An unlabeled antibody method using glucose oxidase-antiglucose oxidase complexes (GAG): a sensitive alternative to immunoperoxidase for the detection of tissue antigens.

Abstract

Immunoenzyme staining for tissue antigens using glucose oxidase as marker enzyme was compared with immunoperoxidase techniques. Both the indirect, conjugated antibody method and the unlabeled antibody procedure employing preformed complexes of glucose oxidase-antiglucose oxidase (GAG) and peroxidase-antiperoxidase (PAP) were used to stain carcinoembryonic antigen (CEA) in paraffin tissue sections. The localization of CEA within a tissue specimen was the same in all cases, and background staining was minimal. The percentage of positive specimens detected with GAG and PAP was similar but was slightly greater with glucose oxidase compared to peroxidase conjugates. The glucose oxidase conjugates and GAG were similar to the comparable immunoperoxidase reagents in enzyme-antibody molar ratio, retention of antibody and enzyme activity, immunohistochemical staining dilutions, and stability. Immunoglucose oxidase and immunoperoxidase were combined to localize CEA and colon-specific antigen-p simultaneously. Excellent contrast and staining separation were shown between the enzymatic reaction products of the two systems. Since immunoglucose oxidase methods are as sensitive as the comparable immunoperoxidase techniques, they should be considered as a reliable alternative to the latter, especially when endogenous peroxidase activity may be a problem. Furthermore, the two methods can be conveniently combined for the simultaneous detection of two antigens.