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. 1974 Feb;26(2):311–321.

Further purification of the mitochondrial inner membrane autoantigen reacting with primary biliary cirrhosis sera

Y Ben-Yoseph, E Shapira, Deborah Doniach
PMCID: PMC1423075  PMID: 4852116

Abstract

The mitochondrial inner membrane autoantigen reacting with sera from patients with primary biliary cirrhosis depends on lipid and protein moieties for complement-fixing activity, but its chemical analysis requires some degree of solubilization. Attemps to achieve this by citraconylation led to anticomplementary effects and inactivation, but treatment with 8 M urea fragmented the membranes sufficiently to allow gel filtration and estimation of its mol. wt at 180,000–200,000. The antigen was further purified by affinity chromatography using Igs from patients with anti-mitochondrial antibodies (AMA) coupled to Sepharose 4B, as immunosorbent. The 8 M urea eluate was about 100 times more active than crude inner membranes and showed a single band on polyacrylamide electrophoresis. Liver and brown fat gave the same band, brown fat having four times the potency of liver. Electron microscopy of the purified antigen from the two organs showed that it reaggregated into membranous vesicles when urea was removed. The purified antigen may be of use if an automated radioimmunoassay were to prove sensitive and specific for the detection of AMA as this antibody is an important marker for `autoimmune' chronic liver disorders.

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317FIG. 5
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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Berg P. A., Muscatello U., Horne R. W., Roitt I. M., Doniach D. Mitochondrial antibodies in primary biliary cirrhosis. II. The complement fixing antigen as a component of mitochondrial inner membranes. Br J Exp Pathol. 1969 Apr;50(2):200–208. [PMC free article] [PubMed] [Google Scholar]
  2. Berg P. A., Roitt I. M., Doniach D., Cooper H. M. Mitochondrial antibodies in primary biliary cirrhosis. IV. Significance of membrane structure for the complement-fixing antigen. Immunology. 1969 Aug;17(2):281–293. [PMC free article] [PubMed] [Google Scholar]
  3. Berg P. A., Roitt I. M., Doniach D., Horne R. W. Mitochondrial antibodies in primary biliary cirrhosis. 3. Characterization of the inner-membrane complement fixing antigen. Clin Exp Immunol. 1969 May;4(5):511–525. [PMC free article] [PubMed] [Google Scholar]
  4. DAVIS B. J. DISC ELECTROPHORESIS. II. METHOD AND APPLICATION TO HUMAN SERUM PROTEINS. Ann N Y Acad Sci. 1964 Dec 28;121:404–427. doi: 10.1111/j.1749-6632.1964.tb14213.x. [DOI] [PubMed] [Google Scholar]
  5. Dixon H. B., Perham R. N. Reversible blocking of amino groups with citraconic anhydride. Biochem J. 1968 Sep;109(2):312–314. doi: 10.1042/bj1090312. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Doniach D., Lindqvist H. L., Berg P. A. Non-organ-specific cytoplasmic antibodies detected by immunofluorescence. Int Arch Allergy Appl Immunol. 1971;41(4):501–513. doi: 10.1159/000230545. [DOI] [PubMed] [Google Scholar]
  7. HUNTER W. M., GREENWOOD F. C. Preparation of iodine-131 labelled human growth hormone of high specific activity. Nature. 1962 May 5;194:495–496. doi: 10.1038/194495a0. [DOI] [PubMed] [Google Scholar]
  8. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  9. Parsons D. F., Williams G. R., Chance B. Characteristics of isolated and purified preparations of the outer and inner membranes of mitochondria. Ann N Y Acad Sci. 1966 Jul 14;137(2):643–666. doi: 10.1111/j.1749-6632.1966.tb50188.x. [DOI] [PubMed] [Google Scholar]
  10. Porath J., Axen R., Ernback S. Chemical coupling of proteins to agarose. Nature. 1967 Sep 30;215(5109):1491–1492. doi: 10.1038/2151491a0. [DOI] [PubMed] [Google Scholar]
  11. Reisfeld R. A., Small P. A., Jr Electrophoretic heterogeneity of polypeptide chains of specific antibodies. Science. 1966 May 27;152(3726):1253–1255. doi: 10.1126/science.152.3726.1253. [DOI] [PubMed] [Google Scholar]
  12. Rizzetto M., Swana G., Doniach D. Microsomal antibodies in active chronic hepatitis and other disorders. Clin Exp Immunol. 1973 Nov;15(3):331–344. [PMC free article] [PubMed] [Google Scholar]
  13. Ward H. A., Nairn R. C. Extraction of gastric parietal cell autoantigen. Clin Exp Immunol. 1967 Sep;2(5):565–571. [PMC free article] [PubMed] [Google Scholar]
  14. Ward H. A., Nairn R. C. Gastric parietal cell autoantigen. Physical, chemical and biological properties. Clin Exp Immunol. 1972 Mar;10(3):435–451. [PMC free article] [PubMed] [Google Scholar]
  15. Wright J. M., Doniach D. The significance of cardiolipin immunofluorescence (CLF). Proc R Soc Med. 1971 Apr;64(4):419–422. [PMC free article] [PubMed] [Google Scholar]

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