Question

In ELISA, what is the purpose of blocking the well with something like the Bovine Serum Albumen? Is it to prevent the primary antibodies from binding to the well? If so, why would the primary antibodies bind to the well instead of the antigen ?

Answer 1

The enzyme-linked immunosorbent assays (ELISA) is an extremely common and powerful laboratory technique for detecting proteins by antibodies. Researchers frequently use bovine serum albumin (BSA) as a blocking agent to prevent non-specific binding of antigens and antibodies to the microtiter well. While studying the interactions of the vaccinia virus complement control protein (VCP) with complement, we found nonspecific binding of VCP to BSA and identify a BSA preparation that did not result in non-specific binding. This work draws attention to the fact that not all BSA preparations are alike. It also highlights the need to perform critical controls to ensure that ELISA reactants do not inappropriately bind to the blocking agent.

BSA is one of the most commonly used blocking agents for ELISA. But since BSA is a serum protein, in certain circumstances it could cause non-specific ELISA signals. For example, in a study that was examining for the presence of human antibodies to Japanese encephalitis virus, antibodies that cross-reacted with BSA were found . Another study found that human antibodies non-specifically bound to BSA . To prevent falsepositive results from either cross reactive antibodies or from non-specific binding of ELISA reagents to BSA, alternative blocking agents can be used. Examples include rabbit serum , casein , heat-denaturing the blocking protein , non-protein blocking solutions like Synblock or Ficoll or polyvinylalcohol . Alternatively, no protein can be included in the blocking buffer , although this too can potentially cause artifactual results .

In this report we show that different BSA preparations used as a blocking agent in an ELISA can give different amounts of non-specific binding of ELISA reactants. A vast majority of published articles that include BSA as a reagent do not provide specific information about the BSA. While the company from which a reagent is purchased is often identified, this still can create difficulties if the vendor sells multiple types of a reagent (e.g., the Sigma-Aldrich catalog lists forty-four BSA products). To investigate if bovine C3 was the contaminant in BSA that caused VCP binding, we directly probed the various BSA preparations with rabbit anti-bovine C3 (Cappel), as well as with goat antihuman C3b antibody (Calbiochem). We found no signal and thus do not think bovine C3 is present in BSA preparations. Given the high homology of human and bovine C3, we were not surprised to find that in control wells coated with human C3b, the anti-bovine C3 antibody recognized human C3b. It is also likely that anti-human C3b would recognize bovine C3 (Boulard, 1989). We were thus unable to easily identify the contaminating substance in the BSA preparations that VCP bound to. Since BSAs that were either globulin free or low endotoxin still bound VCP, we speculate that the process to generate a globulin free, low endotoxin BSA (i.e., BSA-2934) depletes the BSA of this substance.

In immunoassays, a primary antibody is always needed to bind to the target antigen, but a secondary antibody is not necessarily used. Indirect ELISA does not label the primary antibodydirectly, but instead, it labels the secondary antibody with an enzyme .