Production of Antibodies Using Proteins in Gel Bands

Abstract

A number of methods for preparing proteins as antigens have been described (1). These include solubilization of protein samples in buffered solutions (2), solubilization of nitrocellulose filters to which proteins have been adsorbed (3), and emulsification of protein bands in polyacrylamide gels for direct injections (4–8). The latter technique can be used to immunize mice or rabbits for production of antisera or to immunize mice for production of monoclonal antibodies (9–11). This approach is particularly advantageous when protein purification by other means is not practical, as in the case of proteins insoluble without detergent. A further advantage of this method is an enhancement of the immune response, since polyacrylamide helps to retain the antigen in the animal and so acts as an adjuvant (7). The use of the protein directly in the gel band (without elution) is also helpful when only small amounts of protein are available. For instance, in this laboratory, we routinely immunize mice with 5–10 µg total protein using this method; we have not determined the lower limit of total protein that can be used to immunize rabbits. Since polyacrylamide is also highly immunogenic, however, it is necessary in some cases to affinity-purify the desired antibodies from the resulting anti-serum or to produce hybridomas that can be screened selectively for the production of specific antibodies, to obtain the desired reagent.