Interactions between immobilized capture monoclonal antibodies (mAbs), 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.