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A tetrazolium method for peroxidase staining: Application to the antibody‐affinity blotting of α‐fetoprotein separated by lectin affinity electrophoresis

A sensitive staining method of peroxidase labels on nitrocellulose membrane was developed with phenol as an enzyme substrate and NADH as a reducing agent for Nitro Blue tetrazolium to form formazan. The tetrazolium method for peroxidase staining, when applied to the antibody‐affinity blotting of α‐fetoprotein (AFP) after agarose gel electrophoresis, allowed us to detect as little as 3 ng/mL AFP with 4 μL of sample volume, covering a wider linear range of color production up to 200 ng/mL AFP as compared with the conventional diaminobenzidine stain. Accordingly, the proportion of intensities of AFP bands separated by lectin affinity electrophoresis could be determined without employing a calibration curve for each assay.