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MOLECULAR BASIS OF ANTIGENIC SPECIFICITY

抗原特异性的分子基础

基本信息

批准号：
2062022
负责人：
JINDRICH H. KOPECEK
金额：
$ 15.69万
依托单位：
UNIVERSITY OF UTAH
依托单位国家：
美国
项目类别：
财政年份：
1986
资助国家：
美国
起止时间：
1986-06-01 至 1995-06-30
项目状态：
已结题

项目摘要

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.

高亲和力单克隆抗体的初步热力学研究抗荧光抗体（4-4-20）表明其活性位点是一个浅疏水口袋，主要通过熵此外，高分辨率衍射数据（2.5埃）是可用于该抗体的配体化抗原结合片段（Fab），其在16%聚乙二醇中结晶。配体Fab片段还在极性较小的溶剂体系中结晶（46.7%）， 2-甲基-2，4-戊二醇）。完整IgG分子的亲和力为 300-在这种溶剂中会降低一倍相关晶体和溶液研究两个水晶系统都在计划中。我们希望通过这些信息来解释抗原与抗体结合的分子基础结晶目前正在进行其他四种单克隆抗荧光素抗体的试验。与4-4-20独特相关的抗体，但表现出其亲和力在1000倍范围内变化。计划进行解决方案研究来确定熵优势和活性位点 4-4-20的特征对于其它克隆也是共同的。独特型决定因素通常被认为位于或接近活性部位晶体研究应阐明独特型的性质决定因素和独特型试剂是否是有效的工具，相关的活跃网站高分辨率衍射数据（2.0埃）可用于单克隆抗体的未配体Fab片段，其结合单个链DNA（BV 04 -01）。在溶液中，该蛋白质显示出一种碱，对嘧啶的特异性，对胸腺嘧啶的亲和力大于尿嘧啶。这种抗体具有临床重要性，因为它是分离出来的，来自一只患有类似系统性狼疮的自身免疫综合征的老鼠红斑我们已经获得了低分辨率（6埃）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灌注到晶体中以确定观察到的嘧啶特异性。