Using a heavy chain-loss hybridoma 26.4.1LL for studying the structural basis of immunoglobulin chain association.

C Y Yang
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Abstract

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.

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利用重链缺失杂交瘤26.4.1LL研究免疫球蛋白链关联的结构基础。
促成抗体多样性的机制之一是由不同重链和轻链的结合产生的。重链和轻链的可组合性已经在体外链重组和杂交杂交瘤两种系统中进行了研究。本文描述了一种新的体内链结合检测系统,该系统涉及重链缺失变体26.4.1LL,产生两条不同大小的kappa轻链(L(DEX)和L(MPC))。结合DNA转染、免疫沉淀和SDS-PAGE,通过检测一条重链与两条轻链的相对结合趋势,可以系统地阐明重链与轻链之间非共价相互作用的结构基础。为了证明该系统的有效性,我们生成了三个稳定转染的表达gamma2b重链的26.4.1LL细胞系,分别命名为H(DEX), H(CHI)和H(ARS),它们具有结构相关的可变区域:H(DEX)与H(CHI)仅在框架区域不同,而H(CHI)与H(ARS)在互补决定区域不同。在组装的免疫球蛋白分子中,L(DEX)和L(MPC)与重链H(DEX)、H(CHI)和H(ARS)相关的相对量(R值)分别为1.02、0.64和0.05,表明互补决定区和框架区对V(L)-V(H)相互作用的贡献相同。这一结论与前人基于V(L)-V(H)界面埋深面积计算的观测结果一致,证明了该体系的实用性。
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