P53 autoantibody detection by enzyme-linked immunosorbent assay
The ELISA assay for p53 autoantibodies was developed in-house, based on the ELISA procedure of Lubin et al [3] in which the amount of specific p53 autoantibody recorded by ELISA was confirmed by Western blot and immunoprecipitation analyses. Briefly, 96-well assay plates (Falcon 3912; Becton Dickinson and Co, Oxnard, CA, USA) were precoated with baculoviral-derived, purified, wild-type, full-length, human recombinant p53 (0.1 ng/ml phosphate-buffered saline [PBS]) using 30 μl per well. After overnight incubation at 4°C, the plates were washed three times with PBS-0.1% Tween at room temperature. Wells were then blocked for 2 h with 250 μl PBS-0.5% Tween. After five washes with PBS-0.1% Tween, 30 μl of each plasma sample was incubated on ice for 2 h. The plate was again washed five times as above before addition of horse radish peroxidase-linked goat antiserum to human immunoglobulin (Sigma A8667; Sigma Aldrich Co Ltd, Poole, Dorset, UK) and incubation on ice for 1 h. After a further five washes, bound horse radish peroxidase was detected by conventional methods using O-phenylenediamine dihydrochloride (30 min), stopping with 3 mol/l H2SO4, and reading the optical density (OD) at 492 nm.
An OD reading of less than 0.4 was taken as a negative result. This cutoff was determined by titration of positive and negative samples (1/10, 1/50, 1/100 and 1/500) and gave good discrimination between positive and negative samples at 1/500 dilution. All assay runs included the same internal standards of a sample known to be positive at 1/500 dilution and a sample known to be negative at 1/10 dilution, in order to confirm assay reproducibility throughout the series.
The intial screen of all patients was at a plasma dilution of 1/10 in PBS-0.1% Tween. Samples from all patients with a positive OD reading at the 1/10 dilution were then re-assayed at 1/500 (see Sample processing, below). The positive and negative internal standards ensured that all results were directly comparable. Background controls received no p53 protein. Specificity controls used soluble p53 protein added to the positive plasma sample before assay; this resulted in loss of signal.
In one assay series we compared a commercial ELISA kit for p53 autoantibodies (produced by Dianova GmbH; licenced to CalBiochem-Novabiochem Corp; distributed by Oncogene Research Products, Cambridge, MA, USA; cat no. QIA 16) with our in-house ELISA. A total of 20 patients' samples were tested, representing a range of positive and negative readings. Two samples were scored strongly positive by the in-house assay, but only one of these two scored positive by the Dianova assay. Having established sensitivity, specificity and reproducibility of the in-house assay, we judged that this was superior to the commercial assay both in terms of sensitivity and of cost (£1 per test compared with £23 per test). The in-house assay was thus used throughout the study presented here.