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 of 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 polyreactivity of the CB03 antibody as derived from ELISA experiments was confirmed. Interaction of scFv with kappa casein and human myoglobin is strong evidence for the location of polyreactivity within the variable domains of the antibody. Apparent binding constants of the complete antibody to immobilized kappa casein (9.2 x 10(7) M-1) and to human myoglobin (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.
Department of Medical Immunology, Medical School (Charité), Humboldt University of Berlin, Germany.
D
D
Roggenbuck
H
H
König
B
B
Niemann
G
G
Schoenherr
S
S
Jahn
T
T
Porstmann
D018033:Antibodies, Bispecific / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000915:Antibody Affinity; D000937:Antigen-Antibody Reactions / Q000276:immunology*; D001483:Base Sequence; D015374:Biosensing Techniques; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology*; D007075:Immunoglobulin M / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000276:immunology*; D007700:Kinetics; D008969:Molecular Sequence Data
D018033:Antibodies, Bispecific; D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007075:Immunoglobulin M; D007135:Immunoglobulin Variable Region