Reinitz D M, Voss E W
Mol Immunol. 1984 Sep;21(9):775-84. doi: 10.1016/0161-5890(84)90164-0.
Previous studies concerning structure-function relationships of anti-fluorescyl hybridoma proteins utilized primarily high-affinity proteins (Ka greater than 5.0 X 10(7) M-1) possessing distinct idiotypes. Low-affinity anti-fluorescyl monoclonal antibodies, predominantly IgGl or IgG2a possessing kappa light chains were analyzed. Two fusions produced 18 monoclonals, 13 binding fluorescein with a low affinity (less than or equal to 3.0 X 10(6) M-1) and five possessing high affinities (greater than or equal to 5.3 X 10(8) M-1). Solid-phase idiotype assays, utilizing rabbit anti-idiotype reagents against two low-affinity proteins (3-13 and 3-17), showed that all the low-affinity clones (except 2-9 and 2-21) were capable of inhibiting (40-100%) these two idiotype-anti-idiotype interactions while no high-affinity proteins inhibited them. The interactions with 3-13 and 3-17 were inhibited 100 and 88%, respectively, by free fluorescein. When these idiotype-anti-idiotype interactions were inhibited with increasing concns of heterologous hybridoma proteins, three clones inhibited both interactions as effectively as the homologous proteins at all concns tested and inhibition reached 100%. These three clones appeared to possess all the idiotopes that the anti-3-13 and anti-3-17 reagents detected on 3-13 and 3-17. Screening of eight high-affinity anti-fluorescyl proteins previously produced [Kranz and Voss, Molec. Immun. 18, 889-898 (1981a)] identified a single clone [20-4-4 (Ka = 5.0 X 10(7) M-1)] significantly inhibiting the 3-13 and 3-17 interactions (71.0 and 63.6%, respectively). In addition, recombination experiments utilizing H- and L-chains derived from three low-affinity and three high-affinity antibodies resulted in reformation of active sites in all six heterologous combinations when both chains were derived from low-affinity antibodies, and in only one of six combinations when both chains were derived from high-affinity molecules. Thus, the apparent lack of private idiotopes on clones 3-13 and 3-17 and the presence of these idiotopes (or cross-reactive ones) on 11 of 13 low-affinity antibodies and on one of 13 high-affinity antibodies may indicate that clones 3-13 and 3-17 are encoded by germline genes. The H- and L-chain recombination experiments indicated that the idiotype and affinity of parental molecules may be involved in H- and L-chain association.
先前有关抗荧光素杂交瘤蛋白结构-功能关系的研究主要利用了具有独特个体基因型的高亲和力蛋白（亲和常数Ka大于5.0×10⁷ M⁻¹）。分析了主要为具有κ轻链的IgG1或IgG2a的低亲和力抗荧光素单克隆抗体。两次融合产生了18种单克隆抗体，其中13种以低亲和力（小于或等于3.0×10⁶ M⁻¹）结合荧光素，5种具有高亲和力（大于或等于5.3×10⁸ M⁻¹）。利用针对两种低亲和力蛋白（3-13和3-17）的兔抗个体基因型试剂进行的固相个体基因型分析表明，所有低亲和力克隆（2-9和2-21除外）均能够抑制（40%-100%）这两种个体基因型-抗个体基因型相互作用，而高亲和力蛋白则无此抑制作用。游离荧光素分别使与3-13和3-17的相互作用受到100%和88%的抑制。当用浓度不断增加的异源杂交瘤蛋白抑制这些个体基因型-抗个体基因型相互作用时，在所有测试浓度下，有三个克隆对两种相互作用的抑制效果与同源蛋白一样有效，抑制率达100%。这三个克隆似乎具有抗3-13和抗3-17试剂在3-13和3-17上检测到的所有个体基因型决定簇。对先前产生的8种高亲和力抗荧光素蛋白进行筛选[克兰兹和沃斯，《分子免疫学》18，889-898（1981a）]，发现一个单克隆[20-4-4（Ka = 5.0×10⁷ M⁻¹）]能显著抑制3-13和3-17的相互作用（分别为71.0%和63.6%）。此外，利用源自三种低亲和力和三种高亲和力抗体的重链和轻链进行的重组实验表明，当两条链均源自低亲和力抗体时，在所有六种异源组合中均重新形成了活性位点；而当两条链均源自高亲和力分子时，在六种组合中仅有一种重新形成了活性位点。因此，3-13和3-17克隆上明显缺乏私有个体基因型，而13种低亲和力抗体中的11种以及13种高亲和力抗体中的1种上存在这些个体基因型（或交叉反应性个体基因型），这可能表明3-13和3-17克隆是由种系基因编码的。重链和轻链重组实验表明，亲本分子的个体基因型和亲和力可能参与了重链和轻链的缔合。
