Abstract

PIP: 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. The EITB is conducted in 3 stages: 1) the antigen mixture is resolved by gel electrophoresis, 2) the resolved gel is then electrophoretically blotted onto nitrocellulose sheets, and 3) the blotted nitrocellulose is then develped by ELISA. This article details all the procedures involved in this methodology and describes the necessary materials. There is the theoretical danger that not all the antigenic sites can retain the native configuration after SDS treatment to allow recognition by appropriate antibodies. However, experiments with IgG, BSA, and other defined antigens tend to negate this argument. The general use of transfer blotting electrophoretically separated biologic molecules began with the DNA-RNA hybridization studies of Southern, with a technique now referred to as the Southern blot.