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.
1
Citation
0
Reference
10
Related Paper
Abstract:
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.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.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.Purified bovine alpha (IV) NC1 could be used as a substitute for human alpha (IV) NC1 to detect anti-GBM antibodies.Keywords:
Type IV collagen
Goodpasture syndrome
Alpha (finance)
Cite
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.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.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.Purified bovine alpha (IV) NC1 could be used as a substitute for human alpha (IV) NC1 to detect anti-GBM antibodies.
Type IV collagen
Goodpasture syndrome
Alpha (finance)
Cite
Citations (1)
Sera from patients with antiglomerular basement membrane (anti-GBM) antibodies associated with Goodpasture syndrome (GP) or glomerulonephritis were tested by ELISA and electroimmunoblot against whole basement membrane collagen (type IV) isolated from bovine anterior lens capsule (ALC) and bacterial collagenase resistant domains of the collagen molecule, that is, the NC-1 and 7-S domains isolated from either ALC or bovine and human glomerular basement membrane (GBM). Reactivity was high with the NC-1 domain by both the ELISA and the electroimmunoblot techniques. Some of the anti-GBM sera reacted with both the NC-1 and 7-S domains of both human and bovine type IV collagen. At a time when the patients' sera reacted weakly with a collagenase digest of human GBM using a radioimmunoassay, the reactivity with the NC-1 domain was also low, but some of the sera continued to react with the 7-S domain. The data suggest that there may be heterogeneity in the nature of autoantibodies with respect to collagen type IV domain reactivity in the sera of patients with anti-GBM antibody disease.
Type IV collagen
Goodpasture syndrome
Cite
Citations (28)
Nephritis
Type IV collagen
Immunofluorescence
Glomerulopathy
Renal glomerulus
Goodpasture syndrome
Cite
Citations (21)
The glomerular basement membrane antigen in Goodpasture syndrome is a collagenase-resistant molecule with a monomer molecular weight of about 26,000. Type IV collagen isolated from glomerular basement membrane contains collagenase-resistant sequences within its structure. Polyacrylamide gel electrophoresis, enzyme-linked immunosorbent assay, and chemical analysis were used to demonstrate that the collagenase-resistant sequences of type IV collagen contain Goodpasture antigen.
Type IV collagen
Microbial collagenase
Goodpasture syndrome
Goodpasture's syndrome
Cite
Citations (200)
Using Brown Norway (BN) rats, we isolated and characterized the tubular basement membrane (TBM) antigens that are immunologically common to humans. The renal basement membrane (RBM) of BN rat, as an antigen source, was solubilized with 8 M urea instead of collagenase followed by extraction with 0.5 M NaCl. On frozen section-immunohistochemistry, the autoantibody obtained from BN rats, which had been immunized with human RBM and showed tubulointerstitial nephritis, bound to the TBM, the basement membrane of the Bowman's capsule, and the brush border of the proximal tubules, but not to the GBM of the normal BN rat kidney. Nephritogenic antigens were isolated by immunoaffinity chromatography using Sepharose-bound purified autoantibody. By Western blot analysis of the eluate, bands with molecular weight of 200 kDa and 180 kDa were positively reacted to anti-FX1A (brush border antigen) antibody and were apparently different from the major bands with molecular weight of 145 kDa and 130 kDa. The bands with molecular weight of 145 and 130 kDa showed major cross reactivity with antibodies to fibronectin and laminin. In contrast with these high molecular weight (HMW) bands, the major 60 kDa band with three minor bands showed no reactivity with any type of antibody tested. These results indicated that the non-enzymatic solubilization of RBM is one of the possible procedures for isolating the HMW form of antigens. These antigens may be epitopically modified pre-existing constitutions of the basement membrane and may play a role in the induction of tubulointerstitial nephritis.
Type IV collagen
Cite
Citations (0)
Goodpasture syndrome
Pepsin
Nephritis
Cite
Citations (46)
Cite
Citations (30)
Sera from patients with poststreptococcal glomerulonephritis (PSGN) known to have antibodies to proteoglycans were studied for the presence of antibodies against other basement membrane (BM) components. BM collagen (type IV) was isolated in the native state by extracting bovine anterior lens capsule (ALC) with 0.5 M acetic acid. The 7-S (collagenous) domain and the NC-1 (noncollagenous) domain of type IV collagen were obtained after bacterial collagenase digestion of ALC followed by gel filtration. Laminin was isolated from the mouse EHS tumor and fibronectin from human plasma. Immunologic studies, using an ELISA and electroimmunoblot, revealed the presence of antibodies that reacted with intact, native type IV collagen and the 7-S collagenous domain of this molecule. Reaction with the NC-1 (noncollagenous) domain was minimal, and not higher than that obtained with control sera. Laminin reaction strongly with the patients' sera, but fibronectin did not. Unlike sera from patients with Goodpasture syndrome, which contain antibodies primarily against the NC-1 (noncollagenous) domain of type IV collagen, sera from patients with acute PSGN contain antibodies against all the major macromolecular components of BM. This difference in immunologic reactivity may account for the observed differences in the pathologic picture at the glomerular level.
Goodpasture syndrome
Type IV collagen
Cite
Citations (85)
The immune nephritis antibody response against the collagen and glycoprotein portions of the glomerular basement membrane (GBM) has been monitored by using either type IV collagen prepared from pepsin digests or a collagenase digest of GBM. Sheep immunized with GBM, according to Steblay, respond by developing antibodies directed against the collagen and the glycoprotein portions, respectively. Circulating antibodies directed against sheep GBM structures were not demonstrated until overt clinical disease with high serum creatinine values and proteinuria. Such antibodies could, however, be eluted from the kidneys, where they adsorbed in a linear fashion as demonstrated by immunofluorescence microscopy. In spontaneous human nephritis in Goodpasture’s syndrome, circulating antibodies were present at the time of diagnosis. These antibodies reacted only with the glycoprotein portion of the basement membrane, and not with the type IV collagen.
Nephritis
Type IV collagen
Immunofluorescence
Goodpasture syndrome
Cite
Citations (3)
Collagenase-digests of human glomerular (GBM), alveolar (ABM), and placenta basement membranes (PBM) were separated by gel filtration columns and the pools rich in Goodpasture antigens (GP) were identified by an antibody inhibition-ELISA. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting on nitrocellulose membranes was performed with each basement membrane preparation. Sera from patients with florid GP-syndrome and antibodies to glomerular basement membrane (anti-GBM antibodies) were incubated with nitrocellulose strips of GBM, ABM, and PBM. Immunoperoxidase staining revealed reactivity with target antigens of 24, 26, 44, and 50 kD in the GBM and of 44 and 50 kD in the ABM and PBM, respectively. No corresponding reactivity was observed using convalescent GP-sera, sera from patients with other immunological diseases or sera from healthy blood donors. The antigens were sensitive to reduction. We conclude, that antigens of similar molecular-weights can be identified by anti-GBM positive sera in human glomerular, alveolar and placenta basement membranes.
Goodpasture syndrome
Renal glomerulus
Cite
Citations (15)