应用免疫印迹技术检测天疱疮 抗体方法的建立

目的　建立应用免疫印迹技术检测天疱疮抗体方法。方法　环切包皮分离表皮后提取天疱疮抗原,进行印迹反应。对27份寻常型天疱疮（pemphigusvulgaris,PV）患者血清进行检测,并与间接免疫荧光（indirectimmunofluorescence,IIF）比较。结果　在26份血清中检出与表皮分离抗原结合的IgG抗体(96.3%),其中24份与相对分子质量（Mr）为130×10

The method for detection of circulatory pemphigus vulgaris antibodies with immunoblot

Objective To establish the immunoblotting method for detect theantibodies of pemphigus vulgaris (PV). Methods The epidermis of circumcised foreskin was separated, from which the PV antigen was extracted. The protein of PV antigen was separated with SDS polyacrylamide gel electrophoresis. The separated antigen protein was then transferred onto nitrocellulose sheet to make 8×0.3cm slips. The slips were used to react with PV patients′ sera. Finally HRP conjugated mouse anti human IgG was added for immunoblotting. Indirect immunofluorescence was used as comparison. Results Twenty six of 27 sera of PV patients demonstrated IgG antibodies combining with epidermis-separated antigens. Among the 26 patients, 24 had antibodies combining with 130kD protein polypeptide (classic PV antigen), 13 also combined with 150kD antigen polypeptide. In healthy controls, no PV related specific antibodies were found. By indirect immunofluorescence (IIF) method, 25 of 27 were positive. Through χ2 test, the consistency of immunoblot and IIF was significant. Conclusion Immunoblot method is really a practical and reliable diagnostic technique in diagnosis of PV.