Journal of Immunological Methods, 114 (1988) 49-52 49 Elsevier JIM04915 An immunisation protocol which enhances the frequency of antigen-specific monoclonal antibody production O. Shibier, S.M. Hampton and V. Marks Department of Biochemistry, Division of Clinical Biochemistry, Universityof Surrey, Guildford, Surrey GU2 5XH, U.K. (Received21 April 1988,revisedreceived13 May 1988, accepted17 May 1988) An immunisation protocol has been developed for small molecular weight antigens which results in a high percentage of specific hybridomas being produced after cell fusion. An enzyme-linked immunosor- bent assay (ELISA) was developed for screening the desired antibodies in the culture supernatants. A conventional immunisation regimen was followed by doses of antigen in sterile water on each of the last 4 days before fusion. A range of antigen doses was used and the specific efficiency of fusion was increased by selection of the optimum amount. The antigen used as a model antigen in these experiments was biosynthetic human insulin. Key words: Immunization; Monoclonal antibody;Insulin Introduction The technique of hybridising spleen cells from immunised mice with myeloma cells has become a powerful tool for producing monoclonal antibod- ies (KiShler and Milstein, 1975). The success of hybridoma technology depends primarily on the availability of antigen-specific lymphocytes to act as the fusion partners (Rathyen and Underwood, 1985). Stahli et al. (1980) investigated immunisa- tion protocols and showed that injecting animals with large amounts of antigens caused blast cell proliferation with a corresponding increase in the specific efficiency of fusion. We were interested in modifying this protocol for haptens and at the same time reducing the amount of antigen re- quired whilst maintaining a high specific effi- ciency. Correspondence to: O. Shibier, Department of Biochem- istry, Divisionof Clinical Biochemistry, Universityof Surrey, Guildford, SurreyGU2 5XH, U.K, Insulin has been well characterised both physi- cally and chemically (Blundell et al., 1972; Crumpton, 1974; De Weck, 1974; Reichlin, 1975; Atassi, 1978) and has been the object of intense study by immunologists for several years (Schroer, 1981). In this study we used human insulin as the antigen since in addition to its other advantages it is both easy to obtain and inexpensive. Materials and methods Antigen Biosynthetic human insulin was supplied by Eli Lilly, Basingstoke. Preparation of immunogen Human biosynthetic insulin conjugated to egg albumin. Insulin (80 mg) was dissolved in 20 ml distilled water. To this was added 13 ml 2.5% glutaraldehyde solution and 160 mg egg albumin (Sigma, Poole, Dorset) dissolved in 27 ml distilled 0022-1759/88/$03.50 © 1988 ElsevierSciencePublishers B.V.(BiomedicalDivision)