EPITOPE MAPPING OF FACTOR VIII AND VON WILLEBRAND FACTOR

因子 VIII 和冯·维勒布兰德因子的表位作图

• 批准号: 3770445
• 负责人: W FRICKE
• 金额: --
• 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/3770445
• 关键词: SDS polyacrylamide gel electrophoresis; autoantibody; autoradiography; coagulation factor VIII; endopeptidases; epitope mapping; hemophilia As; human tissue; immunoprecipitation; protein structure; recombinant proteins; synthetic peptide; thrombin; trypsin; von Willebrand factor; western blottings

Antibodies to factor VIII are present in about 15% of treated patients with hemophilia A and occur as autoantibodies rarely in previously normal people. Antibodies to von Willebrand factor (vWF) have been reported in several patients with severe von Willebrand disease (vWD) and as autoantibodies in normal people on rare occasions. We are studying the factor VIII autoantibodies which developed in two previously normal people in order to determine the epitope specificities. Factor VIII was digested with either thrombin or trypsin and the proteolytic fragments immunoprecipitated with patient antibody coupled to protein A-sepharose beads. The precipitated fragments were detected by immunoblotting or by autoradiography of 125I-labelled factor VIII. These experiments demonstrated that one antibody, a high-titer, type 1 inhibitor recognized epitopes on the A1, A2, and light chain fragments whereas the other, a type 2 inhibitor, recognized the A2 region and the light chain. Binding to the A2 region was weak. Both antibodies precipitated recombinant 35S-labelled C2 region fragments. We have also studied an autoantibody to vWF. 125I- labelled vWF fragments generated by digestion with Staphylococcal V8 protease (the major principle fragments generated by S V8 are 170 kDa and 100 kDa) were immunoprecipitated by patient IgG bound to staphylococcal protein A-sepharose beads. The precipitated fragments were separated by PAGE and the proteins detected by autoradiography. The majority of the radioactivity was located in a band of molecular weight of about 170 kDa, indicating that this fragment contains the epitope recognized by the patient's antibody. Studies are in progress to further localize this epitope.

因子VIII的抗体存在于约15%的治疗患者中， 血友病A和自身抗体很少发生在以前正常 人 血管性血友病因子（vWF）的抗体已在2009年的文献中报道。 几名严重血管性血友病（vWD）患者， 自身抗体在正常人中的罕见情况。 我们正在研究 在两个以前正常的人身上产生的因子VIII自身抗体 以确定表位特异性。 因子VIII被消化 用凝血酶或胰蛋白酶和蛋白水解片段 用偶联至蛋白A-琼脂糖的患者抗体免疫沉淀 珠 沉淀的片段通过免疫印迹或免疫荧光法检测。 125 I标记的因子VIII的放射自显影。 这些实验 证明了一种抗体，一种高滴度的1型抑制剂， A1、A2和轻链片段上的表位，而另一个，A型 2抑制剂，识别A2区和轻链。 结合至 A2区较弱。 两种抗体均沉淀重组35 S标记的 C2区片段。 我们还研究了vWF的自身抗体。 125 I- 用葡萄球菌V8消化产生的标记vWF片段 蛋白酶（由SV 8产生的主要主要片段为170 kDa， 100 kDa）通过与葡萄球菌结合的患者IgG免疫沉淀 蛋白A-琼脂糖珠。 沉淀的碎片通过离心分离。 PAGE和放射自显影检测蛋白质。 大多数 放射性位于分子量约170 kDa的条带中， 这表明该片段含有由所述抗体识别的表位。 患者的抗体。 研究正在进行中，以进一步将其本地化。 表位