抗去唾液酸糖蛋白受体酶联免疫检测法的建立及临床初评

Establishment of enzyme linked immunosorbent assay of autoantibodies to asialoglycoprotein receptor and its clinical application

目的 建立肝脏特异性自身抗体的ELISA检测方法 ,辅助自身免疫性肝炎的诊断。方法 利用亲和层析法从人肝脏组织中直接纯化去唾液酸糖蛋白受体 (ASGPR) ,经免疫学鉴定后以此纯化抗原包被微孔板 ,酶联免疫固相吸附试验检测去ASGPR的自身抗体 (抗 ASGPR)。结果 本研究从10g肝组织纯化ASGPR ,总量为 1.6mg。经SDS PAGE和Dot ELISA验证 ,纯化ASGPR纯度高、具有抗原活性。ASGPR预吸附可阻断血清抗体与ASGPR间的免疫反应 ,类风湿因子、梅毒阳性血清不干扰本ELISA法的抗原抗体反应。 33例自身免疫性肝炎 (AIH )患者中检出抗 ASGPR 2 4例 ,阳性率为72 .7%。在临床上高度怀疑AIH的 10例患者中 ,抗 ASGPR阳性者有 7例。原发性胆汁性肝硬化、病毒性肝炎、肝炎后肝硬化、肝癌、胆囊炎患者中阳性检出率分别为 2 1.4 % (9/42 )、16 .8% (16 /95 )、16 .1%(10 /6 2 )、10 .7% (3/2 8)、14 .3% (1/7)。药物性肝炎、酒精性肝炎、系统性红斑狼疮和类风湿性关节炎患者中未检测到抗 ASGPR。 2 0 0名健康体检者阳性率为 4 .6 %。抗 ASGPR检测对AIH诊断的特异性为89 .6 %。结论 本研究所建立的抗 ASGPRELISA检测方法可靠、特异性好。抗 ASGPR检测有助于AIH的诊断 ,尤其是对那些抗核抗体、平滑肌抗体、肝肾微粒体

Objective To establish an immune enzyme assay to detect liver specific autoantibodies autoantibodies against asialoglycoprotein receptor(anti ASPGR) in human sera for diagnosis of autoimmune hepatitis(AIH). Methods ASGPR were purified from normal human liver tissue by affinity chromatography. The identified and purified antigens were coated with 96 well microplates, and anti ASGPR was detected by enzyme linked immunoadsordent assay(ELISA). Results The yield of purified receptor was 1.6 mg/10 g wet wt liver. The purified ASGPR showed high purity and antigenicity testified with SDS PAGE and Dot ELISA. The immune reaction in ELISA might be blocked by pre absorption with ASGPR, and was not interfered by rheumatoid factor or syphilis positive sera. 24 of 33 patients with AIH were anti ASGPR positive (72.7%) . Of 10 AIH patients with antinuclear antibodies(ANA) at low titer or negative autoantibodies, 7 cases were anti ASGPR positive. The prevalence of anti ASGPR in primary biliary cirrhosis, viral hepatitis, posthepatitic cirrhosis, hepatic cancer, cholecystitis were 21.4%(9/42)、 16.8% (16/95)、16.1%(10/62)、10.7%(3/28), and 14.3%(1/7), respectively. Anti ASGPR was not detected in drug induced hepatitis, alcoholic hepatitis and other autoimmune diseases, including systemic lupus erythematosus, and rheumatic arthritis. Positive ASGPR was only 4.6% in 200 normal individuals. The specificity of anti ASGPR for diagnosis of AIH were 89.6%. Conclusions ELISA established in this study was a reliable method with high specificity for detecting anti ASGPR. Our results showed that anti ASGPR was a helpful methods for diagnosis of AIH, especially for suspected patients with negative ANA/SMA/ LKM 1.