SEQUENCE, SHAPE AND SPECIFICITY OF ANTIBODIES

抗体的序列、形状和特异性

• 批准号: 3166429
• 负责人: MICHAEL N MARGOLIES
• 金额: $ 20.17万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-09-01 至 1991-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3166429
• 关键词: affinity chromatography; antibody formation; antibody specificity; autoradiography; biochemical evolution; clone cells; gel electrophoresis; genetic mapping; high performance liquid chromatography; hybridomas; immunochemistry; radioimmunoassay; thin layer chromatography

In certain strains of inbred mice immune responses are dominated by antibodies that share common variable region structures (idiotypes) detected serologically by anti-idiotypic reagents. Idiotypic determinants characterizing certain antibody specificities have proven valuable structural and genetic markers in studies of antibody diversity and regulation. Jerne proposed that interactions between idiotype and anti-idiotype regulate immune responses through recognition of idiotypic determinants, rather than by regulation of specific antigen combining sites. The major cross-reacting idiotype (IdCR) in strain A/J mice immunized with p-azophenylarsonate (Ars) is heritable and encoded by germline genes. The Ars system is a model for examining regulations defining the structural epitopes responsible for idiotypy and antibody structure changes involved in enhanced antigen affinity occurring temporally during the immune response (affinity maturation). A second idiotype family (Id36-60) among anti-Ars antibodies shared in A/J and BALB/c strains is serologically and structurally distinct from IdCR, representing an independent marker for studies of regulation. The germline VH genes for both IdCR and Id36-60 were cloned and sequenced. By appropriate selection methods, the feasibility of isolating molecules in which idiotype and antigen binding are dissociated, as well as obtaining variants with enhanced affinity was demonstrated. Further the 3-dimensional structure of an IdCR Fab fragment is at hand. Therefore, we will 1) Define the molecular site of IdCR by determining V region structures of anti-Ars molecules which lack idiotype despite use of IdCR associated gene segments, 2) Characterize the site and temporal pattern of somatic mutation and its contribution to affinity changes among IdCR+ HP, utilizing a) IdCR+ Ars-binding HP obtained during primary and secondary immune responses, b) Spontaneous mutants in cell culture which do not bind Ars or demonstrate enhanced binding. 3) Characterize the pattern of structural changes seen during affinity maturation among Id36-60 antibodies to assess the role of antigen driven selection in explaining why IdCR dominates Id36-60, 4) Examine the contribution of gene junctional diversity to Ars-binding by selecting of unmutated primary response antibodies with junctional changes. 5) These studies are correlated with alterations in idiotype and Ars-binding induced by site-directed mutagenesis and with x-ray crystallographic analysis.

在某些品系的近交系小鼠中，免疫应答主要由 具有共同可变区结构的抗体（独特型） 通过抗独特型试剂进行血清学检测。 独特型决定簇 表征某些抗体特异性已被证明是有价值的 抗体多样性研究中的结构和遗传标记， 调控 Jerne提出，独特型和 抗独特型抗体通过识别独特型抗体来调节免疫应答。 决定因素，而不是通过调节特异性抗原结合 网站. A/J系小鼠的主要交叉反应独特型（IdCR） 用对偶氮苯胂酸盐（Ars）免疫是可遗传的，由 生殖系基因 Ars系统是审查法规的模型 确定负责独特型和抗体的结构表位 与增强的抗原亲和力有关的结构变化 在免疫应答期间暂时地（亲和力成熟）。 第二 A/J中共有的抗Ars抗体中的独特型家族（Id 36 -60）， BALB/c菌株在血清学和结构上不同于IdCR， 代表了调控研究的独立标志物。 种系 对IdCR和Id 36 -60的VH基因进行克隆和测序。 通过 适当的选择方法，分离分子的可行性， 其独特型和抗原结合是分离的，以及获得 证明了具有增强的亲和力的变体。 进一步 3-IdCR Fab片段的三维结构在手边。 所以我们 将1）通过确定V区来定义IdCR的分子位点 尽管使用了IdCR但仍缺乏独特型的抗Ars分子的结构 相关基因片段，2）表征的网站和时间模式， 体细胞突变及其对IdCR+ HP之间亲和力变化的贡献， a）在初次和二次免疫中获得的IdCR+ Ars结合HP 免疫应答，B）细胞培养物中不结合 或显示增强的结合。 3)描述...的模式 在Id 36 -60抗体之间的亲和力成熟期间观察到的结构变化 评估抗原驱动选择在解释IdCR为什么 主导Id 36 -60，4）检查基因连接多样性的贡献 通过选择未突变的初级应答抗体， 连接变化。 5)这些研究与以下改变相关： 独特型和Ars结合诱导的定点诱变和与 X射线晶体学分析。