Blocking agents for ELISA quantification of compounds coming from bovine muscle crude extracts

Abstract

Non-specific binding (NSB) of compounds other than the capture antigen or antibody to the solid support is one of the most important error sources occurring in ELISA. Undesirable NSB may be minimized by saturating the remaining binding sites of the plastic surface with different protein compounds (blocking agents) that must not interfere with the immunoassay. This work had as main objective the evaluation of different blocking agents for their ability to avoid unwanted NSB in ELISA using bovine muscle crude extracts and a polyclonal antibody raised against purified cathepsin L. Results obtained showed that skimmed milk, casein, BSA, goat and rabbit serum proved to be efficient in suppressing NSB. In the case of skimmed milk and casein, an effective blocking effect was observed even in absence of the surcoating step. However, these two compounds and BSA interfered considerably the immunochemical reaction, making their use inadequate. Of the two assayed serums, rabbit serum would be a more suitable blocking agent than goat serum when working with bovine crude extracts because it did not interfere in great manner with the antigen–antibody reaction and, at the same time, did not increase background.