointing in that only two positive hybridomasupernatants were identified by ELISAs oncells infected with VL1RK. Of these, only oneretained activity following two rounds of clon-ing by limiting dilution. This hybridoma wasstable to subsequent passage and subcloningand was designated CAMVIR-1.

PROPERTIES OF CAMVIR-1Tissue culture supernatant from CAMVIR-1reacted with a 56 kilodalton protein in Westernblots of cell lysates infected with VL1RK (figla). A minor species of 64 kilodaltons was alsodetected. This probably represents a glycosy-lated form ofthe protein as it is absent in lysatesof cells infected in the presence oftunicamycin,an inhibitor of N-glycosylation. Similar resultswere reported using a polyclonal rabbit serumraised against an HPV-16 LI fusion protein."No reactivity was observed against cells infec-ted with wild type vaccinia. The antibody alsoreacted with the fusion protein used for im-

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Figure I Western blotanalysis of reactivity ofCAMVIR-1 with LIfusion protein and LIvaccinia recombinant. (a)CVI cells were infectedwith Li-vaccinia (lane a)or wild type vaccinia (laneb) at 30 pfu/cell, and lysedafter 20 hours. Sampleswere subjected to SDS-PAGE electrophoresis,and after transfer tonitrocellulose the blots wereincubated withCAMVIR-I supernatantdiluted at l /I0,followedby affinity purified 125I-protein A at aconcentration of 0 1pCi/ml. After washing, theblots were dried andautoradiographed. (b) 01pg of the purifiedf-galactosidase LI fusionprotein (pHX2,containing amino acids221-C terminus (lane d)and 0)1 pg or an irrelevantfusion protein (containingamino acids 1-220 of theHPV-16 Ll protein, (lanec) were subjected toelectrophoresis on a 125%SDS-acrylamide gel andtransferred tonitrocellulose. The blotswere reacted withCAMVIR-1 at a dilutionof 1/20,followed byperoxidase-linked anti-mouse IgG (AmershamInternational, UK) at adilution of 1/200. Bandswere visualised usingdiaminobenzidinetetrahydrochloride(DAB) at a concentrationof I mg/ml.

a b c d

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munisation but not with an irrelevaprotein containing the N-terminal pthe HPV-16 Li protein (ammno acidsTo determine the cellular localisati

antigen detected by ChAMVIR 1, cinfected with recombinant or WTafter formaldehyde fixation readtCAMVIR-1 and fluorescein-labelimouse immunoglobullin. In cells mnfelLi vaccinia strong nuclear flulorescqobserved, while no reaction coulld bein cell monolayers infected with WT f2g and h). An irrelevant monoclonalof the same subclass (IgG2a) did notxthe recombinant infected cells.

REACTIVITY OF CAMVIR-1 ON TISSUE SE(The ability of CAMVIR-l to detecprotein in formalin fixed, paraffinbedded tissue sections was examina biotin-streptavidin-peroxidasemethod. Sections were obtained I

cervical biopsy specimens of patieabnormal cervical smears, attendingclinic for colposcopy and examinatiolesions were histologically graded asIII. The sections were tested for theof Li protein using CAMVIR-1 tissusupernatant at a concentration of 1/ 102a shows a lesion in which the nucleicells in the superficial layers arestained. These cells have the morp}koilocytes. A parallel section of this 14subjected to in situ hybridisation tothe presence and type of viral DNAThe cells with the maximum copy nHPV 16 DNA are found in the samethe section as the cells containing LI Ithat is, in the superficial layers ofthe 1Iexamined a substantial number of lesi50), and in those cases where stainCAMVIR-1 was observed, in situ htion showed high copy numbers ofDNA in that region of the section.posite was not true, however-that iority of lesions were positive for HPVsitu hybridisation but showed no staiiCAMVIR-1. In one instance we fou

lesion staining positive with CAMVIR-1 con-tained HPV31/33DNA. In most cases lesionscontaining HPV 6 orHPV 11 did not stain withCAMVIR-1, but occasionally a few cells in the

- pH X2 superficial layers of these sections would shownuclear staining. We also compared the sen-sitivity of CAMVIR-1 in detecting LI proteinto that ofa commercially available anti-BPV- l -antibody (Dako). On sections positive forHPV-16 or -33 little or no staining wasobtained with the Dako antibody, althoughCAMVIR-1 gave a strongly positive nuclearreaction (figs 2c and d).

DETECTION OF LI PROTEIN IN CERVICAL SMEARSThe ability ofCAMVIR-1 to detect L1 proteinin routine cervical smears was investigated in ablind trial. Samples were taken from 50 generalpractice patients. DNA extractions were per-formed on exfoliated cells which were sub-jected to dot blot hybridisation with probesspecific for HPV 6, 11, 16 or 18. The smears

nt fusion were examined cytologically and -stained with*ortion of CAMVIR-1 tissue culture supernatant at a1-197). 1/100 dilution using the biotin-streptavidin-ion of the* peroxidase system. The cells were fixed brieflye.lls were in 5% formaldehyde in PBS, and trypsin wasvirus and added before reaction. In 14 of the 50 cases an:ed with insufficient number of cells was present on theled anti- smear to determine if positively stained cellscted with were present. Of the remaining 36 cases, nine:ence was contained cells showing nuclear staining and

bdetected were of koilocytic morphology (figs 2e and f).virus (figs Decoding showed that two of these containedantibody HPV-16 DNA by dot blot hybridisation (be-react with tween 2 x 105'106 genomic copies). In four

cases no HPV DNA could be detected (lessthan 105 genomic copies). In one case HPV

CTIONS DNA was present at low copy number but it:t the LI was not possible to determine which type. Inwax em- two cases where positive staining withled using CAMVIR-1 was observed a very high copystaining number ofHPV-1 1 was present (more than 106

from the genomic copies a cell). In three cases hybridisa-bnts with tion showed the presence ofHPV-16 DNA, buta routine no staining with CAMVIR-1 was observedn. These because the smear was inadequate.

presenceie culture Discussion10. Figure The absence ofa suitable in vitro system for theof several propagation of human papillomaviruses, to-strongly gether with the fact that in clinical lesions only

hology of very low levels of virus are present, has neces-esion was sitated the use of alternative strategies for theestablish production of immunological reagents to these(fig 2b). viruses. In the production of monoclonal anti-umber of bodies suitable targets for the screening ofregion of hybridoma fluids are an important require-protein- ment. To date, the monoclonal antibodiesesion. We reported have been raised and screened againstions (n = bacterial derived proteins.'6"8 We used cellsiing with infected with a vaccinia virus recombinantLybridisa- expressing the HPV 16 LI open reading frameHPV-16 as a target for the selection of monoclonalThe op- antibodies. This system provided a very con-

is, a min- venient and plentiful source of viral protein,!-16 by in free of potentially crossreactive bacterialning with elements.md that a The antibody against the HPV-16 LI pro-

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Figure 2Immunocytochemicalstaining with CAMVIR-1of sections crf cervicalbiopsy specimenscontaining HPV-16 orHPV-33, routine cervicalsmears, and cells infectedwith the Li vacciniarecombinants. (a)Staining with CAMVIR-1 at 1/100 dilution onformolfixed, paraffin waxembedded section of acervical lesion containingHPV-16. Sections weredewaxed and rehydratedthrough a xylene alcoholseries, incubated in 0 100trypsin at 37°Cfor fiveminutes, quenched in 033%H202for 15 minutes, andstained using theVectastain ABC complex.(b) In situ hybridisationusing35S-labelled total HPV-16DNA on the sectionadjacent to that in fig 2a.(c) Staining withCAMVIR-1 at 1/100 ona cervical lesion containingHPV-33 using the sameprotocol as in fig 2a.(d) Staining of the samelesion as in fig 2c, using acommercially availablepolyclonal anti-BPV-antibody (Dako) at1/1 00,followed by theVectastain ABC complex.(e and f) Staining withCAMVIR-1 at 1/100 ofroutine cervical smearsfixed in 500formaldehyde-PBS forfive minutes, permeabilisedin l °O Triton, andsubsequently treated withtrypsin, quenched, andstained in the same way asthe cervical sections.(g and h) Reactivity ofCAMVIR-1 at 1/100dilution on CV-1 cellsinfected with 30 pfu/cellLi vaccinia (g) or wildtype vaccinia (h). Cellswere fixed in 50oformaldehyde-PBS forfive minutes, washed inPBS, and after incubationwith CAMVIR-1 for 30minutes at roomtemperature, washed againin PBS, and reacted withFITC-labelled anti-mouseIgG at 1/70 (Dako).

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tein reacts with a protein of the expectedmolecular weight in recombinant vaccinia virusinfected cells and detects the protein in apredominantly nuclear location, correspondingto earlier findings using polyclonal anti-Ll-antisera. 13We also obtained nuclear staining of

morphologically koilocytic cells on formalinfixed, paraffin wax embedded tissue sectionsusing CAMVIR-1. It was clear from the sub-sequent typing of these sections using in situhybridisation that CAMVIR-1 binds stronglyto cells containing HPV-16 or -33. Veryoccasionally, however, a number of cells in thesuperficial layer of an HPV-6 containing lesion

would show nuclear staining. From the resultson cervical smears it is clear that cells contain-ing very high copy numbers of HPV-1 1 alsogive positive results with CAMVIR- 1. A sub-stantial number of lesions containing HPV-6,however, were totally negative when reactedwith CAMVIR- 1. The antibody probablybinds, to a much lesser extent, to cells contain-ing HPV-6 or -1 1, so that only cells with a veryhigh viral DNA copy number show positivestaining. The epitope to which CAMVIR-1 isdirected was identified as GFGAMDF (a.a.n-umber 230-236) using overlapping peptides inan ELISA (Cason J, McCance D, personalcommunication). This region is highly con-

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served among the different HPV types, andthere is one amino acid substitution betweenHPV16 and HPV-6 or -11 in this region,namely, D to N (GFGAMNF), which wouldexplain the weak cross reactivity with thesevirus types. CAMVIR-1 identifies an epitopeon HPV-1 6 LI that is strongly reactive againstHPV-16 and -33 and, to a much lesser extent,with HPV-6 and -11, and thus is able toidentify a subgroup ofhuman papillomaviruseswith malignant oncogenic potential.

Expression of the L1 capsid protein, asidentified with this monoclonal antibody, isstrongly focal, and in any one lesion only a fewcells with high copy viral DNA synthesise LIprotein. This situation differs from that seen insome other lesions induced by papillomavirus,particularly cutaneous warts'9 and bovinepapillomas20 in which many cells express viralcapsid proteins and in which viral particles arepresent in substantial numbers. The focality ofLi expression in the cervical lesions may berelated to the paucity of viral particles in lesionscontaining HPV 16. Alternatively, it may berelated to the stage of infection at which thecervical lesion has been biopsied. The naturalhistory of HPV- 16 related disease is virtuallyunknown. It is evident from studies on otherpapillomaviruses, particularly CRPV andBPV-1, that the development of a lesion inwhich viral capsid protein and viral particlesare produced requires a considerable timeperiod-of the order of months.2' Anecdotalevidence in human disease suggests that asimilar time period is involved in the develop-ment of genital condylomata."2 In manypatients biopsy specimens may be taken at astage in the natural history of the disease whenviral particles are not produced, either becausethe lesion is at an early stage or because aquiescent or persistent phase is another featureof the disease process. Whatever the explana-tion it is evident that L1 capsid protein issynthesised in a few cells containing highnumbers of viral DNA copies in the superficial,terminally differentiated surface layers of theepithelium.

Detection of viral DNA only informs aboutpresence of the genome and gives little infor-mation on virus replication and pathogenesis.This is particularly important in that papillo-maviruses are strictly epitheliotropic, and theavailable information indicates that permissiveviral replication only occurs in keratinocytesand specifically in the keratinocyte undergoingthe normal process of differentiation in theepithelium. The intra-epithelial neoplasticlesions of the cervix are characterised by aprogressive loss and distortion of the normaldifferentiation process, and these features areincluded in the diagnostic criteria of theselesions.2" Changes in the expression of viralproteins may be a reflection of neoplasticphenotype and may give useful information onprogression and prognosis, and the antibody

CAMVIR-1 may be a useful reagent in thiscontext.

We are grateful to Drs Lynn Kennedy and Ian Frazer of theDepartment of Medicine and Lions Immunology Laboratory,University of Queensland, Australia, for the provision of HPVtyped smears; the Histopathology Department, Addenbrooke'sHospital, Cambridge, for tissue sections of cervical biopsies; andto Mrs Mary Wright for typing the manuscript.

zur Hausen H. Human papillomaviruses and their possiblerole in squamous cell carcinomas. Curr Top MicrobiolImmunol 1977;78:1-30.

2 Durst M, Gissmann L, Ikenburgh H, zur Hausen H. Apapillomavirus DNA from a cervical carcinoma and itsprevalence in cancer biopsy samples from differentgeographic regions. Proc Natl Acad Sci USA 1983;80:3812-5.

3 Campion MJ, McCance DJ, Cuzick J, Singer A. Theprogressive potential of mild cervical atypia: a prospectivecytological, colposcopic and virological study. Lancet1986;ii:237-40.

4 Schneider A, Kraus H, Schuhmann R, Gissmann L. Papil-lomavirus infection of the lower genital tract: detection ofviral DNA in gynaecological swabs. Int J Cancer 1985;35:443-8.

5 Meanwell CA, Cox MF, Blackledge G, Maitland NJ. HPV-16 DNA in normal and malignant cervical epithelium:implications for the aetiology and behaviour of cervicalneoplasia. Lancet 1987;i:703-7.

6 Wickenden C, Steele A, Malcolm ADB, Coleman DV.Screening for wart virus infection in normal and abnormalcervices by DNA hybridisation of cervical scrapes. Lancet1 987;i:65-7.

7 Young LS, Bevan IS, Johnson MA, et al. The polymerasechain reaction: a new epidemiological tool for investi-gating cervical human papillomavirus infection. Br Med J1989;298:14-8.

8 Li CH, Shah KV, Seth A, Gilden R. Identification of thehuman papillomavirus type 6bLl open reading frameprotein in condylomas and corresponding antibodies inhuman sera. J Virol 1987;61:2684-90.

9 Matlashewski G, Banks L, Wu-Liao J, Spence P, Pim D,Crawford L. The expression of human papillomavirustype 18 E6 protein in bacteria and the production of anti-E6 antibodies. J Gen Virol 1986;67:1909-16.

10 Banks L, Spence P, Androphy E, et al. Identification ofhuman papillomavirus type 18 E6 polypeptide in cellsderived from human cervical carcinomas. J Gen Virol1987;68:1351-9.

11 Doorbar J, Gallimore PH. Identification of proteins encodedby the LI and L2 open reading frames of human papillo-mavirus la. J Virol 1987;61:2793-9.

12 Doorbar J, Campbell D, Grand RJA, Gallimore PH. Iden-tification of the human papillomavirus la E4 geneproduct. EMBO J 1984;5:355-62.

13 Browne HM, Churcher MJ, Stanley MA, Smith GL,Minson AC. Analysis of the LI gene product of humanpapillomavirus type 16 by expression in a vaccinia virusrecombinant. J Gen Virol 1988;69:1263-73.

14 Koher C, Howe SC, Milstein C. Fusion between immuno-globulin-secreting and non-secreting myeloma cell lines.Eur J Immunol 1976;6:292-5.

15 McLean C, Buckmaster A, Hancock D, Buchan A,Fuller A, Minson A. Monoclonal antibodies to three non-glycosylated antigens ofherpes simplex virus type 2. J GenVirol 1982;63:297-305.

16 Gowans EJ, Burrell CJ, Jilbert AR, Marmion BP. Cytoplas-mic (but not nuclear) hepatitis B virus (HBV) core antigenreflects HBV DNA synthesis at the level of the infectedhepatocyte. Intervirology 1985;24:220-5.

17 Oltersdorf T, Seedorf K, Rowekamp W, Gissmann L.Identification ofhuman papillomavirus type 16 E7 proteinby monoclonal antibodies. J Gen Virol 1987;68:2933-8.

18 Patel D, Shepherd PS, Naylor JA, McCance DJ. Reactivitiesof polyclonal and monoclonal antibodies raised to themajor capsid protein of human papillomavirus type 16.J Gen Virol 1989;70:69-77.

19 Roseto A, Pothier P, Guillemin M-C, et al. Monoclonalantibodies to the major capsid protein of HPV1. J GenVirol 1984;65:1319-24.

20 Jenson AB, Rosenthal C, Olson C, Pass F, Lancaster WD,Shah K. Immunological relatedness of papilloma virusesfrom different species. JNCI 1980;64:495-500.

21 Olson C. Animal papillomaviruses: historical perspectives.In: Salzman NP, Howley PM, eds. The PapovaviridaeVol. 2. The Papillomaviruses. New York: Plenum Press,1987:39-66.

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23 Buckley CH, Butler EB, Fox H. Cervical intraepithelialneoplasia. J Clin Pathol 1982;35:1-13.

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