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.
Conclusion: Purified bovine alpha (IV) NC1 could be used as a substitute for human alpha (IV) NC1 to detect anti-GBM antibodies.