Immunoaffinity layering enhances the sensitivity of antigen detection on nitrocellulose strips

A simple strategy to remarkably increase the sensitivity of detection of antigens applied as dot or western blot on nitrocellulose membrane using human serum albumin as model antigen has been described. This involves subjecting the antigen bearing nitrocellulose strips to multiple incubation cycles with primary antibody and enzyme conjugated secondary antibody prior to staining for enzyme activity. The sensitivity of detection could be increased up to a thousand fold after three incubation cycles. Aggregation of human serum albumin could be detected by the multiple incubation procedure at very low protein concentration after electrophoresis and transfer onto nitrocellulose.