Subclass analysis and purification of monoclonal antibodies

Once hybridoma lines have been established that secrete monoclonal antibodies of the desired specificity, it is useful to characterize the antibody subclass before mass-producing the antibody (1,2). IgG isotypes may be separated by chromatography on protein A-sepharose, and such affinity columns are useful for concentrating as well as purifying antibody from tissue culture supernatants. When the isotype has been determined, using commercial isotype-specific antisera conjugated to different fluorochromes and monoclonal antibodies of different isotype and specificity, double indirect immunofluorescence can allow similarities in antigen distribution to be assessed. Ideally, antibody should be purified by affinity chromatography using insolubilized antigen columns (see Chapter 3 ), although such a technique obviously requires adequate supplies of purified antigen. For many practical purposes, a crude globulin fraction from ascitic fluid is quite adequate, however.