Diffusion-in-gel enzyme-linked immunosorbent assay (DIG-ELISA). Quantification of antigen by diffusion over an...

The diffusion-in-gel enzyme-linked immunosorbent assay (DIG-ELISA) has previously been used for quantification of antibodies. In the present study, using bovine serum albumin as antigen and a corresponding rabbit antiserum as antibody source, the DIG-ELISA principle was used for quantification of antigen. Antibodies were immobilized on a plastic surface, over which the antigen was allowed to diffuse in an agarose gel. The antigen-antibody reaction zones were then detected by binding of an enzyme-labelled antibody directed against the antigen. The enzyme-substrate reaction, yielding a coloured reaction product, was visualized in a substrate-containing gel. The density and quality of immobilized antibody on the surface was found to be critical. Reproducible results were obtained only when antibodies purified with affinity chromatography on immobilized antigen were used. After 46 h of diffusion the diameter of the reaction zones was directly proportional to the logarithm of the antigen concentration in the range 0.4 mg/1-1 g/1. The variability of the method (CV) was 3%.