Journal of Immunological Methods, 99 (1987) 173-177 173 Elsevier JIM 04330 A rapid method of epitope analysis using Superose 12 gel filtration A study with monoclonal antibodies to chicken riboflavin carrier protein Anjali A. Karande, Sandhya S. Visweswariah and P. Radhakantha Adiga Department of Biochemistry, lndian Institute of Science, Bangalore 560 012, India (Received 3 October 1986, accepted 12 January 1987) A novel and rapid method is described for determining the epitope specificities of monoclonal antibodies. The method utilises a highly reproducible, wide range gel filtration medium, Superose 12. nmol of monoclonal antibodies are incubated with pmol quantities of radiolabelled antigen, and the mixture filtered through Superose 12. Fractions are collected and radioactivity monitored. The size of the resultant immune complex indicates whether two monoclonal antibodies (mixed with the antigen) recognise the same or different epitope(s) of the antigen. The applicability of the above analytical method is illustrated with monoclonal antibodies raised against chicken egg riboflavin carrier protein. Key words: Epitope analysis; Superose gel filtration; Monoclonal antibody; Riboflavin cartier protein, chicken Introduction The method most commonly employed for characterising the epitope specificities of different monoclonal antibodies (MAbs) to an antigen is a blocking solid-phase RIA (Stahli et al., 1983). For this purpose it is mandatory to radiolabel the MAbs, which in turn demands as a prerequisite, purification of the MAbs from either the culture supernatants or ascitic fluids. Much time and ef- fort are needed to obtain sufficient quantities of Correspondence to: P.R. Adiga, Department of Biochem- istry and U.G.C. Centre of Advanced Study, Indian Institute of Science, Bangalore 560 012, India. Abbreviations: BSA, bovine serum albumin; cRCP, chicken riboflavin carrier protein; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; FPLC, fast protein liquid chromatography; IgG, immunoglobulin G; MAb, monoclonal antibody; Mr, relative molecular mass; PBS, phosphate- buffered saline; RIA, radioimmunoassay. the highly purified IgG to discriminate one MAb from another. With a view to finding a quicker and easier alternative, we have developed a novel method for characterising the MAbs which re- quires very small amounts of culture supernatants and thus can be employed soon after subcloning, so that only clones of potential interest can be selected and cultured. Furthermore, it has been frequently observed that after most successful cell fusions, the maximum number of hybrids ob- tained secrete antibodies to the immunodominant epitopes on an antigen (Retegui and Paladini, 1986). Consequently, a number of clones secreting similar antibodies are identified only after exten- sive labour. With the new and quicker method of analysis described below, it is anticipated that the redundant culturing of too many hybrids secreting antibodies directed to the same epitope can be avoided early in the process of developing a bat- tery of MAbs to any antigen. 0022-1759/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)