MOLECULAR BASIS OF ANTIGENIC SPECIFICITY

抗原特异性的分子基础

• 批准号: 3134552
• 负责人: James N Herron
• 金额: $ 16.18万
• 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 1993-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3134552
• 关键词: X ray crystallography; antibody specificity; antigen antibody reaction; autoantibody; calorimetry; chemical binding; computer simulation; enzyme linked immunosorbent assay; epitope mapping; fluoresceins; fluorescent dye /probe; haptens; hybridomas; immunoglobulin structure; immunoglobulins; infrared spectrometry; interferometry; isomorphous substitution; laboratory mouse; molecular dynamics; monoclonal antibody; nuclear magnetic resonance spectroscopy; peptide chemical synthesis; protein sequence; protein structure function; solutions; synthetic antigens; systemic lupus erythematosus; 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.

一种高亲和力单抗的热力学初步研究 抗荧光素抗体(4-4-20)表明其活性部位为 浅疏水口袋，主要通过以下途径结合荧光素 信息量。此外，高分辨率的衍射数据(2.5埃)是 可用于该抗体的连接抗原结合片段(FAB)， 结晶在16%的聚乙二醇中。连接的Fab片段也 在低极性溶剂体系中结晶(46.7% 2-甲基-2，4-戊二醇)。完整免疫球蛋白分子的亲和力为 在这种溶剂中降低300倍。相关晶体和溶液的研究 这两个水晶系统都在计划之中。有了这些信息，我们希望能解释 抗原与这种抗体结合的分子基础。结晶 目前正在对另外四种抗荧光素的单抗进行试验 与4-4-20独特型相关的抗体，但表现为 亲和力在1000倍的范围内变化。计划进行解决方案研究 确定是否具有熵优势和活跃部位 4-4-20的特性也是其他无性系共有的。 独特型决定因素通常被认为位于 活动站点。水晶研究应该澄清独特型的本质 决定因素和独特型试剂是否是识别 相关活动站点。 高分辨率的衍射数据(2.0埃)可用于 与单个抗体结合的单抗的未连接Fab片段 搁浅的DNA(BV04-01)。在溶液中，蛋白质表现出一种碱基 对嘧啶的特异性，与胸腺嘧啶的亲和力比 尿嘧啶。这种抗体具有临床重要性，因为它是被分离出来的。 来自患有类似系统性红斑狼疮的自身免疫综合征的小鼠 红斑狼疮。我们得到了一个低分辨率(6埃)的结构 并计划将该解决方案扩展到更高的分辨率。我们还计划 将寡核苷酸注入晶体以确定其分子基础 观察到的嘧啶专一性。