Occurence and Mechanisms of Antibody-Antigen Allosteric Binding Behavior

抗体-抗原变构结合行为的发生和机制

• 批准号: 6727039
• 负责人: ROBERT C BLAKE
• 金额: $ 8.64万
• 依托单位: XAVIER UNIVERSITY OF LOUISIANA
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6727039
• 关键词: allosteric site; antigen antibody reaction; binding sites; conformation; fluorescence spectrometry; fluorescent dye /probe; fluorimetry; immunofluorescence technique; immunoglobulin G; immunoglobulin structure; monoclonal antibody; protein binding; proteolysis; surface plasmon resonance

The widespread exploitation of monoclonal antibodies for medical research activities, therapeutic treatments, and medical diagnostic immunoassays constitutes a multi-billion dollar health care industry. This project will investigate two types of novel synergistic binding reactions recently described for selected antibody-antigen pairs: those where the affinity of the antibody appeared to increase as the antigen concentration increased (positive cooperativity); and those where the affinity of an antibody directed toward one epitope on a protein antigen appeared to increase in the presence of a second antibody directed toward a different, separate epitope on the same protein antigen. The long-term goal of this project is to understand the molecular mechanisms of these novel synergistic binding reactions. Experiments proposed for the current grant period will focus primarily on functional studies using intact antibodies and corresponding Fab fragments derived from proteolytic cleavage of the parent structures. Functional studies will include kinetic exclusion assays on a KinExA flow fluorimeter and competition assays on a BIAcore surface plasmon resonance spectrometer. Specific aim #1 is to conduct detailed binding studies on monoclonal antibodies that exhibit synergistic binding behavior with their respective antigens. One goal of this aim is to determine whether certain antibodies possess more than two antigen binding sites per intact IgG molecule. Specific aim #2 is to conduct binding studies on protein antigens that bind monoclonal antibodies to separate epitopes in a synergistic manner. One goal of this aim is to quantify the relative contributions of both avidity effects and binding-dependent protein conformation changes to the apparent synergy of binding. In both aims, the unifying hypothesis is that the underlying molecular mechanisms are protein conformation changes that occur as a consequence of the antibody-antigen binding interaction. Any attempts to exploit antibodies that exhibit novel synergistic binding properties to enhance current clinical diagnostic or therapeutic applications must be dependent on existing knowledge concerning the molecular mechanisms whereby the novel binding is expressed. This project is expected to contribute fundamental knowledge toward manipulating these unexpected activities for diagnostic and therapeutic applications. It will also contribute to a basic understanding of a heretofore unrecognized aspect of antibody function.

单克隆抗体广泛用于医学研究活动、治疗治疗和医学诊断免疫测定，构成了数十亿美元的医疗保健产业。该项目将研究最近针对选定的抗体-抗原对描述的两种新型协同结合反应：抗体的亲和力似乎随着抗原浓度的增加而增加（正协同性）；而那些抗体对蛋白质抗原上的一个表位的亲和力似乎在针对同一蛋白质抗原上不同的单独表位的第二个抗体的存在下增加。该项目的长期目标是了解这些新型协同结合反应的分子机制。目前资助期的实验将主要集中在使用完整抗体和相应Fab片段的功能研究上