抗BP230单克隆抗体的制备、鉴定和应用

Preparation,Identification and Application of Monoclonal Antibody of Anti-BP230

目的探讨大疱性类天疱疮抗原1(BP230)作为皮肤与脑组织的共同抗原在大疱性类天疱疮(BP)合并神经系统疾病(ND)中的作用,制备抗BP230单克隆抗体(mAb)并进行鉴定和应用。方法采用抗原免疫Wistar大鼠,采用细胞融合技术制备并纯化mAb,检测效价和亲和常数,以免疫组化、免疫荧光法和免疫印迹进行鉴定。结果抗BP230 mAb效价在45000~139000之间;亲和常数为2.8×108 L/mol;抗BP230 mAb可与鼠脑反应,主要反应部位为大脑皮质;抗BP230 mAb可与小鼠皮肤基底膜带反应;免疫印迹检测到抗BP230 mAb可与鼠皮肤及鼠脑230 kDa蛋白结合。结论成功制备了可识别小鼠大脑皮质、皮肤的抗鼠BP230 mAb,为进一步研究BP230在BP和ND中的作用提供了研究工具。

Objective In order to study the role of BP230 as a shared autoantigen expressed in skin and brain in bullous pemphigoid(BP)patients with neurologic diseases(ND),and to study the preparation,identification and application of monoclonal antibody against BP230.Methods The anti-BP230 mAb was prepared and purified with standard hybridoma technique in wistar rats.Wistar rats were immunized,and mAb was prepared by cell fusion technique and purified.The titer and affinity constant were measured.The identification were tested by immunohistochemistry,immunofluorescence assay and immunoblotting.Results The titer of the mAb was 45000~139000.The affinity constant was 2.8×108 L/mol.The mAb could react with mouse's the cerebral cortex of brain and the basement membrane zone of skin.By western blotting,the mAb could recognized the 230 kDa protein both in the mouse epidermal extract and the mouse brain extract.Conclusion The successful synthesis of monoclonal antibody of anti-BP230 is an useful tool to study furtherly BP230 as the shared autoantigen to bullous pemphigoid and neurological diseases.