J Clin Pathol 1997;50:143-147 143 A novel quantitative immunoassay system for p53 using antibodies selected for optimum designation J Clin Pathol: first published as 10.1136/jcp.50.2.143 on 1 February 1997. Downloaded from of p53 status M D Thomas, G G McIntosh, J J Anderson, D M McKenna, A H Parr, R Johnstone, T W J Lennard, C H W Horne, B Angus Abstract The prediction of likely outcome in cancer Aim-To develop a highly sensitive and would permit allocation of patients to appro- specific enzyme linked immunosorbent priate aggressive therapeutic regimens in in- assay (ELISA) system for analysis of p53 stances where prognosis is poor; and con- protein in cancer lysates. versely, patients with good prognosis could be Methods-The anti-p53 monoclonal anti- spared dangerous and debilitating treatment. bodies D07, 1801, BP53.12, and 421, and Mutations of the tumour suppressor gene p53 anti-p53 polyclonal antiserum CM1 were can result in dysfunction, with accumulation of assessed by immunohistochemistry and the inactive/mutated protein which increases genomic instability leading to the expression western blot analysis to identify those and progression of the metastatic phenotype.' most suitable for determining p53 status Accumulation ofp53 has been shown in several of cancer celis. Antibodies with desired different cancers.2" In breast cancer, we and characteristics were used to develop a others have shown that accumulation of p53 is non-competitive sandwich type ELISA associated with poor clinical prognosis.57 system for analysis of p53 expression in Although p53 overexpression can be detected cancer cytosols. Using the ELISA, p53 immunohistochemically, only a qualitative esti- protein concentrations were measured in mation can be made. It has, however, been a small series of breast cancers, and the proposed that quantitative analysis of p53 pro- quantitative values compared with p53 tein concentrations in cancer will provide addi- immunohistochemical data of the same tional objective prognostic information.8 Sev- cancers. eral groups have described non-competitive Results-DO7 and 1801 gave the most sandwich type immunological based assay sys- specific and reliable results on immuno- tems, using monoclonal/polyclonal antibody http://jcp.bmj.com/ histochemisry and western blot analysis. combinations, for quantitative analysis of p53 Using these two antibodies, a non- in tumour cytosol.9-' Using immunohisto- competitive sandwich type ELISA system chemistry, we have recently reported that was developed to p53 assessment of p53 status in breast cancer tissue analyse quantita- sections is critically dependent upon the tively. Analysis of the breast cancer series antibodies used for screening, and influences showed a good correlation between immu- the significance of the prognostic data on September 23, 2021 by guest. Protected copyright. nohistochemistry and the ELISA- generated.5 Similarly, the choice of antibodies tumours were generally positive using to be applied in quantitative p53 assays may both techniques. Discrepancies were exert a notable effect on the final estimate of noted however: some cancers were immu- tumour p53 concentrations. Selection of the Department of nohistochemically negative but ELISA Pathology, Royal most appropriate antibodies will assure the Victoria Infirmary, positive. One explanation for this may be specificity and reproducability of the data on University of that the ELISA is more sensitive than which subsequent assessments of patient prog- Newcastle upon Tyne, immunohistochemistry. nosis may be based. Newcastle upon Tyne Conclusion-The p53 ELISA system is a NEl 4LP double monoclonal M D Thomas non-competitive anti- Methods sandwich G G McIntosh body method, using D07 and TUMOUR TISSUE J J Anderson 1801 which have been shown to be highly Samples of mammary carcinoma tissue were D M McKenna specific for p53 protein by immunohisto- collected at surgery. The tumour was dissected A H Parr and western blot The R Johnstone chemistry analysis. free of superfluous tissue and representative C H W Horne lower threshold of the assay is 0.1 ng/nd samples processed for the production of frozen B Angus analyte in an enriched recombinant p53 sections and cytosols. preparation. As p53 is now regarded as a Department of Clinical protein associated with prognosis in Surgery CYTOSOL PREPARATION T W J Lennard breast and other cancers, the assay may p53 was quantified in cytosol extracts prepared have clinical applications. for routine estimation of oestrogen/ Correspondence to: (7 Clin Pathol 1997;50:143-147) progesterone receptor analysis. Frozen tumour Dr B Angus. tissue (approximately 5 mm2) was powdered Accepted for publication Keywords: p53; p53 antibodies; immunohistochemis- using a micro-dismembranator and resus- 5 November 1996 try; western blot; quantitative ELISA. pended in 1 ml HEPES/EDTA buffer (20 mM 144 Thomas, McIntosh, Anderson, McKenna, Parr, J7ohnstone, et al HEPES, 1.5 mM EDTA, pH 7.4) containing WESTERN BLOT ANALYSIS protease inhibitors: aprotinin (2 jg/ml), leu- Specificity of the p53 antibodies was assessed J Clin Pathol: first published as 10.1136/jcp.50.2.143 on 1 February 1997. Downloaded from peptin (2 jg/ml), phenylmethylsulphonyl fluo- using western blot analysis against a cell lysate ride (PMSF) (50 jig/ml), and pepstatin (1 jg/ of the breast adenocarcinoma cell line MDA- ml). Further disruption was achieved using MB-231. Cells mechanically harvested from sonication. Owing to the heat lability of the confluent 250 ml tissue culture flasks were proteins to be assayed, the samples were main- washed three times with PBS and lysed in tained on ice throughout the process. The 1.5 ml ice cold Laemmli buffer (0.125 M Tris, tissue/cell lysate was clarified by ultracentrifu- 4% SDS, 40% glycerol,1 % bromophenol blue, gation, 50 000 x g at 4°C for 40 minutes. and 5% mercaptoethanol, pH6.8). The lysate Cytosol fractions were collected, and stored at was held on ice, sonnicated using a single five -70°C prior to analysis for p53. Samples were second burst, and stored at -70°C until analysed within two weeks of preparation. required. Samples of lysate were fractionated by discontinuous SDS-polyacrylamide gel electrophoresis, using a 10% T, 2.6% C, poly- ESTIMATION OF TOTAL PROTEIN CONCENTRATION acrylamide gel. Proteins were transferred onto Total protein concentration of the cytosol nitrocellulose membrane using a semi-dry preparation was determined in relation to electroblotting system. Blots were probed with bovine serum albumin (BSA) using bicin- p53 antibodies D07, 1801, BP53.12, 421, choninic acid (BCA) protein assay kits (Pierce each at 0.5 jig/ml and with CM1 at dilutions of and Warriner Ltd). 1 in 1000 and 1 in 2000. Reacted p53 antibod- ies were detected using alkaline phosphatase conjugated goat anti-mouse immunoglobulin p53 ANTIBODIES or goat anti-rabbit immunoglobulin polyclonal Anti-p53 monoclonal antibodies D07, 1801 anti-sera. Blots were developed with 0.1 mg/ml and BP53.12, and the rabbit anti-p53 polyclo- nitroblue tetrazolium/0.15 mg/ml BCIP (5- nal antiserum CM1 were all obtained from bromo-4-chloro-3-indolyl phosphate), in de- Novocastra Laboratories Ltd, Newcastle upon velopment buffer (0.1 M Tris buffer, 0.1 M Tyne, UK. Anti-p53 monoclonal antibody 421 NaCl and 50 mM MgCl2, pH 9). was obtained from Oncogene Science Ltd (Sera Laboratories Ltd, Crawley Down, UK). SELECTION OF p53 ANTIBODIES FOR ELISA The concentration of monoclonal antibodies DEVELOPMENT 1801, D07, and BP53-12 was determined p53 antibodies for the development of the using IgG radial immunodiffusion. ELISA were selected on the basis of their per- formance in immunohistochemistry, for opti- IMMUNOHISTOCHEMISTRY mum assignment of p53 status, and their To assess the immunohistochemical perform- specificity for p53 in western blot analysis. http://jcp.bmj.com/ ance of antibodies directed against p53 for the Using these criteria, monoclonal antibodies assignment of p53 status, we stained serial 1801 and D07 were selected for use in assay cryostat sections of breast cancers with test development. monoclonal antibodies D07, 1801, BP53.1 and 421, and the rabbit polyclonal antiserum PREPARATION OF BIOTINYLATED Do7 CM1. Sections, 3 jim thick, of cryopreserved Monoclonal antibody D07 was purified from hybridoma culture supernatant by absorption on September 23, 2021 by guest. Protected copyright. tumour tissue were cut and air dried onto with protein A (Sigma). Antibody was eluted at microscope slides. Endogenous tissue peroxi- pH 3 and dialysed against 0.1 M borate buffer, dase activity was quenched using 0.6% hydro- pH 8.8. Purified antibody was then reacted gen peroxide in methanol, for five minutes. with ester of biotinamidocaproate N-hydroxy- Subsequently, non-specific immunostaining succinimide, in dimethyl sulphoxide (DMSO), was blocked by incubating the sections with (100 jg ester/mg monoclonal antibody) for 5% lamb serum/phosphate buffered saline four hours at room temperature. The conjuga- (PBS) for 30 minutes. Test p53 monoclonal tion reaction was terminated by the addition of antibodies at 0.5 jg/ml, or rabbit anti-serum 1 M ammonium chloride (259 l/mg mono- CM1 at a dilution of 1 in 1000 were applied clonal antibody), and the products extensively and incubated with sections for one hour at dialysed against 0.1 M Tris/HCl, pH 7.4. room temperature. Binding of p53 antibody was detected using biotinylated goat anti- ELISA PROCEDURE mouse or goat anti-rabbit polyclonal anti- A sandwich type ELISA system was developed serum and streptavidin/biotinylayed horseradish for the quantitative analysis of p53. Solid phase peroxidase complex (Dako, High Wycombe, monoclonal antibody 1801 was used as the p53 UK). Sections were developed with 3,3- immobiliser, and biotinylated D07 as the p53 diaminobenzidine tetrahydrochloride (Sigma, detector. In brief, Immulon-1 96-well ELISA Poole, Dorset, UK) in Tris buffered saline trays (Dynatech Laboratoies, Billinghurst, UK) (TBS) containing 0.3% hydrogen peroxide, were coated overnight at 4°C with 50 jl/well counterstained with Meyer's haematoxylin, de- 10 jg/ml 1801 in PBS.