Purification of peroxidase-conjugated antibody for enzyme immunoassay by affinity chromatography on concanavalin A

Preparation of peroxidase-conjugated antibody contaminated with very small amounts of unconjugated antibody for highly sensitive enzyme immunoassays is described. The purification process is dependent upon the differential affinity of antibody and peroxidase-conjugated antibody for concanavalin A (Con A). Application of peroxidase and radioactive labelled IgG alone or as a mixture to Con A-Sepharose columns showed that 62-83% of IgG and 0% of peroxidase could be eluted under starting conditions, while 70-83% of peroxidase and about 6% of IgG could be eluted with appropriate buffer. The contamination of the conjugate with unconjugated antibody is dependent on the ratio of conjugated to unconjugated antibody in the mixture to be purified. Using this procedure on IgG conjugated with peroxidase with a ratio of unconjugated to conjugated IgG of about 2 : 1 a preparation was obtained where the ratio was changed to 0.1 : 1.