REAL-TIME BIOSPECIFIC INTERACTION ANALYSIS OF A NATURAL HUMAN POLYREACTIVE MONOCLONAL IGM ANTIBODY AND ITS FAB AND SCFV FRAGMENTS WITH SEVERAL ANTIGENS

Surface plasmon resonance (SPR) was used to investigate the kinetics o f interactions between the human monoclonal polyreactive IgM antibody CB03, its Fab as well as its single-chain variable fragment (scFv) and different antigens. From these experiments apparent binding constants were determined and compared with binding constants obtained by ELISA experiments. In SPR studies with the complete antibody, the polyreact ivity of the CB03 antibody as derived from ELISA experiments was confi rmed. Interaction of scFv with kappa casein and human myoglobin is str ong evidence for the location of polyreactivity within the variable do mains of the antibody.Apparent binding constants of the complete antib ody to immobilized kappa casein (9.2 x 10(7) M(-1)) and to human myogl obin (1.6 x 10(7) M(-1)) are up to 83 times higher than those of Fab. The binding constants of the scFv to the above mentioned antigens are again about 10 times lower when compared with Fab, which is mainly due to the lower association rates of the complexes formed by the scFv.