Preparation of biotinylated, affinity-purified antibodies for enzyme-linked immunoassays using blotting membrane as an antigen support

A method combining the affinity purification and biotinylation of antibodies has been developed utilizing the high-capacity binding of blotting membranes and glutaraldehyde treatment to immobilize antigen. Following reaction of the antisera with the membrane-bound antigen, the biotinylation of the antibodies is performed in situ, before release of the IgG-biotin complex with low pH buffer. The antibody is able to recognize antigen in subsequent reactions in enzyme-linked immunosorbent assays, and preliminary results have shown that the antibody can also be used in immunocytochemical localization of antigen in frozen sections. Biotinylation in situ may protect the variable region of the IgG compared to antibodies biotinylated in solution, thus increasing their antigen recognition. The method has the advantage of being suitable for multiple use of the antigen-coated membrane, making it particularly attractive where only small amounts of antigen may be available. (C) 1996 Academic Press, Inc.