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 resonance analysis (BIAcore) of culture supernatants showed that the entire cloned Tg10 antibody displays an affinity comparable to that of the parental antibody. This approach also permitted determination of the probable role of each chain to the recognition of the cognate epitope due to the ability of COS-7 cells to secrete independently each of the two constituting immunoglobulin chains. Tg10 heavy chain recognizes hTg in the absence of the light chain, but with a ten-fold lower affinity mainly due to an increase in kappaoff. In contrast, the light chain is unable to bind hTg on its own. This suggests that the latter is probably involved in stabilization rather than in initiating the formation of the antibody/antigen complex and that the specificity of Tg10 is mostly, if not exclusively, carried by the heavy chain. The potential applications of combined cell transfection and surface plasmon resonance to our understanding of antigen/antibody interactions are discussed.
Noel
D
D
CNRS UMR 9921, UFR Sciences Pharmaceutiques et Biologiques, Montpellier, France.
Bernardi
T
T
Navarro-Teulon
I
I
Marin
M
M
Martinetto
J P
JP
Ducancel
F
F
Mani
J C
JC
Pau
B
B
Piechaczyk
M
M
Biard-Piechaczyk
M
M
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D001666:Binding Sites, Antibody; D015374:Biosensing Techniques; D002460:Cell Line; D005822:Genetic Vectors / Q000276:immunology; D006801:Humans; D015151:Immunoblotting; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry* / Q000302:isolation & purification; D007147:Immunoglobulin Light Chains / Q000737:chemistry* / Q000302:isolation & purification; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000276:immunology; D013057:Spectrum Analysis / Q000379:methods; D013816:Thermodynamics; D013954:Thyroglobulin / Q000737:chemistry / Q000276:immunology*; D014162:Transfection / Q000276:immunology*
D000911:Antibodies, Monoclonal; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D011994:Recombinant Proteins; D013954:Thyroglobulin