Isolation And Purification Of Non-Collagen Region 1 Of Type Iv Collagen ¦¡ Chain Of Bovine Glomerular Basement Membrane And Its Application In The Treatment Of Glomerular Basement Membrane-Related Diseases
Keywords
Antibodies/Isolation And Purification, Glomerular Membranes/Immunology, Pulmonary Hemorrhage Nephritis Syndrome
Abstract
Purpose: Isolate and purify the specific target antigen of anti-glomerular basement membrane (gbm) antibodies, and evaluate its application value in the detection of anti-gbm antibodies. Method: Extract bovine glomeruli from bovine kidneys through screens with different pore sizes , use modified 40g*l-1 sodium deoxycholate to destroy glomerular cells to obtain gbm. After digestion with collagenase, collect the soluble bovine gbm crude antigen. Separate it through monoq anion exchange chromatography column under the condition of ph7.5. The glomerular basement membrane type IV collagen ¦Á chain non-collagen domain 1 [¦Á(IV)nc1] was purified, and its purity and activity were identified by SDS-polyacrylamide gel electrophoresis and westernblot. 2mg*l-1 purified bovine ¦Á(IV)nc1 was coated on an enzyme-labeled plate, and a bovine ¦Á(IV)nc1-elisa method was established to detect anti-gbm antibodies in serum, and a control study was conducted with the classic elisa method using human gbm soluble protein as crude antigen. The serum came from 90 100 patients with known positive anti-human gbm antibodies, 100 normal patients and 50 patients with other kidney diseases. Correlation analysis of the two methods was performed and the sensitivity and specificity of bovine ¦Á(IV)nc1-elisa were calculated. Results: Purification The bovine ¦Á(¢¤)nc1 showed by SDS-polyacrylamide gel electrophoresis as proteins with relative molecular masses of 25¡Á103 and 50¡Á103 and could be recognized by anti-gbm antibody-positive serum; application of bovine ¦Á(¢¤)nc1- The sensitivity and specificity of the ELISA method for detecting anti-gbm antibodies are 100% and 98% respectively, and the correlation with human gbm crude antigen-ELISA is 0.6. Conclusion: The modified sodium deoxycholate method is used to destroy intraglomerular cells. And further separate and purify bovine ¦Á(¢¤)nc1 through monoq ion exchange column to obtain high-purity specific target antigen; the purified bovine ¦Á(¢¤)nc1 can be used as a specific antigen to detect human anti-gbm antibodies.
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Original research was done by Xingang, Zhao Minghui, Ding Jiaosheng, Wang Haiyan
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