Mutz M, Hawthorne T, Ferrone S, Pluschke G
Central Research Services, Physics, Ciba-Geigy Ltd., Basel, Switzerland.
Mol Immunol. 1997 Jul;34(10):695-707. doi: 10.1016/s0161-5890(97)00095-3.
The thermodynamic parameters of interactions between six variants of the anti-idiotypic monoclonal antibody (mAb) CGP 60686 produced by the hybridoma MK2-23 with an idiotypic mAb and five different anti-anti-idiotypic mAb were studied with high sensitivity titration calorimetry. CGP 60686 recognizes an epitope in the antigen-combining region of the human high-molecular-weight-melanoma-associated antigen (HMW-MAA)-specific mouse mAb CGP 76873 produced by the hybridoma 763.74. The five HMW-MAA-specific anti-anti-idiotypic mAbs GH 464, GH 518, GH 149, GH 386 and GH 586 were generated from mice immunization with mAb CGP 60686. All interactions between the anti-idiotypic mAb and the idiotypic mAb or the anti-anti-idiotypic mAb showed large exothermic binding enthalpies between -15 and -23 kcal/mol and binding affinities larger than 6 x 10(9) M-1. Four of the five anti-anti-idiotypic mAbs tested exhibited significantly higher binding enthalpies for the interaction with the anti-idiotypic than the idiotypic mAbs. Replacement of either the heavy or the light chain variable region of the anti-idiotypic mAbs with an unrelated variable region abolished the idiotype to anti-idiotype interaction. Thus, both the heavy and the light chain variable region of the anti-idiotypic mAbs are required for binding to the idiotype. The values of the binding enthalpy showed only small variations when binding of the idiotypic mAb CGP 76873 to four variants of the anti-idiotypic mAb CGP 60686 with different immunoglobulin constant regions, but identical variable regions were compared. Furthermore, Fab fragments of the idiotypic mAbs showed almost the same binding enthalpy per binding site as the whole IgG molecules. Immunoglobulin constant regions thus had little influence on the idiotype to anti-idiotype interactions. Taken together, the observed thermodynamic parameters suggest that the idiotype to anti-idiotype interactions studied here are enthalpy-driven processes with only minor entropic contributions. High sensitivity titration calorimetry was used to monitor protein-protein interactions within an anti-idiotypic antibody cascade. It was found that the direct measurement of the interaction enthalpy allowed a quantitative characterization of the binding processes studied.
利用高灵敏度滴定量热法研究了杂交瘤MK2 - 23产生的抗独特型单克隆抗体（mAb）CGP 60686的六种变体与一种独特型mAb以及五种不同的抗抗独特型mAb之间相互作用的热力学参数。CGP 60686识别杂交瘤763.74产生的人高分子量黑色素瘤相关抗原（HMW - MAA）特异性小鼠mAb CGP 76873抗原结合区域中的一个表位。这五种HMW - MAA特异性抗抗独特型mAb GH 464、GH 518、GH 149、GH 386和GH 586是用mAb CGP 60686免疫小鼠产生的。抗独特型mAb与独特型mAb或抗抗独特型mAb之间的所有相互作用均显示出较大的放热结合焓，介于 - 15至 - 23千卡/摩尔之间，结合亲和力大于6×10⁹ M⁻¹。所测试的五种抗抗独特型mAb中有四种与抗独特型mAb相互作用时表现出的结合焓明显高于与独特型mAb相互作用时的结合焓。用不相关的可变区替换抗独特型mAb的重链或轻链可变区会消除独特型与抗独特型之间的相互作用。因此，抗独特型mAb的重链和轻链可变区对于与独特型的结合都是必需的。当比较独特型mAb CGP 76873与具有不同免疫球蛋白恒定区但可变区相同的抗独特型mAb CGP 60686的四种变体的结合时，结合焓的值仅显示出微小变化。此外，独特型mAb的Fab片段每个结合位点显示出与完整IgG分子几乎相同的结合焓。因此，免疫球蛋白恒定区对独特型与抗独特型之间的相互作用影响很小。综上所述，观察到的热力学参数表明，此处研究的独特型与抗独特型之间的相互作用是由焓驱动的过程，熵的贡献很小。高灵敏度滴定量热法用于监测抗独特型抗体级联中的蛋白质 - 蛋白质相互作用。发现对相互作用焓的直接测量能够对所研究的结合过程进行定量表征。
