The application of glucose oxidase-labeled antibodies for the detection of proteins on nitrocellulose

We have evaluated the sensitivity of immunostaining with glucose oxidase for the detection of monomeric human serum albumin (HSA) and monomeric human immunoglobulin G (IgG). A modification of a histochemical procedure was utilized by replacing phenazine methosulfate (PMS) with 1-methoxyphenazine methosulfate (mPMS) and by replacing Tris-HCl with Tris-citrate to improve the solubility of the tetrazolium compounds tested. mPMS is less sensitive to light, and may be stored for long period in solution; it is now used routinely by histochemists in place of PMS in dehydrogenase cytochemistry. pH values of 6.3–8.3 were tested, with the reaction at pH 8.3 providing a slight increase in sensitivity. The reaction rate increased markedly as the pH became more alkaline. The minimum quantity of HSA detected was 3 ng applied directly to nitrocellulose and 10 ng when blotted. Human IgG was routinely detected at 250 pg and occasionally at 100 pg when dotted on the nitrocellulose.