牛肾小球基底膜Ⅳ型胶原α链非胶原区1的分离纯化及其在抗肾小球基底膜相关疾病中的应用(英文)

Purification of alpha chain NC1 domains of type Ⅳ collagen from bovine kidney and their application in ELISA for detecting anti-glomerular basement membrane antibodies

目的 :分离纯化抗肾小球基底膜 (GBM)抗体的特异性靶抗原 ,并评价其在抗GBM抗体检测中的应用价值。方法 :通过孔径不同的筛网从牛肾脏中提取牛肾小球 ,采用改良的 4 0g·L-1去氧胆酸钠破坏肾小球细胞而获得GBM ,经胶原酶消化后收集可溶性牛GBM粗抗原。在 pH 7.5的条件下经MonoQ阴离子交换层析柱分离纯化肾小球基底膜Ⅳ型胶原α链非胶原区 1[α(Ⅳ )NC1],并经SDS -聚丙烯酰胺凝胶电泳和Westernblot鉴定其纯度及活性。应用 2mg·L-1纯化的牛α(Ⅳ )NC1包被酶标板 ,建立牛α(Ⅳ )NC1-ELISA方法检测血清中抗GBM抗体 ,并与经典的以人GBM可溶性蛋白为粗抗原的ELISA法进行对照研究。血清来自 90例已知抗人GBM抗体阳性的患者、10 0例正常人和 5 0例其他肾脏疾病患者。对两种方法进行相关分析并计算牛α(Ⅳ )NC1-ELISA的敏感性、特异性。结果 :纯化的牛α(Ⅳ )NC1经SDS -聚丙烯酰胺凝胶电泳表现为相对分子质量为 2 5× 10 3 和 5 0× 10 3的蛋白并可被抗GBM抗体阳性血清所识别 ;应用牛α(Ⅳ )NC1-ELISA法检测抗GBM抗体的敏感性和特异性分别为 10 0 %和 98% ,与人GBM粗抗原 -ELISA的相关性为 0 .6。结论 :应用改良的去氧胆酸钠方法破坏肾小球内细胞并进一步经MonoQ离子交换柱分离纯化牛α(Ⅳ )NC1,可以得到高纯度?

Objective: To purify alpha chain NC1 domains of type Ⅳ collagen [α(Ⅳ)NC1] from bovine kidney and to evaluate their application in ELISA for detecting anti glomerular basement membrane(GBM) antibodies. Methods: Glomeruli were isolated by differential sieving from bovine kidney, and GBM was isolated by 40 g·L -1 deoxycholic acid extraction technique. Then the insoluble basement membrane material was digested using collagenase, and the non collagenous domain(NC1) was isolated by Mono Q ion exchange chromatography. The purity and activity of the purified α(Ⅳ)NC1 technique were assessed using SDS-PAGE and Western blot analysis. An ELISA was established using purified bovine α(Ⅳ)NC1 as solid phase antigens to detect anti GBM antibodies. Ninety sera from patients with known anti GBM antibody positive were tested by α(Ⅳ)NC1-ELISA. One hundred sera from healthy blood donors and fifty sera from patients with other renal diseases were used as controls. The specificity and the sensitivity of the method were evaluated. Results: Bovine α(Ⅳ)NC1 was purified with 25×10 3 and 50×10 3 on SDS-PAGE and could be blotted by known anti GBM antibody positive sera. The specificity and the sensitivity of the α(Ⅳ)NC1-ELISA were 98% and 100% respectively. Conclusion: Purified bovine α(Ⅳ)NC1 could be used as a substitute for human α(Ⅳ)NC1 to detect anti GBM antibodies.