Selection and optimization of monoclonal antibody immobilization procedure for its use in IRMA
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Selection and optimization of monoclonal antibody immobilization procedure for its use in IRMA Vrinda P. C. • S. N. Paradkar • N. Sivaprasad
Received: 1 October 2012 / Published online: 13 January 2013 Ó Akade´miai Kiado´, Budapest, Hungary 2013
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 ‘preformed 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. Keywords SPI IRMA Monoclonal antibodies NSB Immobilization and coating
Vrinda P. C. (&) S. N. Paradkar N. Sivaprasad Radiopharmaceuticals Programme, Board of Radiation and Isotope Technology (BRIT), BARC Vashi Complex, Navi Mumbai 400 705, India e-mail: [email protected]
Introduction Solid phase immunoassay (SPI) using immobilized antibody on polystyrene support has become indispensible in medical diagnostics, immunology, pharmacy and other areas of medical and veterinary science [1–3]. The ‘simplicity of use’ is the only criteria responsible for unsaid popularity of SPI technology. This actually overshadows many disadvantages of SPI technology viz. economy (per tube antibody consumption), need for separate immobilization method suitable for specific analyte and the necessity for automation [4, 5]. The last two listed factors become crucial especially for the kit manufacturer. For any immunoassay to be developed the selected antibody has to be evaluated for its usefulness [6]. Antibodies and proteins have varying affinities for polystyrene surface; making it impossible to predict the adsorption behavior of a particular protein without performing experimental studies [3–7]. Several immobilization methods are reported in the literature till the date and can be classified based on nature of the protein, polystyrene and also on the type of usage (research or production). Typically proteins can be adsorbed on polysty
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