Monoclonal antibodies for use in an immunoradiometric assay for α-foetoprotein

Abstract

The advantages offered by a mouse IgG 1 monoclonal antibody to human α-foetoprotein (AFP) for the preparation of [ 125I]antibody for use in an immunoradiometric assay (IRMA) have been investigated. The antibody was isolated from ascites fluid by sodium sulphate precipitation followed by gel filtration on Sephadex G-200. The freeze-dried powder and solutions thereof were stable and were used for iodination to 1 atom 125I/ molecule antibody by the chloramine-T procedure. At high antigen concentrations 70–80% of the added [ 125I]Ab was present in the sandwich. Linear response curves in the range 1–100 μg antigen/l incubate were obtained when [ 125I]Ab was in slight excess. In this region an Ag : Ab ratio of 1.9 : 1 was obtained which is consistent with the saturation of a bifunctional antibody. Although non-specific binding (in the absence of antigen) was consistently <0.1% of added [ 125I]Ab, this was the main factor in determining assay detection limits. The serum AFP levels from both non-pregnant and pregnant subjects as measured by the IRMA using the [ 125I]monoclonal Ab and by radioimmunoassay (RIA) using a sheep antiserum to AFP were in excellent agreement. The IRMA was manipulatively simple, employed a shorter incubation time (2 h), required shorter counting times than the RIA and gave a much wider working range. The provision of a monoclonal antibody for labelling removes the one major practicability barrier which otherwise limits the development and use of the potentially superior IRMA system.