ANALYSIS OF THE INDIVIDUAL CONTRIBUTIONS OF IMMUNOGLOBULIN HEAVY AND LIGHT-CHAINS TO THE BINDING OF ANTIGEN USING CELL TRANSFECTION AND PLASMON RESONANCE ANALYSIS

We have cloned the Tg10 murine monoclonal antibody, which is specific for a human thyroglobulin (hTg) epitope targeted by autoantibodies in several thyroid pathologies. Transfection of COS-7 cells with plasmids expressing Tg10H and -kappa chains combined with surface plasmon reso nance analysis (BIAcore) of culture supernatants showed that the entir e cloned Tg10 antibody displays an affinity comparable to that of the parental antibody. This approach also permitted determination of the p robable role of each chain to the recognition of the cognate epitope d ue to the ability of COS-7 cells to secrete independently each of the two constituting immunoglobulin chains. Tg10 heavy chain recognizes hT g in the absence of the light chain, but with a ten-fold lower affinit y mainly due to an increase in k(off). In contrast, the light chain is unable to bind hTg on its own. This suggests that the latter is proba bly involved in stabilization rather than in initiating the formation of the antibody/antigen complex and that the specificity of Tg10 is mo stly, if not exclusively, carried by the heavy chain. The potential ap plications of combined cell transfection and surface plasmon resonance to our understanding of antigen/antibody interactions are discussed.