Selection and optimization of monoclonal antibody immobilization procedure for its use in IRMA

Abstract

Immobilization of monoclonal antibody (McAb) on polystyrene support is an integral part of any immunoassay (IA) procedure. Two different immobilization approaches (direct and indirect) were evaluated for their effectiveness. In case of direct immobilization, mouse monoclonal antibody was adsorbed ‘passively’ on polystyrene tubes. The same antibody was immobilized as ‘pre-formed complex’ in case of indirect approach via mouse IgG as linker. Both the approaches displayed comparable Bmax using different buffer systems. However, NSB observed via mouse IgG linker was always on the higher side (0.8–4.0 %). This could be significantly reduced (up to <0.4 %) by controlling the concentration of mouse IgG.Theoretical advantages envisaged via indirect approach viz. economy and stability of immobilized antibody were similar to that of simple passive adsorption. Hence, we have selected simple passive adsorption method using bicarbonate buffer (pH 8.4). Our study also confirmed the need for ‘tube maturation’ in order to retain the immunological integrity of the immobilized antibody. The performance of the tubes prepared by direct immobilization method was evaluated in an immunoradiometric assay for human Luteinizing hormone (LH).The developed method is simple, convenient and amenable for large scale production of antibody immobilized polystyrene tubes.