Using a heavy chain-loss hybridoma 26.4.1LL for studying the structural basis of immunoglobulin chain association.
One of the mechanisms contributing to antibody diversity is created by the association of different heavy and light chains. The combinability of heavy and light chains has been studied previously in two systems: in vitro chain recombination and hybrid hybridoma. Here, a novel in vivo chain combination assay system involving a heavy chain-loss variant, 26.4.1LL, producing two kappa light chains (L(DEX) and L(MPC)) different in size is described. In conjunction with DNA transfection, immunoprecipitation and SDS-PAGE, the structural basis of noncovalent interaction between heavy and light chains can be elucidated systematically by examining the relative association tendency of a heavy chain with two light chains. To demonstrate the usefulness of this system, three stably transfected 26.4.1LL cell lines expressing gamma2b heavy chains, designated as H(DEX), H(CHI) and H(ARS), respectively, with structural interrelated variable regions were generated: H(DEX) differs from H(CHI) only in framework regions whereas H(CHI) differs from H(ARS) in complementarity-determining regions. The relative amounts (R values) of L(DEX) and L(MPC) associated with the heavy chains H(DEX), H(CHI) and H(ARS) in the assembled immunoglobulin molecules were found to be 1.02, 0.64 and 0.05, respectively, suggesting that the complementarity-determining regions and framework regions contribute equally to the V(L)-V(H) interaction. This conclusion is consistent with previous observations based on calculation of the buried area in the V(L)-V(H) interface, thus demonstrating the usefulness of this system.
Yang
C Y
CY
Institute of Molecular Biology, National Chung Hsing University, Taichung, Taiwan, ROC.
D000818:Animals; D014644:Genetic Variation; D006825:Hybridomas / Q000276:immunology*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology*; D007147:Immunoglobulin Light Chains / Q000235:genetics / Q000276:immunology*; D007145:Immunoglobulin kappa-Chains / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology; D014162:Transfection
D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007145:Immunoglobulin kappa-Chains; D011993:Recombinant Fusion Proteins