Biochemical analysis challenging Western blot analysis as validation step for antibodies intended for ELISA and immunohistochemistry

Abstract

The relative contributions of the conformation and primary structure of an epitope to overall antigen-antibody interaction (AAI) at denatured, native or formalin fixed (FF) state were compared using six randomly chosen commercial antibodies using Quantitative Dot Blot (QDB) method. AAIs at native and FF states were found ranged 1.3 [~] 10.2 and 0.5 [~] 45.4 folds, respectively, over those at denatured state in cellular and tissue lysates. Using two antibodies against different epitopes of PYGL protein, we showed that PYGL levels in several types of tissues and cell lines were highly correlated (r=0.99 from Pearson, p<0.0001, n=25) when measured with these two antibodies at native state. Yet, one antibody was found to be nonspecific with one type of these tissues using Western blot analysis. These observations suggested that the conformation of an epitope may serve as dominant contributor of overall AAI at native state in general, regardless of linear or conformational epitopes. In many cases, it would override nonspecific interactions formed at denatured state to challenge Western blot analysis as a validation tool for antibodies intended for immunohistochemistry (IHC) and ELISA.