STRUCTURAL REQUIREMENTS OF ANTIBODY COMBINING SITES

本研究的长期目标是了解 抗体可变区氨基酸序列导致抗体 对抗原的特异性和亲和力以及独特型表达。 抗体基因的寡核苷酸定向诱变将用于 在互补决定区（CDRS）中引入突变， 对偶氮苯胂酸盐（Ars）特异性的小鼠抗体。突变抗体 将在将诱变基因转染到 骨髓瘤或杂交瘤细胞系，并且转染瘤将回收， 在液体微培养物中或在软琼脂糖中。分泌的抗体， 将通过标准免疫学测定筛选上清液或原位。 抗体亲和力对变化特别敏感的假设 在某些位置将通过引入所有可能的 已经发现在四个位置中的每一个上的取代影响 抗Ars抗体的亲和力。改善的最低要求 抗体亲和力和改变的特异性将通过 在所述抗体的CDR中一次引入一个或几个随机突变， 抗-Ars抗体，并筛选与抗-Ars抗体的亲和力增加的突变体。 或具有新抗原特异性。为了探索每个人的角色， 抗Ars抗体的6个CDR在抗原结合和独特型 在表达中，CDR将通过替换为 甘氨酸残基。重建一个反Ars结合点将 然后尝试通过将氨基引入到这样的甘氨酸取代的CDRS中， 与Ars接触有关的酸残基。的 甘氨酸取代的CDR也将以低频率随机诱变， 以获得Ars或其它抗原的特异性抗体。见解 从拟议的研究中获得的信息应有助于我们了解 一般的蛋白质折叠和特别的抗体互补， 这将使我们更接近长期目标， 用于研究和医疗目的的抗体。

The long term objective of this study is to attain an understanding of how the amino acid sequence of antibody variable regions leads to antibody specificity and affinity for antigen, and to idiotypic expression. Oligonucleotide-directed mutagenesis of antibody genes will be used to introduce mutations in the complementarity determining regions (CDRS) of a mouse antibody specific for p-azophenylarsonate (Ars). Mutant antibodies will be expressed following transfection of the mutagenized genes into myeloma or hybridoma cell lines, and transfectomas will be recovered either in liquid microcultures or in soft agarose. Secreted antibodies, in supernatants or in situ, will be screened by standard immunological assays. The hypothesis that antibody affinity is particularly sensitive to changes at certain positions will be tested by introducing all possible substitutions at each of four positions that have been found to affect affinity of the anti-Ars antibody. Minimal requirements for improved antibody affinity and for changed specificity will be determined by introducing one or a few random mutations at a time in the CDRs of the anti-Ars antibody, and screening for mutants with increased affinity for Ars or with new antigen specificities. To probe the roles of each of the six CDRs of the anti-Ars antibody in antigen binding and idiotypic expression, the CDRs will be individually "blanked out" by replacement with glycine residues. The reconstruction of an anti-Ars combining site will then be attempted by introducing into such glycine-replaced CDRS, the amino acid residues that have been implicated in Ars contact. The glycine-replaced CDRs will also be randomly mutagenized at low frequency, to obtain antibodies specific for Ars or for other antigens. Insights gained from the proposed studies should further our understanding of protein folding in general and of antibody complementarity in particular, and should bring us a step closer to the long term objective of tailoring antibodies for research and medical purposes.