Skip to main content
NCBI home page
Clinical and Experimental Immunology logoLink to Clinical and Experimental Immunology
. 1987 Dec;70(3):640–648.

Detection of anti-mitochondrial antibodies by ELISA and Western-blot techniques and identification by one and two-dimensional gel electrophoresis of M2 target antigens.

G Dighiero 1, C Magnac 1, J de Saint Martin 1, N Abuaf 1
PMCID: PMC1542188  PMID: 3325204

Abstract

Seven hundred and eleven sera were simultaneously studied by immunofluorescence (IF), complement fixation test (CFT) and ELISA for the detection of anti-mitochondrial antibodies (AMA). One hundred and nineteen of these sera were also studied by Western-blot techniques, while some of them were examined by two-dimensional gel electrophoresis so as to identify the polypeptides recognized by M2 antibodies. The results indicated that: (1) ELISA is a more sensitive technique for detecting type M2 AMA (27 scored positive in 27 primary biliary cirrhosis (PBC), as compared to 21/27 by IF and 16/27 by CFT). (2) Although ELISA appeared to be a promising screening method, some false positive results were observed that necessitated a double confirmation of positive sera by another technique. (3) Western-blot experiments with rat mitochondrial purified preparation indicated that sera from AMA type 2 could recognize eight different polypeptides and that most of them identified 63-60, 48, 44, and 35-33 kD polypeptides, whereas the 54 and 27 kD were less frequently recognized. A trypsin treatment of antigens confirmed the enzyme sensitivity of most of these antigens. These results suggested some heterogeneity among M2 AMA, though this series of PBC was not large enough to relate the heterogeneous pattern noticed in Western-blot to the clinical and histological patterns observed in PBC.

Full text

PDF
640

Images in this article

642Fig. 1
on p.642
645Fig. 2
on p.645
646Fig. 3
on p.646

Selected References

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

  1. Berg P. A., Klein R., Lindenborn-Fotinos J. Antimitochondrial antibodies in primary biliary cirrhosis. J Hepatol. 1986;2(1):123–131. doi: 10.1016/s0168-8278(86)80015-0. [DOI] [PubMed] [Google Scholar]
  2. Crivelli O., Lavarini C., Chiaberge E., Amoroso A., Farci P., Negro F., Rizzetto M. Microsomal autoantibodies in chronic infection with the HBsAg associated delta (delta) agent. Clin Exp Immunol. 1983 Oct;54(1):232–238. [PMC free article] [PubMed] [Google Scholar]
  3. Dighiero G., Lymberi P., Mazié J. C., Rouyre S., Butler-Browne G. S., Whalen R. G., Avrameas S. Murine hybridomas secreting natural monoclonal antibodies reacting with self antigens. J Immunol. 1983 Nov;131(5):2267–2272. [PubMed] [Google Scholar]
  4. Dighiero G., Poncet P., Rouyre S., Mazie J. C. Newborn Xid mice carry the genetic information for the production of natural autoantibodies against DNA, cytoskeletal proteins, and TNP. J Immunol. 1986 Jun 1;136(11):4000–4005. [PubMed] [Google Scholar]
  5. Doniach D., Roitt I. M., Walker J. G., Sherlock S. Tissue antibodies in primary biliary cirrhosis, active chronic (lupoid) hepatitis, cryptogenic cirrhosis and other liver diseases and their clinical implications. Clin Exp Immunol. 1966 Jul;1(3):237–262. [PMC free article] [PubMed] [Google Scholar]
  6. Frazer I. H., Mackay I. R., Jordan T. W., Whittingham S., Marzuki S. Reactivity of anti-mitochondrial autoantibodies in primary biliary cirrhosis: definition of two novel mitochondrial polypeptide autoantigens. J Immunol. 1985 Sep;135(3):1739–1745. [PubMed] [Google Scholar]
  7. Homberg J. C., Andre C., Abuaf N. A new anti-liver-kidney microsome antibody (anti-LKM2) in tienilic acid-induced hepatitis. Clin Exp Immunol. 1984 Mar;55(3):561–570. [PMC free article] [PubMed] [Google Scholar]
  8. Homberg J. C., Stelly N., Andreis I., Abuaf N., Saadoun F., Andre J. A new antimitochondria antibody (anti-M6) in iproniazid-induced hepatitis. Clin Exp Immunol. 1982 Jan;47(1):93–102. [PMC free article] [PubMed] [Google Scholar]
  9. Kenna J. G., Neuberger J., Davies E., Eddleston A. L., Williams R. A simple enzyme-linked immunosorbent assay for detection of anti-mitochondrial antibodies. J Immunol Methods. 1984 Oct 26;73(2):401–413. doi: 10.1016/0022-1759(84)90415-0. [DOI] [PubMed] [Google Scholar]
  10. Klein R., Maisch B., Kochsiek K., Berg P. A. Demonstration of organ specific antibodies against heart mitochondria (anti-M7) in sera from patients with some forms of heart diseases. Clin Exp Immunol. 1984 Nov;58(2):283–292. [PMC free article] [PubMed] [Google Scholar]
  11. Labro M. T., Andrieu M. C., Weber M., Homberg J. C. A new pattern of non-organ- and non-species-specific anti-organelle antibody detected by immunofluorescence: the mitochondrial antibody number 5. Clin Exp Immunol. 1978 Mar;31(3):357–366. [PMC free article] [PubMed] [Google Scholar]
  12. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  13. Lindenborn-Fotinos J., Baum H., Berg P. A. Mitochondrial antibodies in primary biliary cirrhosis: species and nonspecies specific determinants of M2 antigen. Hepatology. 1985 Sep-Oct;5(5):763–769. doi: 10.1002/hep.1840050510. [DOI] [PubMed] [Google Scholar]
  14. Lindenborn-Fotinos J., Sayers T. J., Berg P. A. Mitochondrial antibodies in primary biliary cirrhosis. VI. Association of the complement fixing antigen with a component of the mitochondrial F1-ATPase complex. Clin Exp Immunol. 1982 Nov;50(2):267–274. [PMC free article] [PubMed] [Google Scholar]
  15. Mendel-Hartvig I., Nelson B. D., Löf L., Tötterman T. H. Primary biliary cirrhosis: further biochemical and immunological characterization of mitochondrial antigens. Clin Exp Immunol. 1985 Nov;62(2):371–379. [PMC free article] [PubMed] [Google Scholar]
  16. Mouritsen S., Demant E., Permin H., Wiik A. High prevalence of anti-mitochondrial antibodies among patients with some well-defined connective tissue diseases. Clin Exp Immunol. 1986 Oct;66(1):68–76. [PMC free article] [PubMed] [Google Scholar]
  17. Norberg R., Gårdlund B., Thorstensson R., Lidman K. Further immunological studies of sera containing anti-mitochondrial antibodies, type M 5. Clin Exp Immunol. 1984 Dec;58(3):639–644. [PMC free article] [PubMed] [Google Scholar]
  18. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  19. Sayers T. J., Binder T., Berg P. A. Heterogeneity of anti-mitochondrial antibodies: characterization and separation of the antigen associated with the pseudolupus erythematosus syndrome. Clin Exp Immunol. 1979 Jul;37(1):68–75. [PMC free article] [PubMed] [Google Scholar]
  20. Schultheiss H. P., Berg P., Klingenberg M. The mitochondrial adenine nucleotide translocator is an antigen in primary biliary cirrhosis. Clin Exp Immunol. 1983 Dec;54(3):648–654. [PMC free article] [PubMed] [Google Scholar]
  21. WALKER J. G., DONIACH D., ROITT I. M., SHERLOCK S. SEROLOGICAL TESTS IN DIAGNOSIS OF PRIMARY BILIARY CIRRHOSIS. Lancet. 1965 Apr 17;1(7390):827–831. doi: 10.1016/s0140-6736(65)91372-3. [DOI] [PubMed] [Google Scholar]
  22. Wright D. J., Doniach D., Lessof M. H., Turk J. L., Grimble A. S., Catterall R. D. New antibody in early syphilis. Lancet. 1970 Apr 11;1(7650):740–743. doi: 10.1016/s0140-6736(70)90972-4. [DOI] [PubMed] [Google Scholar]

Articles from Clinical and Experimental Immunology are provided here courtesy of British Society for Immunology

RESOURCES