Antibody‐affinity blotting, a sensitive technique for the detection of α‐fetoprotein separated by lectin affinity electrophoresis in agarose gels

Abstract

Abstract A sensitive technique of antibody‐affinity blotting was developed for the detection of small amounts of human α‐fetoprotein (AFP) in the presence of high background lectins and other serum proteins in agarose‐gel affinity electrophoresis. AFP was specifically transferred by blotting to nitrocellulose membranes which were precoated with affinity‐purified horse or goat polyclonal antibodies to human AFP. The AFP transfers were treated with rabbit immunoglobulins to human AFP, followed by affinity‐purified goat anti‐rabbit IgG(H+L)‐horseradish peroxidase conjugate for color development with 3,3′‐diaminobenzidine. The method allowed us to detect as low as 4 pg/mm 2 AFP (or 4 ng/ml AFP with a 3 μl sample volume applied to a 0.5 × 6 × 1 mm trough). In lectin affinity electrophoresis, AFP bands constituting more than 10% of the total AFP were quantitatively detected with 5 μl of 125–500 ng/ml AFP applied to a 1 × 5 × 1 mm trough. When horse antiserum to AFP or its immunoglobulin fraction was used to precoat nitrocellulose membranes, the background stain due to concanavalin A(Con‐A) in agarose gel became marked. This was eliminated by washing the transfers with 0.2 M α‐methyl‐D‐mannoside before subsequent antibody treatments. The washing with α‐methyl‐D‐mannoside also counteracted the uneven staining of separated AFP bands by Con‐A. The antibody‐affinity blotting suits best for the quantitative transfer of a specific protein to nitrocellulose membrane in the presence of varying amounts of other background proteins.