JOUHAL OF lM~ WlO6ICAl zyxwvutsrqponm ELSEVIER Journal of Immunological Methods 183 (1995) 167-174 Colloidal gold conjugated monoclonal antibodies, studied in the BIAcore biosensor and in the Nycocard immunoassay format Berit Johne * , Kari Hansen, Einar Mgrk, Jostein Holtlund zyxwvutsrqponmlkjihgfed Immunochemistry R&D, Nycomed Pharma AS, P.O. Box 5012 Maj., N-0301 Oslo, Nonvay zyxwvutsrqponmlkjihgfedcba Abstract Interactions between immobilized capture monoclonal antibodies CmAbs), analyte molecules and colloidal gold conjugated second monoclonal antibodies have been investigated in the BIAcore biosensor and in the Nycocard immunoassay format. This report focuses on six monoclonal antibodies against human heart myoglobin, although, results with other antigens are also discussed. The BIAcore was used to screen monoclonal antibodies as antigen capture reagents, and for their function as colloidal gold conjugated second antibodies in the Nycocard. Some antibodies with low affinity caused by a rapid antigen dissociation rate, showed high affinity kinetics when used unlabelled or as gold conjugated detector reagents. One gold conjugated mAb with excellent properties in the Nycocard, showed double binding to one epitope, when tested in the BIAcore. The real time visualization of association and dissociation rates was a unique tool in the elucidation of antigen-antibody interactions. Our study confirmed that good antibody candidates selected with the BIAcore must always be tested in their actual conjugation situation before final optimization. Keywords: Monoclonal antibody; Myoglobin; Immunoassay; Colloidal gold; Biosensor; BIAcore; Real time biospe- cific interaction analysis 1. Introduction The Nycocard immunoassay format is a quan- titative, rapid (2 min) test system where the ana- lyte is captured by a membrane bound antibody Abbreviations: MAb, monoclonal antibody; Ag, antigen; ELISA, enzyme linked immunosorbent assay; EIA, enzyme immunoassay; RAM-Fc, rabbit anti-mouse Fc fragment; RAM-Cl, rabbit anti-mouse IgGl; RU, refractive units; RT BIA, real time biospecific interaction analysis; SPR, surface plasmon resonance. * Corresponding author. Tel.: +47-22-891010; Fax: +47- 22-891200. and detected by colloidal gold conjugated second antibody, providing a coloured signal directly pro- portional to the amount of analyte bound (Hjortdal et al., 1991; Urdal et al., 1992). The highest sensitivity in this format is at present 20 ng/ml (myoglobin) based on selected high affin- ity monoclonal antibodies (Johne et al., 1993), and in-house developed patented colloidal gold conjugation technology. Antibodies with high affinity and optimal binding kinetics are essential to immunoassay sensitivity (Campbell, 1991; Johne et al., 1993; Chappey et al., 1994; Cook and Wood, 1994; Larvor et al., 1994; Wild, 1994). However, various conjugation methods may affect 0022-1759/95/$09.50 Q 1995 Elsevier Science B.V. All rights reserved SSDI 0022-1759(95)00047-X