AUTOIMMUNE THROMBOCYTOPENIA

自身免疫性血小板减少症

• 批准号: 6184649
• 负责人: ROBERT MCMILLAN
• 金额: $ 35.26万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-30 至 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6184649
• 关键词: antigen antibody reaction; autoantibody; autoimmune disorder; calcium binding protein; chimeric proteins; clinical research; epitope mapping; glycoproteins; human subject; immunoglobulin G; peptide library; platelets; quality of life; thrombocytopenia; thrombopoietic factor; transfection /expression vector

DESCRIPTION (Adapted from applicant's abstract) Autoimmune thrombocytopenia is often due to antibodies reactive with platelet glycoprotein (GP) complexes, particularly GPIIb/IIIa. Autoepitopes are often divalent cation-dependent. The investigators will focus on two research areas: autoantibody characterization and antigenic localization. Autoantibody characterization. They will: 1) Demonstrate the presence of platelet-associated and plasma antibodies and focus on antibodies against GPIIb/IIIa, since these are most common; 2) Evaluate antibody clonality by examining light chain and IgG subtype characteristics and 3) Study autoantibody binding to megakaryocytes in vitro and determine if bound antibody effects megakaryocyte maturation, activates complement or induces phagocytosis. Antigen localization. They will: 1) Determine if rabbit antibody to the GPIIb/IIIa calcium-bindings sites will inhibit autoantibody binding; 2) Test autoantibody binding to a series of large recombinant peptides, spanning glycoproteins Iib and IIIa. They will insert specific PCR-generated cDNA fragments into a vector which results in their expression by E. Coli as fusion proteins with a 6X-histidine tag which can then be affinity-purified using a nickel nitrilo-tricacetic acid resin column. Antibodies which bind to one of the large peptides will be studied further by determining binding patterns to smaller peptides within this region; 3) Evaluate epitopes, not identified by this technique, with random peptide libraries and GPIIb/IIIa chimeras. Antibodies are incubated with fUSE5 filamentous phage expressing several million random peptide sequences. Phage expressing sequences which bind to antibodies are separated from non-specific phage by serial 'planning' with staphylococcal-A protein agarose followed by acid elution. The sequence of the specific peptide(s) is determined. In addition, they will develop human/xenopus and human/avian GPIIb/IIIa chimeras which they will use to examine the binding patterns of human antiplatelet autoantibodies. Results of these studies will be correlated with the patients' clinical course and thrombopoietin levels. In addition, the effect of immune thrombocytopenia on the patients' quality of life will be studied.

描述 （改编自申请人的摘要）自身免疫性血小板减少症通常是由于 与血小板糖蛋白（GP）复合物反应的抗体， 特别是 GPIIb/IIIa。 自身表位通常是二价阳离子依赖性的。 研究人员将重点关注两个研究领域：自身抗体 表征和抗原定位。 自身抗体表征。 他们将： 1) 证明血小板相关和血浆的存在 抗体并重点关注针对 GPIIb/IIIa 的抗体，因为这些是最常见的 常见的; 2) 通过检查轻链和 IgG 评估抗体克隆性 亚型特征和3) 研究自身抗体与巨核细胞的结合 体外并确定结合的抗体是否影响巨核细胞成熟， 激活补体或诱导吞噬作用。 抗原定位。 他们 将： 1) 确定兔抗体是否针对 GPIIb/IIIa 钙结合 位点会抑制自身抗体结合； 2) 测试自身抗体与 系列大型重组肽，涵盖糖蛋白 Iib 和 IIIa。 他们将特定的 PCR 生成的 cDNA 片段插入到载体中， 导致它们在大肠杆菌中表达为融合蛋白 6X-组氨酸标签，然后可以使用镍进行亲和纯化 次氮基三乙酸树脂柱。 与其中之一结合的抗体 将通过确定与大肽的结合模式来进一步研究大肽 该区域内较小的肽； 3) 评估未识别的表位 该技术采用随机肽库和 GPIIb/IIIa 嵌合体。 将抗体与表达多种蛋白的 fUSE5 丝状噬菌体一起孵育 百万个随机肽序列。 噬菌体表达序列结合 通过一系列“规划”，将抗体与非特异性噬菌体分离 葡萄球菌-A 蛋白琼脂糖，然后酸洗脱。 的顺序为 确定特定的肽。 此外，他们还将开发 人类/爪蟾和人类/鸟类 GPIIb/IIIa 嵌合体，它们将用于 检查人类抗血小板自身抗体的结合模式。 结果 这些研究将与患者的临床病程相关 血小板生成素水平。 此外，免疫性血小板减少症的影响 将研究患者的生活质量。