Newman B A, Kabat E A
J Immunol. 1985 Aug;135(2):1220-31.
This is the first report of an immunochemical study of the combining site specificities of a set of monoclonal antibodies to dextran B512 from C57BL/6J mice. The results confirm previous observations on antidextran combining sites and reveal specificities not seen earlier extending the observed repertoire of antibody combining sites to the single alpha (1----6)-linked glucosyl antigenic determinant. Eight C57BL/6J anti-dextran B512 hybridomas, four IgM,kappa and four IgA,kappa, were produced by PEG fusion of immune spleen cells with the nonproducer myeloma cell line P3X63Ag8 6.5.3. Antibody combining site specificities were determined by quantitative precipitin assays with 14 dextrans. Native dextrans with high percentages of linear alpha (1----6)-linked glucoses, similar to the immunogen B512, were the best precipitinogens; dextrans with alternating alpha (1----3), alpha (1----6) linkages, and highly branched dextrans were less effective. All antibodies precipitated with a synthetic, unbranched alpha (1----6)-linked dextran, suggesting their combining sites were "groove-like" and directed toward internal sequences of alpha (1----6)-linked residues, rather than "cavity-like" and directed toward a nonreducing terminal glucose. Two of the IgA hybridomas gave biphasic precipitin curves with dextran B512; this was shown to be due to differences in the precipitability of IgA monomers and polymers. Differences were observed in the reactivities of several dextrans considered previously to be structurally similar, and a newly proposed structural model of dextran B1299S was assessed. Quantitative precipitin inhibition studies with alpha (1----6)-linked isomaltosyl (IM) oligosaccharides, IM2 to IM9, showed that maximum inhibition was reached with IM6 or IM7, consistent with earlier estimates of the upper limit for the sizes of anti-B512 combining sites. Two IgM hybridomas showed a unique pattern, with inhibition being obtained only with IM5 or larger IM oligosaccharides. Association constants of the antidextrans for dextran B512 and for IM7, determined by affinity gel electrophoresis, ranged from 10(2) to 10(4) ml/g, comparable to earlier findings with antidextrans and other anticarbohydrate antibodies.
这是对一组来自C57BL/6J小鼠的抗右旋糖酐B512单克隆抗体结合位点特异性进行免疫化学研究的首次报告。结果证实了先前关于抗右旋糖酐结合位点的观察结果，并揭示了以前未见过的特异性，将观察到的抗体结合位点库扩展到单一的α(1→6)连接的葡糖基抗原决定簇。通过免疫脾细胞与非分泌型骨髓瘤细胞系P3X63Ag8 6.5.3进行聚乙二醇融合，产生了8个C57BL/6J抗右旋糖酐B512杂交瘤，其中4个为IgM，κ型，4个为IgA，κ型。通过用14种右旋糖酐进行定量沉淀试验来确定抗体结合位点的特异性。与免疫原B512相似，具有高比例线性α(1→6)连接葡萄糖的天然右旋糖酐是最好的沉淀原；具有交替α(1→3)、α(1→6)连接的右旋糖酐以及高度分支的右旋糖酐效果较差。所有抗体都能与一种合成的、无分支的α(1→6)连接的右旋糖酐沉淀，这表明它们的结合位点是“凹槽样”的，指向α(1→6)连接残基的内部序列，而不是“腔样”的，指向非还原末端葡萄糖。两个IgA杂交瘤与右旋糖酐B512产生双相沉淀曲线；这被证明是由于IgA单体和聚合物的沉淀性差异所致。观察到几种以前被认为结构相似的右旋糖酐的反应性存在差异，并对新提出的右旋糖酐B1299S结构模型进行了评估。用α(1→6)连接的异麦芽糖基(IM)寡糖IM2至IM9进行定量沉淀抑制研究表明，IM6或IM7达到最大抑制，这与早期对抗B512结合位点大小上限的估计一致。两个IgM杂交瘤表现出独特的模式，仅用IM5或更大的IM寡糖才能获得抑制。通过亲和凝胶电泳测定的抗右旋糖酐对右旋糖酐B512和IM7的结合常数范围为10²至10⁴ml/g，与早期抗右旋糖酐和其他抗碳水化合物抗体的研究结果相当。
