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      Isolation of Human Glomerular Basement Membrane Antigens by Affinity Chromatography Utilising Goodpasture’s Kidney Antibody Eluates

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      , ,
      Kidney and Blood Pressure Research
      S. Karger AG

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          Abstract

          The soluble material produced from a 96-hour digest of sonicated human glomerular basement membranes with purified collagenase was shown to contain at least 12 components by sodium dodecyl sulphate-polyacrylamide gel electrophoresis with molecular weights ranging from 20×10<sup>3</sup> to greater than 200 ×10<sup>3</sup> daltons. Five of these components produced antigenic peaks after two-dimensional immunoelectrophoresis into agar plates containing rabbit antiserum to the solubilised glomerular basement membrane. Two groups of antigenic components were demonstrated in these gels by two-dimensional immunoelectrophoresis autoradiographic techniques utilising <sup>125</sup>I-labelled collagenase-digested human glomerular basement membranes reacted with antibodies eluted at acid pH from the kidney of the Goodpasture’s patient. This acid eluate was shown to contain contaminants of α<sub>1</sub>-antitrypsin and albumin co-eluting with the antibodies bound to the glomerular basement membrane. After removal of these contaminants, the antibodies were linked to cyanogen bromide-activated Sepharose and used to affinity purify four antigenic fractions from the collagenase-digested glomerular basement membrane. These fractions were eluted by 0.2 Mglycine pH 2.8 and with 0.5 Mammonium thiocyanate and had molecular weights of 22–25,43–45,65–70 and 205 ×10<sup>3</sup> daltons. The smaller molecular weight components were shown to be common to both included and excluded fractions obtained by Ultragel AcA 44 chromatography of the digested material in 10 m M phosphate pH 8. This suggests that the larger molecular weight component would be an aggregate of a smaller component. Preliminary carbohydrate and amino acid analyses indicated that the different elution procedures elicited antigens with different chemical characteristics.

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          Author and article information

          Journal
          KBR
          Kidney Blood Press Res
          10.1159/issn.1420-4096
          Kidney and Blood Pressure Research
          S. Karger AG
          978-3-8055-2952-5
          978-3-318-05781-2
          1420-4096
          1423-0143
          1980
          1980
          06 November 2008
          : 3
          : 1-6
          : 156-162
          Affiliations
          Department of Pathology, University of Otago, and Clinical School of Medicine, Christchurch, New Zealand
          Article
          172756 Renal Physiol 1980;3:156–162
          10.1159/000172756
          f427933b-8bb5-433e-9556-2ad8fa37d70a
          © 1980 S. Karger AG, Basel

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          History
          Page count
          Pages: 7
          Categories
          Biochemistry

          Cardiovascular Medicine,Nephrology
          Cardiovascular Medicine, Nephrology

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