J Interphase and III Increased HPVin

J Clin Pathol 1991;44:33-38

Interphase cytogenetics using biotin anddigoxigenin labelled probes: III Increasedsensitivity and flexibility for detecting HPV incervical biopsy specimens and cell lines

C S Herrington, A K Graham, J O'D McGee

AbstractA monoclonal antibody to digoxin en-abled sandwich techniques to be used forthe detection of hybridised digoxigeninlabelled probes in cultured cells andparaffin wax sections. This system hasgreater flexibility than alkaline phos-phatase conjugated polyclonal anti-digoxigenin antibody and permits theuse of alternative detector enzymes, suchas horseradish peroxidase and fluores-cence labels. The APAAP detectionsystem that does not require the use ofbiotin can also be used in situationswhere endogenous biotin is a problem.The low level of background stainingcombined with precise substrate deposi-tion of the amplified peroxidase systemgives higher sensitivity and resolution.This permits localisation of closelyadjacent chromosomal loci in interphasenuclei. The most sensitive peroxidasebased digoxigenin detection systemvisualises two and a half to 12 copies ofhuman papillomavirus (HPV) pernucleus. This system is also suitable forthe analysis of low copy number HPVinfection of cervical tissues.

University ofOxfordNuffield DepartmentofPathology andBacteriology, JohnRadcliffe Hospital,Headington, OxfordOX3 9DUC S HerringtonA K GrahamJ O'D McGeeCorrespondence to:Professor McGeeAccepted for publication16 August 1990

Interphase cytogenetics encompasses, amongother things, visual discrimination ofindividual chromosomes or genes in inter-

phase nuclei.'-3 This has previously beenperformed by fluorescence45 and more

recently by enzyme based chromogenicprocedures using biotin and digoxigeninlabelled probes.' During the course of thelatter work, it became apparent that the detec-tion systems for biotin or digoxigenin labelledprobes using alkaline phosphatase baseddetection systems did not give sufficientresolution to discriminate between closelyadjacent chromosomal loci.

Digoxigenin labelled probes have been usedboth as alternatives and in addition to biotinlabelled probes in non-isotopic in situhybridisation (NISH).67 The main advantageof digoxigenin over biotin labelled probes forNISH is that digoxigenin (a plant alkaloidanalogue), unlike biotin, is not present con-

stitutively in mammalian cells. Hence, inimmunohistochemical detection systems,digoxigenin does not generate endogenous

background to the same extent as biotin.Digoxigenin labelled probes, however, are lesssensitive than biotinylated probes using singlestep detection methods.6 The sensitivity ofdetection procedures can be enhanced usingantibody sandwich techniques using a linkerantibody to bridge between antibody directedagainst reporter molecule and an enzyme con-jugate.9 A refinement of this concept is to usebiotinylated antibodies, which can be used tobridge between the first antibody, and anavidin enzyme conjugate.'0 This givesincreased sensitivity due to the high affinity ofavidin for biotin (dissociation constant = I0-`M) and improved specificity when the firstantibody is of monoclonal type.

Digoxigenin, the aglycone derivative of thecardiac glycoside digoxin, has high affinity forantibodies to digoxin."'3 This suggested to usthat the antibodies which have beendeveloped for the monitoring and treatment ofclinical digoxin toxicity would increase thesensitivity of detection of digoxigenin labelledprobes in NISH.The aims of this investigation were: (1) to

develop systems of high sensitivity andresolution for digoxigenin labelled probeswhich are applicable to interphase cyto-genetics-for the latter, CaSki and HeLa cellswere used as a model system; and (2) to adaptdetection systems for digoxigenin labelledprobes so that they are more sensitive andflexible for detection of genes in archivalbiopsy specimens.

MethodsAll chemicals were obtained from Sigma (UK)or BDH (UK) unless otherwise stated.The HPV6 probe was the amp2 fragment

cloned into pBR322,'4 the HPV16 probe thewhole viral genome in pAT153,'5 and HPV18the whole viral genome in pBR322. 16 Biotin anddigoxigenin were incorporated into plasmidDNAs by nick translation, and the degree oflabelling and size of fragments were aspreviously described.6 17

Paraffin wax sections were prepared andnucleic acids unmasked as previouslydescribed.6HeLa 229 cells (ATCC, USA) were grown to

confluence in 80 cm2 flasks in RPMI 1640medium (Gibco, UK) supplemented with L-glutamine (1 x; Gibco, UK); penicillin/

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Herrington, Graham, McGee

streptomycin (1 x; Gibco, UK); and 20% fetalcalf serum (Gibco, UK). Detachment wasachieved using trypsin/EDTA solution(Gibco, UK) and the cells were pelleted at600 x g. After two washes in PBS the cellswere fixed in 10% formalin, embedded in agar,and processed to paraffin wax in the usual way.Sections of 4 ,gm were cut and unmasked usingproteinase K at a concentration of 1 mg/ml inPBS for 15 minutes at 37°C.CaSki cells were grown onto slides, fixed in

paraformaldehyde, and nucleic acids unmaskedas previously described.6

HYBRIDISATIONAliquots of hybridisation mix (8 u1) containing2 ng/pl of biotin or digoxigenin labelled HPV6,HPV 16, or HPV18 were added to each well onmultispot slides which were covered with a14 mm glass coverslip (Chance, UK) and theslides placed in a moist Terasaki plate.Hybridisation mix consisted of 50% forma-mide, 5% dextran sulphate, 2 x SSC, and 0 05mol/l TRIS-HCI, pH 7 3; 1 x SSC = 0 15mol/l sodium chloride, 0-015 mol/l sodiumcitrate. Sections and probes were simul-taneously denatured at 95°C for 15 minutes on asolid stainless steel plate in a hot air oven andthen hybridised at 420C for 2 hours.

DETECTION OF HYBRIDISATION SIGNALAll antibody/avidin incubations were carriedout for 30 minutes at 22°C unless otherwisestated. Slides were washed in three changes of4x SSC at 22°C (five minutes each change),soaked in blocking agent TBT (0.05 mol/lTRIS-HCI, 0-10 mol/l sodium chloride (pH7 2) containing 3% (w/v) bovine serumalbumin and 0-05% Triton 100 (v/v)) at 22°Cfor 10 minutes. Subsequent visualisation ofsignal was determined by the reportermolecule.

Biotin labelled probes(a) Single step procedure Sections wereincubated in avidin-alkaline phosphatase oravidin-peroxidase (Dako, UK) diluted 1 in 100in TBT. Unbound conjugate was removed bywashing for five minutes twice in 50 mMTRIS-HCI, 10 mM NaCl, pH 7 2 (TBS), andthe sections incubated in the appropriatesubstrate.(b) Three step procedure Sections wereincubated first in monoclonal mouse anti-biotin (Dako, UK) diluted 1 in 50 in TBT,washed in TBS (twice at 10 minutes each), andthen in biotinylated rabbit anti-mouse (F(ab')2fragment, Dako, UK). After washing in TBS,the third incubation was in either avidin alka-line phosphatase (bio-3-A/N), or streptavidinperoxidase (Dako, UK) (bio-3-P/C). Theformer was diluted 1 in 50 and the latter 1 in100 in TBT containing 5% non-fat milk. Afterwashing in TBS, sections were incubated in theappropriate substrate (see below).

Digoxigenin labelled probes(a) Single step procedure Sections wereincubated in alkaline phosphatase conjugatedantidigoxigenin (Boehringer, Germany)

diluted 1 in 600 in TBT, washed in TBS, andthe signal was developed using NBT/BCIP(see below).(b) Three step procedure Sections wereincubated in monoclonal antidigoxin (Sigma,UK) diluted 1 in 10 000 in TBT. Subsequentdetection was as for the three step detection ofbiotinylated probes, with second incubation inbiotinylated rabbit anti-mouse (F(ab')2 frag-ment) and the third incubation in avidin-peroxidase (dig-3-P/C) or avidin-alkalinephosphatase (dig-3-A/N).(c) APAAP system Slides were incubated inmonoclonal a4tidigoxin as in (a), followed byrabbit anti-mouse immunoglobulin (Dako,UK), diluted 1 in 50 in TBT, then APAAPcomplex (Dako, UK) diluted 1 in 50 in TBT.Signal was developed using NBT/BCIP.(d) Fluorescence detection Slides were treatedas for the three step detection method in (b)except that streptavidin-fluorescein iso-thiocyanate (Dako, UK) was used in the finalstep. Optimal results were obtained with adilution of .1 in 20 of the fluorescent conjugatein TBT containing 5% non-fat milk.

SUBSTRATE PREPARATIONNitrobluoe tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) and amino-ethylcarbazole (AEC) were made as previouslydescribed.618 Diaminobenzidine (DAB) wasdiluted to 0-5 mg/ml in distilled water andstored in 5 ml aliquots at -20°C. Hydrogenperoxide was added to DAB (final concentra-tion 03%) immediately before use. After probedetection slides were air dried at 42°C andmounted in glycerol jelly (for enzyme/chromogen reactions). For fluorescencepreparations, PBS/glycerol (1:9 v/v) contain-ing 2-3% diazobicyclo-octane (DABCO)(Sigma, UK) was used.Frequency distributions were compared

using the two tailed Mann-WhitneyU test witha correction for tied values.

ResultsThe results of single step detection methods forbiotin and digoxigenin probes have previouslybeen reported.67 These were less sensitive thanthe three step procedures described below.

COMPARATIVE SENSITIVITY AND RESOLUTION OFDIGOXIGENIN AND BIOTIN IN INTERPHASE CELLSThe three step peroxidase AEC detectionmethod for digoxigenin labelled probes (dig-3-P/C) produced very low background stainingand enhanced resolution in CaSki and HeLacells (figs 1 and 2). Discrete HPV signals wereconsiderably more easily resolved when com-pared with the three step alkaline phosphataseNBT/BCIP detection system for digoxigenin(dig-3-A/N) (fig 1); the latter is the counterpartof the most sensitive system we have developedhitherto."An estimate of the absolute sensitivity of the

dig-3-P/C method (fig 3) can be made byanalysis of the frequency distribution of dotsper interphase nucleus in CaSki cells. Themedian number of dots per nucleus is nine. Up

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Digoxigenin probe detection

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A BFigure 1 CaSki cells hybridised with digoxigenin labelled HPV16 probe and detected using (A) three step alkalinephosphatase NBT/BCIP (dig-3-A/N) and (B) three step peroxidase AEC (dig-3-P/C). The nuclei are lightlycounterstained with haematoxylin in (B). Note the clear resolution of closely adjacent dots within individual nuclei in(B), but the high level of background with staining of nucleoli (small arrows) and coalescence of dots (large arrow) in(A).

to four discrete dots were also detected in thenuclei of HeLa cells (which contain 10-50copies of HPV1820), which had been fixed in.formalin and embedded in paraffin wax (fig 2).

COMPARATIVE SENSITIVITY OF DIGOXIGENIN ANDBIOTIN LABELLED PROBES ON BIOPSY SPECIMENSThe relative sensitivities of digoxigenin andbiotinylated probes were assessed on routinelyprocessed, paraffin wax embedded archivalcervical biopsy specimens. These showed thecriteria of wart virus infection,21 with or with-out CIN1-3.

Figure 2 HeLa cells fixed informalin and embedded in paraffin wax were hybridisedwith digoxigenin labelled HPV18 and detected using dig-3-P/C. Four discrete signals(large arrow) can be seen within a single nucleus. Note that signals are present withinother nuclei but that some are out of the plane offocus.

Digoxigenin labelled probesThe amplified procedure for the detection ofdigoxigenin using alkaline phosphatase withNBT/BCIP as substrate (dig-3-A/N) gave asignal in a greater number of cells comparedwith the comparable three step alkaline phos-phatase system for biotin detection (bio-3-A/N) (fig 4). The background produced by dig-3-A/N is significantly less than that produced bybio-3-A/N and the signal intensity is enhancedto a greater degree using digoxigenin comparedwith biotin (fig 4). Clear signal within almost allparabasal cells is present with digoxigeninlabelled probes (fig 4A). The signal to noiseratio of digoxigenin labelled probes is superiorto that ofbiotinylated probes-compare figs 4Aand 4B.Using the three step peroxidase AEC detec-

tion system for the detection of digoxigeninlabelled HPV16 (dig-3-P/C) in lesions definedhistopathologically as CIN3, signal can be seenin all layers of the epithelium, including thebasal layer (fig 5). Notably, the morphology ofthis signal is different from that obtained byNISH analysis of wart virus infection withmild atypia (fig 4). The combination of thesubstrate AEC with haematoxylin counter-staining aids interpretation.The APAAP procedure produced a clear

signal with little background (data not shown),but it was less sensitive than the procedure usedin fig 4 as judged by both the number ofpositive cells and the fact that parabasal cellswere not labelled.

Fluorescence detection of digoxigeninlabelled probes also produced satisfactory

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Figure 3 Frequencydistributions of dot numberper nucleus for (A) dig-3-P/C and (B) bio-3-P/C.Dig-3-P/C is significantlymore sensitive than bio-3-P/C (p < 0 001), withmedian dot numbers ofnine and seven,respectively. 0~

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results, with low background staining ofsurrounding connective tissue (data notshown). This procedure, however, was lesssensitive as signal was not detected withinparabasal cells.

DiscussionDigoxigenin has been used to label probesby terminal labelling of oligonucleotides,22

random priming,23 or nick translation.6 Theseprobes have been used on nitrocellulosefilters2223 and for in situ hybridisation.67 Thesimplest way to detect digoxigenin labelledprobes is by using an alkaline phosphataseconjugated polyclonal antidigoxigeninantibody. This system is less sensitive than thecorresponding avidin based single step detec-tion system for biotin,6 a difference likely to bedue to the higher affinity of avidin for biotin(KD 10-1') compared with the affinity ofantibody for antigen (KD 10-12 at best).To improve the sensitivity of digoxigenin

labelled probes we have used a monoclonalantibody to digoxin.- As shown here, thisproduces considerably greater sensitivity. Thesignal also develops within minutes with NBT/BCIP andAEC compared with an hour or morefor single step digoxigenin detection and up to16 hour incubations in NBT/BCIP in somedetection systems for biotin.24 These methodsincrease the sensitivity of these probes asdetermined by the number ofHPV signals percell in cultured cells and by the number ofHPVpositive cells in clinical samples. The detectionsystems for biotin produce more backgroundstaining than the corresponding systems fordigoxigenin. This is likely to be due toamplification of signal from endogenous biotin.By increasing the number of rounds ofamplification of digoxigenin, the signal can beenhanced still further but, as this requires theuse of an anti-biotin antibody and an avidinconjugate, the background staining is corres-pondingly increased. Nevertheless, amplifieddetection systems for digoxigenin amplifyendogenous biotin by one round fewer than thecorresponding system for biotin.Advantages of the peroxidase-AEC system

are both its high degree of resolution and theease of interpretation of preparations counter-

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Figure 4 Sections of a condyloma acuminatum wereprobed with (A) digoxigenin labelled and (B) biotinlabelled HPV6, detected using the three step methodsdig-3-A/N and bio-3-A/N, respectively. Parabasal cellsare positive only with the digoxigenin labelled probe(arrows). Note the low level ofbackground staining inthe subjacent stroma in (A). The biotinylated probeproduces greater background staining in the region of thebasement membrane rendering interpretation impossible.

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Digoxigenin probe detection

Figure 5 A biopsy specimen of CIN 3 probed with digoxigenin labelled HPV16 anicktected using the peroxidase AEC system (dig-3-P/C). Note that signal is present ibasal cells (large arrow).

stained with haematoxylin. Thus cleadjacent dots can be readily distinguisheCaSki and HeLa cell nuclei and in arc}biopsy specimens. Both these cell lines contheir respective HPV types integrated intchost genome at multiple sites.25 26 i

produces better resolution and colour con

than DAB, making it more suitable for siItaneous double labelling of genes.7 I

resolution is not possible using NBT/B(because diffusion of substrate leads to loEdefinition and hence loss of spatial resoluiThus closely adjacent gene loci can becriminated with AEC: this is not possiblealkaline phosphatase based systems.The three step peroxidase AEC detec

system (dig-3-P/C) is more sensitive thancomparable biotin based system (bio-3-J(fig 3). Analysis of the frequency distributicdot numbers per nucleus for these two metlshows that many more cells containing r

than 10 dots per nucleus are detectable u

digoxigenin. Given that these systems i

applied to similar cell populations, thisference is likely to have been due to the grcresolution of signal using digoxigenin as plabel (fig 1). Calculation of sensitivity fronrmedian values of these distributions giveabsolute sensitivity of 40 viral genomesbiotin and 30 for digoxigenin detection u

the amplified peroxidase methods, giventhe total number of copies of HPV per Ccell nucleus is about 270.6 This measur

however, an underestimate as it asserelative sensitivity more accuratelyabsolute sensitivity,6 and as can be seen fror2, the presence of multiple signals in HeLanuclei using this system suggests thatsensitivity is greater. Indeed, it has 1

estimated that HeLa cells contain 10-50 ccofHPV18.20 As four NISH signals were visised in paraffin wax embedded HeLa celh;estimate of the sensitivity of this detecsystem is two and a half to 12 viral copicroutinely processed material.

In routine cervical biopsy specimens, thethree step digoxigenin system is clearly themost sensitive developed for clinical practice.An HPV signal is evident in almost allparabasal cells. It is of note that the mor-phology of the signal (single dots) obtained inthe biopsy specimen shown in fig 5 resemblesthat seen in CaSki and HeLa cells, in which theviral genomes are known to be integrated.2526 Itis tempting to speculate that this NISH signalmorphology is indicative of viral integrationand this is under investigation by routinemolecular biological techniques in thislaboratory.APAAP and fluorescence detection of digoxi-

genin labelled probes can be performed byminor modification to the three step detectionsystem. These may be useful in clinicallaboratories for routine screening by NISH ofHPV in cervical biopsy specimens but they are

d less sensitive than the methods described in'n detail above. It should be noted, however, that

the APAAP system has the advantage that itdoes not require biotin. This may be useful intissues where endogenous biotin creates high

)sely background.d in Overall, the preferred methods are therefore:hival (i) three step alkaline phosphatase NBT/BCIPitain detection of digoxigenin labelled probes where) the high sensitivity and low background but not\EC high signal resolution are required; (ii) threetrast step peroxidase AEC detection of digoxigeninmul- where high sensitivity, high resolution, and lowsigh background are required in addition to nuclearCIP, counterstaining.ss of In conclusion, HPV probes labelled withtion. digoxigenin can be detected using long estab-dis- lished monoclonal antibodies to digoxin. Thiswith permits amplification ofthe signal produced by

these probes to a degree that detects low copy-tion number viral infection in cell lines and routine1 the clinical samples. Flexibility is also enhanced inP/C) that digoxigenin detection can be achieved withan of horseradish peroxidase, fluorescence andhods APAAP systems. The latter has the advantagenore that it does not require biotin. The enhancedIsing sensitivity and resolution also make digoxi-were genin an attractive label for interphasedif- cytogenetics and for the detection of HPV incater cervical disease.,robea the CSH is a CRC Clinical Research Fellow and holds a Juniors an Research Fellowship at Green College, Oxford. This work was

for supported by grants to J O'D McG from the Cancer Researchor Campaign(UK).

singthataSkie is, 1 Burns J, Chan VT-W, Jonasson JA, Fleming KA, Taylor S,-sses McGee JO'D. Sensitive system for visualising bio-

tinylated DNA probes hybridised in situ: rapid sexthan determination in intact cells. J Clin Pathol 1985;38:

m fig ~ 1085-92.m fig 2 Cremer T, Landegent J, Bruckner A, et al. Detection of1 cell chromosome aberrations in the human interphase nucleusthe by visualisation of specific target DNAs with radioactive

and non-radioactive in situ hybridisation techniques:been diagnosis of trisomy 18 with probe L1.84. Hum Genet)pies 1986;74:346-52.

3 Bhatt B, McGee JO'D. Chromosomal assignment of genes.iual- In: Polak J, McGee JO'D, eds. In situ hybridisation:s an principles and practice. Oxford: OUP, 1990:149-64.'.ion 4 Hopman AHN, Ramaekers FCS, Raap AK, et al. In situ:tion hybridisation as a tool to study numerical chromosome-s in aberrations in solid bladder tumours. Histochemistry 1988;

89:307-16.5 Pinkel D, Landegent J, Collins C, et al. Fluorescence in situ

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hybridisation with human chromosome specific libraries:Detection oftrisomy 21 and translocations ofchromosome4. Proc Natl Acad Sci USA 1988;85:9138-47.

6 Herrington CS, Burns J, Graham AK, Bhatt B, McGeeJO'D. Interphase cytogenetics using biotin and digoxi-genin labelled probes I: relative sensitivity of both repor-ter molecules for the detection of HPV16 in CaSki cells. JCell Pathol 1989;42:591-600.

7 Herrington CS, Burns J, Graham AK, Bhatt B, McGeeJO'D. Interphase cytogenetics using biotin and digoxi-genin labelled probes II: simultaneous detection of viraland mammalian nucleic acids in individual nuclei. J ClinPathol 1989;42:601-6.

8 Herrington CS, McGee JO'D. Interphase cytogenetics.Neurochem Res 1990;15:467-74.

9 Guesdon J-L, Ternynck T, Avrameas S. The use of avidin-biotin interaction in immunoenzymatic techniques.J Histochem Cytochem 1979,27:1 131-9.

10 Hsu S-M, Raine L, Fanger H. Use of avidin-biotin-per-oxidase complex (ABC) in immunoperoxidase techniques.J Histochem Cytochem 198 1;29:577-80.

11 Smith JW, Butler VP, Haber E. Characterisation ofantibodies of high affinity and specificity for the digitalisglycoside digoxin. Biochemistry 1970;9:331-7.

12 Monji N, Ali H, Castro A. Quantification of digoxin byenzyme immunoassay: synthesis of a maleimide derivativeof digoxigenin succinate for enzyme coupling. Experientia1980;36:1141-3.

13 Valdes R, Brown B, Graves SW. Variable cross-reactivity ofdigoxin metabolites in digoxin immunoassay. Am J ClinPathol 1984;82:210-13.

14 De Villiers E-M, Gissmann L, Zur Hausen H. Molecularcloning of viral DNA from human genital warts. J Virol1981;40:932-5.

15 Durst M, Gissmann L, Ikenberg H, Zur Hausen H. Apapillomavirus DNA from a cervical carcinoma and itsprevalence in cancer biopsy samples from differentgeographical regions. Proc Natl Acad Sci USA 1983;80:3812-15.

16 Boshart ML, Gissmann L, Ikenberg H, Kleinheinz A,Scheurlen W, Zur Hausen H. A new type of

papillomavirus DNA, its presence in genital cancerbiopsies and in cell lines derived from cervical cancer.EMBO J 1984;3:1151-7.

17 Chan VT-W, Fleming KA, McGee JO'D. Detection ofsubpicogram quantities of specificDNA sequences on blothybridisation with biotinylated probes. Nucleic Acids Res1985;13:8083-91.

18 Burns J, Graham AK, Frank C, Fleming KA, Evans MF,McGee JO'D. Detection of low copy human papillomavirus DNA and mRNA in routine paraffin sections ofcervix by non-isotopic in situ hybridisation. J Clin Pathol1987;40:858-64.

19 Hcrrington CS, Burns J, Graham AK, McGee JO'D.Discrimination of closely homologous HPV types bynonisotopic in situ hybridisation: definition and deriva-tion of tissue lIms. Histochem J 1990;22:545-54.

20 Schwarz E, Freese UK, Gissmann L, et al. Structure andtranscription of human papillomavirus sequences incervical carcinoma cells. Nature 1985;314:111-14.

21 Coleman DV, Evans DMD. Biopsy pathology and cytology ofthe cervix. London: Chapman and Hall, 1988.

22 Schafer R, Zischler H, Epplen JT. DNA fingerprintingusing non-radioactive oligonucleotide probes specific forsimple repeats. Nucleic Acids Res 1988;16:9344.

23 Dooley S, Radtke J, Blin N, Unteregger G. Rapid detectionof DNA-binding factors using protein-blotting anddigoxygenine-dUTP marked probes. Nucleic Acids Res1988;16: 11839.

24 Mougin C, Guitteny AF, Fouque B, Viennet G, Teoule R,Bloch B. Histochemical detection of the messenger RNAscoding for calcitonin and calcitonin gene-related peptidein medullary carcinomas of the thyroid with radioactiveand biotinylated oligonucleotide probes. J Pathol 1990;160:187-94.

25 Popcscu NC, DiPaolo JA, Amsbaugh SC. Integration sitesofhuman papillomavirus 18 DNA sequences on HeLa cellchromosomes. Cytogenet Cell Genet 1987;44:58-62.

26 Mincheva A, Gissmann L, Zur Hausen H. Chromosomalintegration sites of human papillomavirus DNA in threecervical cancer cell lines mapped by in situ hybridisation.Med Microbiol Immunol 1987;1 76:245-56.

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