Production of monoclonal antibodies to placental alkaline phosphatase: Preliminary characterisation includes identification of one antibody reactive with routinely fixed histological preparations

Abstract

We describe the production and preliminary characterisation of a set of monoclonal antibodies (MAbs) raised against placental alkaline phosphatase (PLAP). Different forms of antigen presentation, PLAP or PLAP-like expressing whole cells, placental membranes or purified PLAP were used to immunise BALB/c mice. Initial screening was carried out against the immunising material by ELISA, against fresh frozen placental sections by immunostaining and against purified PLAP using an enzyme capture assay. The most successful fusions were those following whole cell immunisation, producing 27 antibodies that all reacted with both the placental and testicular form of enzyme. These all showed a broadly similar pattern of reactivity when tested against a range of human malignant cell lines. Further characterisation identified one antibody, 8B6, as strongly reactive with formal in-fixed paraffin-embedded placental sections. This antibody also performed well when tested against a range of normal and malignant routinely fixed tissue sections. Of 14 antibodies analysed for immunoglobulin isotype, 10 were of the IgG, subclass. In competitive binding studies with 7 antibodies to discriminate epitopes, at least 4 distinct binding sites were identified. By Scatchat-d analysis on 4 of these antibodies, binding constants of 3 were within the range 3.5-5.3 × 10⁻⁹m. Unusually the 4th antibody appeared to recognise 2 separate antigen sites with binding constants of 2.1 and 7.5 × 10-9^m. In a preliminary study to compare patterns of reactivity of a selection of the new antibodies with a limited number of sera from smokers and seminoma patients, results indicate their potential for further typing within the placental group of enzymes.