SEQUENCE, SHAPE, AND SPECIFICITY OF ANTIBODIES

抗体的序列、形状和特异性

• 批准号: 3166432
• 负责人: MICHAEL N MARGOLIES
• 金额: $ 26.12万
• 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/3166432
• 关键词: affinity chromatography; antibody formation; antibody specificity; autoradiography; biochemical evolution; clone cells; gel electrophoresis; genetic mapping; high performance liquid chromatography; hybridomas; immunochemistry; immunoglobulin idiotypes; laboratory mouse; laboratory rabbit; protein sequence; radioimmunoassay; site directed mutagenesis; 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，免疫反应由 具有共同可变区结构(独特型)的抗体 用抗独特型试剂进行血清学检测。独特型决定因素 确定某些抗体的特异性已被证明是有价值的 抗体多样性和抗体多样性研究中的结构和遗传标记 监管。杰恩提出，独特型和 抗独特型通过识别独特型调节免疫反应 决定因素，而不是通过调节特定的抗原结合 网站。A/J系小鼠的主要交叉反应独特型(IdCR) 用对偶氮苯亚硝酸盐(Ars)免疫是可遗传的，并由 生殖系基因。ARS系统是审查法规的典范 确定独特型和抗体的结构表位 与抗原亲和力增强有关的结构变化 在免疫反应(亲和力成熟)期间暂时性的。一秒钟 抗Ars抗体独特型家系(Id36-60)在A/J和J BALB/c株在血清学和结构上与IdCR不同， 代表了监管研究的一个独立的标志。生殖系 对IdCR和Id36-60的VH基因进行了克隆和测序。通过 选择合适的方法，分离分子的可行性。 哪些独特型和抗原结合是分离的，以及获得 展示了亲和力增强的变异体。再往前走 IdCR Fab片段的三维结构近在咫尺。因此，我们 将1)通过确定V区来确定IdCR的分子位置 使用IdCR但缺乏独特型的抗ARs分子的结构 相关基因片段，2)表征位置和时间模式 IdCR+Hp的体细胞突变及其对亲和力变化的贡献 利用在初级和次级期间获得的IdCR+Ars结合HP 免疫反应，b)细胞培养中不结合的自发突变体 ARS或演示增强的结合。3)描述……的模式 Id36-60抗体亲和力成熟过程中的结构变化 评估抗原驱动的选择在解释IdCR原因中的作用 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反应抗体与Ars结合 交界处的变化。5)这些研究与 定点突变诱导的独特型和Ars结合 X射线结晶学分析。