[29] Enzyme-linked immunoelectrotransfer blot techniques (EITB) for studying the specificities of antigens and antibodies separated by gel electrophoresis

This chapter describes the enzyme-linked immunoelectrotransfer blot techniques (EITB) for studying the specificities of antigens and antibodies separated by gel electrophoresis. The enzyme-linked immunoelectrotransfer blot technique (EITB) combines the high resolving power of gradient sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and the high sensitivity of the enzyme-linked immunosorbent assay (ELISA) to produce an extremely powerful qualitative tool for studying antigen–antibody pairs. Using this procedure, antigens electrophoretically resolved on SDS–PAGE are transferred onto nitrocellulose or diazo sheets and identified by ELISA methods. The chapter discusses the general comments about the technique. The EITB is conducted in three stages: (a) the antigen mixture that can be extremely complex, such as solubilized whole cells, is first resolved by gel electrophoresis (two-dimensional gels can also be used); (b) the resolved gel is then electrophoretically blotted onto nitrocellulose sheets; and (c) the blotted nitrocellulose is then developed by ELISA.