Journal of Immunological Methods, 50 (1982) 133--144 133 Elsevier Biomedical Press MONOCLONAL ANTIBODIES FOR USE IN AN IMMUNORADIOMETRIC ASSAY FOR a-FOETOPROTEIN W.M. HUNTER *, J.G. BENNIE *, D.J.H. BROCK ** and V. VAN HEYNINGEN *** * MRC Immunoassay Team, 2 Forrest Road, Edinburgh EH1 2QW, ** Department of Human Genetics, Edinburgh University, Western General Hospital, Crewe Road, Edin- burgh EH4 2XU and * ** MRC Clinical and Population Cy togenetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, U.K. (Received 6 July 1981, accepted 30 October 1981) The advantages offered by a mouse IgGl monoclonal antibody to human a-foetopro- tein (AFP) for the preparation of [12SI]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 12sI/ molecule antibody by the chloramine-T procedure. At high antigen concentrations 70-- 80% of the added [12SI]Ab was present in the sandwich. Linear response curves in the range 1--100 pg antigen/1 incubate were obtained when [12SI]Ab was in slight excess. In this region an Ag : Ab ratio of 1.9 : 1 was obtained which is consistent with the satura- tion of a bifunctional antibody. Although non-specific binding (in the absence of antigen) was consistently <0.1% of added [ 12 Si]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 [ 12si ]monoclonal Ab and by radioimmunoassay (RIA) using a sheep antiserum to AFP were in excellent agreement. The IRMA was manipula- tively 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 practica- bility barrier which otherwise limits the development and use of the potentially superior IRMA system. Key words: monoclonal antibodies -- o~-foetoprotein -- immunoradiometric assay INTRODUCTION The radioimmunoassay (RIA) procedure which involves the competition between unlabelled and labelled antigen for a fixed and limited amount of antibody (Yalow and Berson, 1960) is in widespread use for the microdeter- mination of proteins. Miles and Hales (1968) described an immunoradio- metric assay (IRMA) which employs similar reagents and techniques but is fundamentally different in that a radiolabelled antibody (*Ab) is used to 0022-1759/82/0000--0000/$02.75 © 1982 Elsevier Biomedical Press