EPITOPE MAPPING OF ANTIBODIES TO FACTOR VIII

两名抗第VIII因子抗体患者的血浆正在检测中 研究以确定第VIII因子上识别的表位 抗体。通常的方法是用酶裂解。 因子VIII，然后免疫沉淀得到的片段使用 患者免疫球蛋白共价偶联蛋白A-琼脂糖凝胶 珠子。凝血酶消化第VIII因子后， 50kD(A1区--进一步切割成30kD和20kD 片段)、43kD(A2区)和73kD(由 片段A3、C1和C2)被生成。患者B的血浆 免疫共沉淀轻链以及A1和A2片段， 而患者P的血浆只沉淀轻链。作为一名 控制具有已知抗原表位特异性的单抗 使用A2片段的氨基末端。免疫沉淀法 胰酶裂解第VIII因子未获成功。目前还不清楚 为什么，但可能的解释包括 消化和蛋白质不足，无法通过免疫印迹检测。

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.