J Clin Pathol 1991;44:33-38 33 Interphase cytogenetics using biotin and digoxigenin labelled probes: III Increased J Clin Pathol: first published as 10.1136/jcp.44.1.33 on 1 January 1991. Downloaded from sensitivity and flexibility for detecting HPV in cervical biopsy specimens and cell lines C S Herrington, A K Graham, J O'D McGee Abstract background to the same extent as biotin. A monoclonal antibody to digoxin en- Digoxigenin labelled probes, however, are less abled sandwich techniques to be used for sensitive than biotinylated probes using single the detection of hybridised digoxigenin step detection methods.6 The sensitivity of labelled probes in cultured cells and detection procedures can be enhanced using paraffin wax sections. This system has antibody sandwich techniques using a linker greater flexibility than alkaline phos- antibody to bridge between antibody directed phatase conjugated polyclonal anti- against reporter molecule and an enzyme con- digoxigenin antibody and permits the jugate.9 A refinement of this concept is to use use of alternative detector enzymes, such biotinylated antibodies, which can be used to as horseradish peroxidase and fluores- bridge between the first antibody, and an cence labels. The APAAP detection avidin enzyme conjugate.'0 This gives system that does not require the use of increased sensitivity due to the high affinity of biotin can also be used in situations avidin for biotin (dissociation constant = I0-` where endogenous biotin is a problem. M) and improved specificity when the first The low level of background staining antibody is of monoclonal type. combined with precise substrate deposi- Digoxigenin, the aglycone derivative of the tion of the amplified peroxidase system cardiac glycoside digoxin, has high affinity for gives higher sensitivity and resolution. antibodies to digoxin."'3 This suggested to us This permits localisation of closely that the antibodies which have been adjacent chromosomal loci in interphase developed for the monitoring and treatment of nuclei. The most sensitive peroxidase clinical digoxin toxicity would increase the http://jcp.bmj.com/ based digoxigenin detection system sensitivity of detection of digoxigenin labelled visualises two and a half to 12 copies of probes in NISH. human papillomavirus (HPV) per The aims of this investigation were: (1) to nucleus. This system is also suitable for develop systems of high sensitivity and the analysis of low copy number HPV resolution for digoxigenin labelled probes infection of cervical tissues. which are applicable to interphase cyto- genetics-for the latter, CaSki and HeLa cells on September 30, 2021 by guest. Protected copyright. were used as a model system; and (2) to adapt Interphase cytogenetics encompasses, among detection systems for digoxigenin labelled other things, visual discrimination of probes so that they are more sensitive and individual chromosomes or genes in inter- flexible for detection of genes in archival phase nuclei.'-3 This has previously been biopsy specimens. performed by fluorescence45 and more recently by enzyme based chromogenic procedures using biotin and digoxigenin Methods labelled probes.' During the course of the All chemicals were obtained from Sigma (UK) latter work, it became apparent that the detec- or BDH (UK) unless otherwise stated. tion systems for biotin or digoxigenin labelled The HPV6 probe was the amp2 fragment probes using alkaline phosphatase based cloned into pBR322,'4 the HPV16 probe the detection systems did not give sufficient whole viral genome in pAT153,'5 and HPV18 resolution to discriminate between closely 16 University of Oxford the whole viral genome in pBR322. Biotin and Nuffield Department adjacent chromosomal loci. digoxigenin were incorporated into plasmid of Pathology and Digoxigenin labelled probes have been used DNAs by nick translation, and the degree of Bacteriology, John both as alternatives and in addition to biotin Radcliffe Hospital, labelling and size of fragments were as Headington, Oxford labelled probes in non-isotopic in situ previously described.6 17 OX3 9DU hybridisation (NISH).67 The main advantage Paraffin wax sections were prepared and C S Herrington of digoxigenin over biotin labelled probes for nucleic acids unmasked as previously A K Graham NISH is that digoxigenin (a plant alkaloid J O'D McGee described.6 analogue), unlike biotin, is not present con- Correspondence to: HeLa 229 cells (ATCC, USA) were grown to Professor McGee stitutively in mammalian cells. Hence, in confluence in 80 cm2 flasks in RPMI 1640 Accepted for publication immunohistochemical detection systems, medium (Gibco, UK) supplemented with L- 16 August 1990 digoxigenin does not generate endogenous glutamine (1 x; Gibco, UK); penicillin/ 34 Herrington, Graham, McGee streptomycin (1 x; Gibco, UK); and 20% fetal diluted 1 in 600 in TBT, washed in TBS, and calf serum (Gibco, UK). Detachment was the signal was developed using NBT/BCIP achieved using trypsin/EDTA solution (see below). (Gibco, UK) and the cells were pelleted at (b) Three step procedure Sections were J Clin Pathol: first published as 10.1136/jcp.44.1.33 on 1 January 1991. Downloaded from 600 x g. After two washes in PBS the cells incubated in monoclonal antidigoxin (Sigma, were fixed in 10% formalin, embedded in agar, UK) diluted 1 in 10 000 in TBT. Subsequent and processed to paraffin wax in the usual way. detection was as for the three step detection of Sections of 4 ,gm were cut and unmasked using biotinylated probes, with second incubation in proteinase K at a concentration of 1 mg/ml in biotinylated rabbit anti-mouse (F(ab')2 frag- PBS for 15 minutes at 37°C. ment) and the third incubation in avidin- CaSki cells were grown onto slides, fixed in peroxidase (dig-3-P/C) or avidin-alkaline paraformaldehyde, and nucleic acids unmasked phosphatase (dig-3-A/N). as previously described.6 (c) APAAP system Slides were incubated in monoclonal a4tidigoxin as in (a), followed by HYBRIDISATION rabbit anti-mouse immunoglobulin (Dako, Aliquots of hybridisation mix (8 u1) containing UK), diluted 1 in 50 in TBT, then APAAP 2 ng/pl ofbiotin or digoxigenin labelled HPV6, complex (Dako, UK) diluted 1 in 50 in TBT. HPV 16, or HPV18 were added to each well on Signal was developed using NBT/BCIP. multispot slides which were covered with a (d) Fluorescence detection Slides were treated 14 mm glass coverslip (Chance, UK) and the as for the three step detection method in (b) slides placed in a moist Terasaki plate. except that streptavidin-fluorescein iso- Hybridisation mix consisted of 50% forma- thiocyanate (Dako, UK) was used in the final mide, 5% dextran sulphate, 2 x SSC, and 0 05 step. Optimal results were obtained with a mol/l TRIS-HCI, pH 7 3; 1 x SSC = 0 15 dilution of .1 in 20 of the fluorescent conjugate mol/l sodium chloride, 0-015 mol/l sodium in TBT containing 5% non-fat milk. citrate. Sections and probes were simul- taneously denatured at 95°C for 15 minutes on a SUBSTRATE PREPARATION solid stainless steel plate in a hot air oven and Nitrobluoe tetrazolium/5-bromo-4-chloro- then hybridised at 420C for 2 hours. 3-indolyl phosphate (NBT/BCIP) and amino- ethylcarbazole (AEC) were made as previously DETECTION OF HYBRIDISATION SIGNAL described.618 Diaminobenzidine (DAB) was All antibody/avidin incubations were carried diluted to 0-5 mg/ml in distilled water and out for 30 minutes at 22°C unless otherwise stored in 5 ml aliquots at -20°C. Hydrogen stated. Slides were washed in three changes of4 peroxide was added to DAB (final concentra- x SSC at 22°C (five minutes each change), tion 03%) immediately before use. After probe soaked in blocking agent TBT (0.05 mol/l detection slides were air dried at 42°C and mounted in TRIS-HCI, 0-10 mol/l sodium chloride (pH glycerol jelly (for enzyme/ http://jcp.bmj.com/ 7 2) containing 3% (w/v) bovine serum chromogen reactions). For fluorescence albumin and 0-05% Triton 100 (v/v)) at 22°C preparations, PBS/glycerol (1:9 v/v) contain- for 10 minutes. Subsequent visualisation of ing 2-3% diazobicyclo-octane (DABCO) signal was determined by the reporter (Sigma, UK) was used. molecule. Frequency distributions were compared using the two tailed Mann-Whitney U test with Biotin labelled probes a correction for tied values. on September 30, 2021 by guest. Protected copyright. (a) Single step procedure Sections were incubated in avidin-alkaline phosphatase or avidin-peroxidase (Dako, UK) diluted 1 in 100 Results in TBT. Unbound conjugate was removed by The results of single step detection methods for washing for five minutes twice in 50 mM biotin and digoxigenin probes have previously TRIS-HCI, 10 mM NaCl, pH 7 2 (TBS), and been reported.67 These were less sensitive than the sections incubated in the appropriate the three step procedures described below. substrate. (b) Three step procedure Sections were COMPARATIVE SENSITIVITY AND RESOLUTION OF incubated first in monoclonal mouse anti- DIGOXIGENIN AND BIOTIN IN INTERPHASE CELLS biotin (Dako, UK) diluted 1 in 50 in TBT, The three step peroxidase AEC detection washed in TBS (twice at 10 minutes each), and method for digoxigenin labelled probes (dig-3- then in biotinylated rabbit anti-mouse (F(ab')2 P/C) produced very low background staining fragment, Dako, UK). After washing in TBS, and enhanced resolution in CaSki and HeLa the third incubation was in either avidin alka- cells (figs 1 and 2). Discrete HPV signals were line phosphatase (bio-3-A/N), or streptavidin considerably more easily resolved when com- peroxidase (Dako, UK) (bio-3-P/C). The pared with the three step alkaline phosphatase former was diluted 1 in 50 and the latter 1 in NBT/BCIP detection system for digoxigenin 100 in TBT containing 5% non-fat milk. After (dig-3-A/N) (fig 1); the latter is the counterpart washing in TBS, sections were incubated in the ofthe most sensitive system we have developed appropriate substrate (see below).