Improvement in the binding of cobrotoxin to microtiter plates by glutaraldehyde at neutral pH

The coating of the wells of microtiter plates with cobrotoxin in Tris (pH 9.8) or PBS (pH 7.2) buffer was assessed by enzyme-linked immunoassay (ELISA). It was found that the poor binding in neutral buffer was improved by adding glutaraldehyde (GA), and the bound amount reached the same extent as that measured with alkali buffer. The optimal concentration of GA was approx. 0.02%. A decrease in optical density was observed with GA concentration higher than 0.02%. This may result from the modification of cobrotoxin by GA, which induced a decrease in the antigenicity of the cobrotoxin as revealed by competitive immunoassay. This study implies that the coating of physiological samples with GA for ELISA may be carried out with physiological buffers without the need to change the buffer to one of alkaline pH.