A non-denaturing enzyme linked immunosorbent assay with protein preadsorbed onto aluminum hydroxide

Abstract

A method has been developed which prevents denaturation of proteins used for coating of plastic surfaces in enzyme linked immunosorbent assays (ELISA). The system takes advantage of the use of aluminum hydroxide (Al(OH) 3) as an adsorbent for proteins. A model protein has been analyzed, and monoclonal antibodies specific for either the native form or the denatured form of the protein were used to monitor the extent of denaturation. Adsorption of the proteins to Al(OH) 3 in carbonate buffer, pH 9.3, before coating the ELISA plate abolished the denaturation otherwise observed after direct adsorption of protein to plastic surfaces. The protection against denaturation was dependent on the buffer system and was not observed when phosphate buffers were used, due to elution of protein from Al(OH) 3 or lack of binding to Al(OH) 3 in the presence of phosphate. There is evidence that protein adsorbed onto the Al(OH) 3 is required for binding of Al(OH) 3 onto the plastic surface. This system may be useful in assay systems where discrimination between the native and denatured forms of proteins is important.