A tetrazolium method for staining peroxidase labels in blotting assays.
A sensitive staining method of horseradish peroxidase-labeled immunoglobulins on nitrocellulose membrane was established by employing a reaction chain leading to formazan formation with phenol as a substrate of peroxidase and NADH as a hydrogen donor to reduce nitro blue tetrazolium. Higher concentrations of NADH relative to phenol were necessary to increase the intensity of staining and to ensure a wide dose-response range of color production with respect to the applied enzyme activities. By an optimized tetrazolium method in combination with antibody-affinity blotting, as low as 4 ng/ml alpha-fetoprotein was detected and 3-4-fold greater color intensities in a working assay range as compared with those of existing methods were obtained. The present technique of peroxidase staining may prove to have a wide application for the enzyme immunoassay using blotting modalities.
D007124:Immunoenzyme Techniques; D009243:NAD; D009580:Nitroblue Tetrazolium; D019800:Phenol; D010636:Phenols; D013194:Staining and Labeling; D013778:Tetrazolium Salts; D000509:alpha-Fetoproteins / Q000032:analysis
D010636:Phenols; D013778:Tetrazolium Salts; D000509:alpha-Fetoproteins; D009243:NAD; D009580:Nitroblue Tetrazolium; D019800:Phenol