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.
OBJECTIVE
To purify alpha chain NC1 domains of type IV collagen [alpha (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 alpha (IV) NC1 technique were assessed using SDS-PAGE and Western blot analysis. An ELISA was established using purified bovine alpha (IV) NC1 as solid phase antigens to detect anti-GBM antibodies. Ninety sera from patients with known anti-GBM antibody positive were tested by alpha (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 alpha (IV) NC1 was purified with 25 x 10(3) and 50 x 10(3) on SDS-PAGE and could be blotted by known anti-GBM antibody positive sera. The specificity and the sensitivity of the alpha (IV) NC1-ELISA were 98% and 100% respectively.


CONCLUSIONS
Purified bovine alpha (IV) NC1 could be used as a substitute for human alpha (IV) NC1 to detect anti-GBM antibodies.
Department of Nephrology, Peking University First Hospital, Beijing 100034, China.
Gang
G
Xin
Minghui
M
Zhao
Jiaosheng
J
Ding
Haiyan
H
Wang
D000818:Animals; D019867:Anti-Glomerular Basement Membrane Disease; D001323:Autoantibodies / Q000302:isolation & purification*; D002417:Cattle; D003094:Collagen; D024141:Collagen Type IV; D004797:Enzyme-Linked Immunosorbent Assay; D050533:Glomerular Basement Membrane; D006801:Humans; D007668:Kidney; D012680:Sensitivity and Specificity
D001323:Autoantibodies; D024141:Collagen Type IV; C051951:antiglomerular basement membrane antibody; D003094:Collagen