BSA blocking in enzyme-linked immunosorbent assays is a non-mandatory step: a perspective study on mechanism of BSA blocking in common ELISA protocols

Abstract

BSA blocking is a routine practice among clinicians and researchers working on immunoassays throughout the world. The primary role of BSA is to prevent the non-specific binding by blocking the leftover spaces over solid surface after immobilization of a capture biomolecule. However, the acquired diversity of BSA blocking has remained conflicted on nature of the solid surfaces used, antigen–antibody combinations, and their concentrations. Here, we investigate the necessity of BSA blocking in common ELISA protocols by performing ELISA detection of human-IgG, rabbit-IgG, human-IgE, concanavalin A and hepatitis C core antigen with and without BSA blocking on different microplates and with different concentrations of analytes. We found that irrespective of solid surfaces or analyte concentrations, the ELISA protocols with and without-BSA blocking produce similar outcomes when performed with PBST washing. However, if PBS instead of PBST is used for washing in assays with BSA blocking, the chances of wrong predictions enhance significantly. Further, by using FITC-tagged BSA, we have found that BSA binds weakly to microplate surface and escapes during PBST washing. Again, if PBS rather than PBST is used in combination to BSA blocking, case-dependent non-specificity is added to ELISA results. Based on these observations, we suggest to empirically determine the absolute necessity of BSA-blocking, as majority of ELISA protocols do not need BSA-blocking.