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Journal of Clinical Laboratory Analysis logoLink to Journal of Clinical Laboratory Analysis
. 2002 Mar 15;16(2):103–108. doi: 10.1002/jcla.10026

Specificity of autoantibodies to SS‐A/Ro on a transfected and overexpressed human 60 kDa Ro autoantigen substrate

Marvin J Fritzler 1,, Cheryl Hanson 1, Joan Miller 1, Theophany Eystathioy 1
PMCID: PMC6808092  PMID: 11948800

Abstract

The objective of this study was to analyze apparently discrepant results that arose during the use of an indirect immunofluorescence (IIF) assay using transfected HEp‐2 cells to detect anti‐SS‐A/Ro autoantibodies in human sera. Fourteen sera that had SS‐A/Ro antibodies as detected on this commercial substrate, but did not have antibodies to SS‐A/Ro as determined by double immunodiffusion (ID) or enzyme‐linked immunosorbent assay (ELISA), were studied by immunoprecipitation (IP) of radiolabeled cell extracts and full‐length recombinant SS‐A/Ro. A multi‐antigen strip immunoblotting (IB) assay containing both the 52‐ and 60‐kDa antigens was included in the analysis. We confirmed that 12 of 14 of the sera under study had antibodies to SS‐A/Ro protein antigens as determined by at least one other immunoassay. One serum had antibodies to hyRNA but no detectable reactivity with the 52‐ or 60‐kDa antigens. One serum remained negative in all assays for SS‐A/Ro autoantibodies. J. Clin. Lab. Anal. 16:103–108, 2002. © 2002 Wiley‐Liss, Inc.

Keywords: autoantibodies, Sjögren’s syndrome, systemic lupus erythematosus, indirect immunofluorescence, transfected cells

REFERENCES

  • 1. Anderson JR, Gray KG, Beck JS, Kinnear WF. 1961. Precipitating autoantibodies in Sjögren’s disease. Lancet 2:456–460. [DOI] [PubMed] [Google Scholar]
  • 2. Clark G, Reichlin M, Tomasi TB. 1969. Characterization of a soluble cytoplasmic antigen reactive with sera from patients with systemic lupus erythematosus. J Immunol 102:117–122. [PubMed] [Google Scholar]
  • 3. Alspaugh MA, Tan EM. 1975. Antibodies to cellular antigens in Sjogren’s syndrome. J Clin Invest 55:1067–1073. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4. Alspaugh M, Maddison P. 1979. Resolution of the identity of certain antigen‐antibody systems in systemic lupus erythematosus and Sjögren’s syndrome: an interlaboratory collaboration. Arthritis Rheum 22:796–798. [DOI] [PubMed] [Google Scholar]
  • 5. Lerner MR, Boyle JA, Hardin JA, Steitz JA. 1981. Two novel classes of small ribonucleoproteins detected by antibodies associated with lupus erythematosus. Science 211:400–402. [DOI] [PubMed] [Google Scholar]
  • 6. Ben‐Chetrit E. 1993. The molecular basis of the SSA/Ro antigens and the clinical significance of their autoantibodies. Br J Rheumatol 32:396–402. [DOI] [PubMed] [Google Scholar]
  • 7. Chan EKL, Andrade LEC. 1992. Antinuclear antibodies in Sjögren’s syndrome. Rheum Dis Clin North Am 18:551–570. [PubMed] [Google Scholar]
  • 8. Van Venrooij WJ, Slobbe RL, Pruijn GJM. 1993. Structure and function of La and Ro RNPs. Mol Biol Rep 18:113–119. [DOI] [PubMed] [Google Scholar]
  • 9. Gaither KK, Fox OF, Yamagata H, Mamula MJ, Reichlin M, Harley JB. 1987. Implications of Anti‐Ro/Sjögren’s syndrome‐A antigen autoantibody in normal sera for autoimmunity. J Clin Invest 79:841–846. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10. Harmon CE, Deng JS, Peebles CL, Tan EM. 1984. The importance of tissue substrate in the SS‐ARo antigen‐antibody system. Arthritis Rheum 27:166–173. [DOI] [PubMed] [Google Scholar]
  • 11. Xia P, Fritz KA, Geoghegan WD, Jordon RE. 1987. The particulate (speckled‐like thread) nuclear staining pattern: species and cellular distribution of the Ro/SS‐A antigen. J Clin Lab Immunol 22:101–105. [PubMed] [Google Scholar]
  • 12. Takano S, Matsushima H, Hiwatashi T, Miyachi K. 1989. Detection of anti‐SS‐A/Ro antibody by using pig spleen supernatant and characterization of corresponding antigen. Japan J Rheumatol 2:67–78. [PubMed] [Google Scholar]
  • 13. Wolin SL, Steitz JA. 1984. The Ro small cytoplasmic ribonucleoproteins: identification of the antigenic protein and its binding site on the Ro RNAs. Proc Natl Acad Sci U S A 81:1996–2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14. Forman MS, Nakamura M, Mimori T, Gelpi C, Hardin JA. 1985. Detection of antibodies to small nuclear ribonucleoproteins and small cytoplasmic ribonucleoproteins using unlabeled cell extracts. Arthritis Rheum 28:1356–1361. [DOI] [PubMed] [Google Scholar]
  • 15. Ben‐Chetrit E, Chan EKL, Sullivan KF, Tan EM. 1988. A 52‐kD protein is a novel component of the SS‐A/Ro antigenic particle. J Exp Med 167:1560–1571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16. Rader MD, O’Brien C, Liu Y, Harley JB, Reichlin M. 1989. Heterogeneity of the Ro/SSA antigen: different molecular forms in lymphocytes and red blood cells. J Clin Invest 83:1293–1298. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17. Buyon JP, Slade SG, Chan EKL, Tan EM, Winchester R. 1990. Effective separation of the 52 kDa SSA/Ro polypeptide from the 48 kDa SSB/La polypeptide by altering conditions of polyacrylamide gel electrophoresis. J Immunol Methods 129:207–210. [DOI] [PubMed] [Google Scholar]
  • 18. Slobbe RL, Pruijn GJM, Damen WGM, Van Der Kemp JWCM, Van Venrooij WJ. 1991. Detection and occurrence of the 60‐and 52‐kD Ro‐(SS‐A) antigens and of autoantibodies against these proteins. Clin Exp Immunol 86:99–105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19. Manoussakis MN, Kistis KG, Liu X, Aidinis V, Guialis A, Moutsopoulos HM. 1993. Detection of anti‐Ro(SS‐A) antibodies in autoimmune diseases: comparison of five methods. Br J Rheumatol 32:449–455. [DOI] [PubMed] [Google Scholar]
  • 20. Meilof JF, Bantjes I, de Jong J, Van Dam AP, Smeenk RJT. 1990. The detection of anti‐Ro(SS‐A) and anti‐La(SS‐B) antibodies. A comparison of counterimmunoelectrophoresis with immunoblot, ELISA, and RNA‐precipitation assays. J Immunol Meth 133:215–226. [DOI] [PubMed] [Google Scholar]
  • 21. Bossuyt X, Meurs L, Mewis A, Marien G, Blanckaert N. 2000. Screening for autoantibodies to SS‐A/Ro by indirect immunofluorescence using HEp‐2000TM cells. Ann Clin Biochem 37:216–219. [DOI] [PubMed] [Google Scholar]
  • 22. Boire G, Craft J. 1990. Human Ro ribonucleoprotein particles: characterization of native structure and stable association with the La polypeptide. J Clin Invest 85:1182–1190. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23. Boire G, Lopez‐Longo F‐J, Lapointe S, Menard H‐A. 1991. Sera from patients with autoimmune disease recognize conformational determinants on the 60‐kd Ro/SS‐A protein. Arthritis Rheum 34:722–729. [DOI] [PubMed] [Google Scholar]
  • 24. Chan EKL, Buyon JP. 1994. The SS‐A/Ro antigen In: Van Venrooij W, Maini RN, editors. Manual of biological markers of disease. Dordecht, The Netherlands: Kluwer Academic Publishers; p 1–18. [Google Scholar]
  • 25. Tan EM, Smolen JS, McDougal JS, Klippel JH, Fritzler MJ, Gordon TP, Hardin JA, Kalden JR, Lahita RG, Maini RN, Rothfield NF, Takasaki Y, Wiik A, Wilson MR, Koziol JA. 2002. A critical evaluation of enzyme immunoassay kits for the detection of antinuclear antibodies of defined specificities. II. Potential for quantitation of antibody content. J Rheumatol 29:68–74. [PubMed] [Google Scholar]
  • 26. Hendrick JP, Wolin SL, Rinke J, Lerner MR, Steitz JA. 1981. Ro small cytoplasmic ribonucleoproteins are a subclass of La ribonucleoproteins: further characterization of the Ro and La small ribonucleoproteins from uninfected mammalian cells. Mol Cell Biol 1:1138–1148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27. Keech CL, McCluskey J, Gordon TP. 1994. Transfection and overexpression of the human 60‐kDa Ro/SS‐A autoantigen in HEp‐2 cells. Clin Immunol Immunopathol 73:146–151. [DOI] [PubMed] [Google Scholar]
  • 28. Fritzler MJ, Miller BJ. 1995. Detection of autoantibodies to SS‐A/Ro by indirect immunofluorescence using a transfected and overexpressed human 60 kD Ro autoantigen in HEp‐2 cells. J Clin Lab Anal 9:218–224. [DOI] [PubMed] [Google Scholar]
  • 29. Keech CL, Howarth S, Coates T, Rischmueller M, McCluskey J, Gordon TP. 1996. Rapid and sensitive detection of anti‐Ro (SS‐A) antibodies by indirect immunofluorescence of 60 kDa Ro HEp‐2 transfectants. Pathology 28:54–57. [DOI] [PubMed] [Google Scholar]
  • 30. Pollock W, Toh BH. 1999. Routine immunofluorescence detection of Ro/SS‐A autoantibody using HEp‐2 cells transfected with human 60 kDa Ro/SS‐A. J Clin Pathol 52:684–687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31. Morozzi G, Bellisai F, Simpatico A, Pucci G, Bacarelli MR, Campanella V, Marcolongo R, Galeazzi M. 2000. Comparison of different methods for the detection of anti‐Ro/SSAm antibodies in connective tissue diseases. Clin Exp Rheumatol 18:729–731. [PubMed] [Google Scholar]
  • 32. Peene I, Van Ael W, Vandenbossche M, Vervaet T, Veys E, De Keyser F. 2000. Sensitivity of the HEp‐2000 substrate for the detection of anti‐SSA/Ro60 antibodies. Clin Rheumatol 19:291–295. [DOI] [PubMed] [Google Scholar]
  • 33. Tan EM, Feltkamp TEW, Alarcon‐Segovia D, Dawkins RL, Homma M, Kalden JR, Lambert PH, Lange A, Maini RN, McDuffie FC, McDougal JS, Norberg R, Wilson M. 1988. Reference reagents for antinuclear antibodies. Arthritis Rheum 31:1331. [Google Scholar]
  • 34. Smolen JS, Butcher B, Fritzler MJ, Gordon T, Hardin J, Kalden JR, Lahita R, Maini RN, Reeves W, Reichlin M, Rothfield N, Takasaki Y, Van Venrooij WJ, Tan EM. 1997. Reference sera for antinuclear antibodies. II. Further definition of antibody specificities in international antinuclear antibody reference sera by immunofluorescence and Western blotting. Arthritis Rheum 40:413–418. [DOI] [PubMed] [Google Scholar]
  • 35. Fritzler MJ, Pauls JD, Kinsella TD, Bowen TJ. 1985. Antinuclear, anticytoplasmic and anti‐Sjögren’s syndrome antigen‐A (SS‐A/Ro) antibodies in female blood donors. Clin Immunol Immunopathol 36:120–128. [DOI] [PubMed] [Google Scholar]
  • 36. Fritzler MJ, Hamel JC, Ochs RL, Chan EKL. 1993. Molecular characterization of two human autoantigens: unique cDNAs encoding 95‐ and 160‐kD proteins of a putative family in the Golgi complex. J Exp Med 178:49–62. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37. Harley JB, Scofield RH, Reichlin M. 1992. Anti‐Ro in Sjögren’s syndrome and systemic lupus erythematosus. Rheum Dis Clin North Am 18:337–353. [PubMed] [Google Scholar]

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