MOLECULAR INTEGRITY OF FVIII IN COMMERCIAL FVIII CONCENTRATES

商业 FVIII 浓缩物中 FVIII 的分子完整性

• 批准号: 3804911
• 负责人: M A LAMB
• 金额: --
• 依托单位: CENTER BIOLOGICS EVALUATION RESEARCH HEMATOLOGY
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3804911
• 关键词: SDS polyacrylamide gel electrophoresis; antibody; chromatography; coagulation factor VIII; immunoglobulin G; immunoprecipitation; laboratory rabbit; protein degradation; proteolysis; western blottings

Since FVIII is highly susceptible to proteolysis, purification processes used in the manufacture of FVIII may result in fragmentation, activation and/or degradation of the FVIII molecule. The molecular integrity of a variety of currently available FVIII concentrates as well as several preparations which are in development is being determined. An immunoprecipitation procedure has been developed for isolating FVIII from commercial concentrates. Polyclonal rabbit IgG against recombinant FVIII has been purified by Protein A chromatography and absorbed with immobilized PPF to remove antibodies to other proteins. The resulting IgG preparation has been coupled to CNBr activated agarose. This solid phase immunoabsorbent is being used to immunoprecipitate FVIII from commercial concentrates. Immunoprecipitates are being electrophoresed on SDS PAGE. FVIII protein is being visualized using the Western blot technique with enzyme linked antibodies. Western blots of FVIII from the various concentrates are being compared with regard to the size and relative distribution of FVIII polypeptides.

由于FVIII对蛋白水解高度敏感，因此纯化工艺 用于生产FVIII可能导致片段化、活化 和/或FVIII分子的降解。 一个分子的完整性 各种目前可用的FVIII浓缩物以及几种 正在确定正在开发的制剂。 一个 已经开发了用于分离FVIII的免疫沉淀方法 商业浓缩物。 抗重组兔IgG多克隆抗体 FVIII已通过蛋白A色谱法纯化，并用 固定化PPF以去除针对其他蛋白质的抗体。 所得 IgG制备物已偶联至CNBr活化的琼脂糖。 该固体 相免疫吸附剂用于免疫沉淀FVIII， 商业浓缩物。 免疫沉淀物正在被 SDS-PAGE。 使用蛋白质印迹法观察FVIII蛋白 酶联抗体技术。 FVIII的蛋白质印迹来自 各种浓缩物在尺寸方面进行比较， FVIII多肽的相对分布。