EPITOPE MAPPING OF ANTIBODIES TO FACTOR VIII

来自两名具有因子VIII抗体的患者的血浆正在被 研究以鉴定因子VIII上被 抗体。 一般的方法是酶促切割 因子VIII，然后用免疫沉淀法 患者免疫球蛋白与蛋白A共价偶联-琼脂糖凝胶 珠 凝血酶消化因子VIII后， 50 kD（A1区-进一步裂解为30 kD和20 kD 片段）、43 kD（A2区）和73 kD（轻链， 片段A3、C1和C2）。 患者B的血浆 免疫沉淀轻链以及A1和A2片段， 而患者P的血浆仅沉淀轻链。 作为 控制对表位具有已知特异性的单克隆抗体， 使用A2片段的氨基末端。 的免疫沉淀 胰蛋白酶裂解的因子VIII是不成功的。 目前还不清楚 为什么，但可能的解释包括表位的破坏， 消化和免疫印迹检测不到足够的蛋白质。

Plasma from two patients with antibodies to factor VIII are being studied to identify the epitopes on factor VIII that are recognized by the antibodies. The general approach has been to enzymatically cleave factor VIII and then immunoprecipitate the resulting fragments using patient immunoglobulin covalently coupled to protein A - Sepharose beads. Following digestion of factor VIII by thrombin, fragments of 50 kD (the A1 region - which is further cleaved to 30 kD and 20 kD pieces), 43 kD (the A2 region), and 73 kD (the light chain consisting of fragments A3, C1, and C2) are generated. Patient B's plasma immunoprecipitates the light chain and both the A1 and A2 fragments, whereas patient P's plasma precipitates the light chain only. As a control a monoclonal antibody with known specificity for an epitope in the aminoterminus of the A2 fragment is used. Immunoprecipitation of trypsin cleaved factor VIII has been unsuccessful. It is unclear as to why, but possible explanations include destruction of the epitopes by digestion and insufficient protein to be detected by immunoblotting.