MOLECULAR BASIS OF ANTIGENIC SPECIFICITY

抗原特异性的分子基础

• 批准号: 3445763
• 负责人: James N Herron
• 金额: $ 5.09万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-06-01 至 1989-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3445763
• 关键词: X ray crystallography; antigen antibody reaction; antigens; autoimmune disorder; biotechnology; fluoresceins; genetic manipulation; hybridomas; immunoglobulin G; immunoglobulin structure; monoclonal antibody; pyrimidines; solutions; thermodynamics

Preliminary thermodynamic studies of a high affinity monoclonal anti-fluorescyl antibody (4-4-20) indicate that its active site is a shallow hydrophobic pocket, which binds fluorescein primarily through entropy. Moreover, high resolution diffraction data (2.5 Angstrom) is available for the liganded antigen binding fragment (Fab) of this antibody, which crystallizes in 16% polyethyleneglycol. Liganded Fab fragments also crystallize in a less polar solvent system (46.7% 2-methyl-2,4-pentanediol). The affinity of the intact IgG molecule is 300-fold lower in this solvent. Correlated crystal and solution studies of both crystal systems are planned. With this information we hope to explain the molecular basis of antigen binding to this antibody. Crystallization trials are currently in progress with four other monoclonal anti-fluorescyl antibodies which are idiotypically related to 4-4-20, but exhibit affinities which vary over a 1000-fold range. Solution studies are planned to determine whether the entropy predominance and active site characteristics of 4-4-20 are common to the other clones as well. Idiotypic determinants are generally thought to be located at or near the active site. Crystal studies should clarify the nature of idiotypic determinants and whether idiotypic reagents are valid tools for identifying related active sites. High resolution diffraction data (2.0 Angstrom) is available for the unliganded Fab fragment of a monoclonal antibody which binds single stranded DNA (BV04-01). In solution, the protein exhibited a base specificity for pyrimidines, with greater affinity for thymine than uracil. This antibody is of clinical importance because it was isolated from a mouse with an autoimmune syndrome similar to systemic lupus erythematosus. We have obtained a low-resolution (6 Angstrom) structure and plan to extend this solution to higher resolution. We also plan to perfuse oligonucleotides into crystals to determine the molecular basis of the observed pyrimidine specificity.

高亲和力单克隆的初步热力学研究 抗氟烯酰基抗体（4-4-20）表明其活性位点是一个 浅疏水口袋，主要通过荧光素结合 熵。 此外，高分辨率衍射数据（2.5 Angstrom）为 可用于该抗体的配体抗原结合片段（FAB）， 它在16％聚乙烯中结晶。 配体的晶圆厂碎片 在较小的极性溶剂系统中结晶（46.7％ 2-甲基-2,4-戊二醇）。 完整的IgG分子的亲和力是 该溶剂中的300倍。 相关的晶体和溶液研究 计划了两个晶体系统。 有了这些信息，我们希望解释 抗原与该抗体结合的分子基础。 结晶 目前，试验正在进行中，其他四个单克隆抗氟乙烯 与4-4-20相关的愚蠢抗体，但展示 在1000倍范围内变化的亲和力。 计划进行解决方案研究 确定熵优势和活动位置是否 4-4-20的特性也是其他克隆的共同点。 通常认为，白痴型的决定因素位于或附近 活性站点。 水晶研究应阐明惯用型的性质 决定因素以及白痴型试剂是否是识别的有效工具 相关的活动站点。 高分辨率衍射数据（2.0 Angstrom）可用于 单克隆抗体的非配体的Fab片段，该抗体结合单个抗体 滞留的DNA（BV04-01）。 在溶液中，蛋白质表现出碱 嘧啶的特异性，对胸腺素的亲和力比 乌拉西尔。 该抗体具有临床重要性，因为它是分离的 从具有自身免疫性综合征的鼠标类似于全身性狼疮 红斑。 我们获得了低分辨率（6埃斯特罗姆）结构 并计划将该解决方案扩展到更高的分辨率。 我们还计划 灌注寡核苷酸成晶体，以确定 观察到的嘧啶特异性。