Skip to main content
NCBI home page
Journal of Clinical Laboratory Analysis logoLink to Journal of Clinical Laboratory Analysis
. 2004 Jan 13;18(1):19–26. doi: 10.1002/jcla.20003

Methods to detect antifibrillarin antibodies in patients with systemic sclerosis (SSc): A comparison

Josefina Huerta García 1, Monica Delgado Osuna 1, Filiberto Martinez Castrejon 1, Laura Guzman Enriquez 1, Pedro A Reyes 2, J Jesus Cortes Hermosillo 1,✉
PMCID: PMC6808019  PMID: 14730553

Abstract

Autoantibodies against nucleolar antigens are common in systemic sclerosis (SSc). They include autoantibodies against fibrillarin (Fb), which are serological markers for SSc. Fb is associated with the evolutionally‐conserved box C/D of small nucleolar RNAs (snoRNAs). We compared indirect immunofluorescence (IIF), Western blot (WB), and immunoprecipitation (IPP) of total small RNAs assays to determine which of these techniques is most specific for the detection of snoRNPs. We also examined the frequency and specificity of autoantibodies from SSc patients to snoRNAs, snRNAs, and scRNAs, and concluded that 1) IIF can not determine autoantibody specificity against Fb, 2) 36% of SSc sera were false‐negative by WB, and 3) by IPP, anti‐Fb autoantibodies from SSc patients can bind U3, U8, U13, U15, and U22 snoRNAs. J. Clin. Lab. Anal. 18:19–26, 2004. © 2004 Wiley‐Liss, Inc.

Keywords: immunofluorescence, Western blot, immunoprecipitation snoRNAs, rheumatic diseases

REFERENCES

  • 1. Tan EM. Antinuclear antibodies: diagnostic markers for autoimmune diseases as probes for cell biology. Adv Immunol 1989;44:93–151. [DOI] [PubMed] [Google Scholar]
  • 2. Tan EM, Rodnan GP, Garcia I, Moroi Y, Fritzler MJ, Peebles C. Diversity of antinuclear antibodies in progressive systemic sclerosis: anti‐centromere antibody and its relationship to CREST syndrome. Arthritis Rheum 1980;23:617–625. [DOI] [PubMed] [Google Scholar]
  • 3. Pollard KM, Reimer G, Tan EM. Autoantibodies in scleroderma. Clin Exp Rheumatol 1989;7:S57–S62. [PubMed] [Google Scholar]
  • 4. Kipnis RJ, Craft J, Hardin JA. The analysis of antinuclear and antinucleolar autoantibodies of scleroderma by radioimmuno‐precipitation assays. Arthritis Rheum 1990;33:1431–1437. [DOI] [PubMed] [Google Scholar]
  • 5. Fritzler MJ. Autoantibodies in scleroderma. J Dermatol 1993; 20:257–268. [DOI] [PubMed] [Google Scholar]
  • 6. Vazquez Abad D, Rothfield NF. Autoantibodies in systemic sclerosis. Intern Rev Immunol 1995;12:145–157. [DOI] [PubMed] [Google Scholar]
  • 7. Chang M, Wang RJ, Yangco DT, Sharp GC, Komatireddy GR, Hoffman RW. Analysis of autoantibodies against RNA polymerases using immunoaffinity purified RNA polymerase I, II and III antigen in an enyme‐linked immunosorbent assay. Clin Immunol Immunopathol 1998;89:71–78. [DOI] [PubMed] [Google Scholar]
  • 8. Harvey GR, Butts S, Rands AL, Patel Y, McHugh NJ. 1999. Clinical and serological associations with anti‐RNA polymerases antibodies in systemic sclerosis. Clin Exp Immunol 117:395–402. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9. Yu Y, Scharl EC, Smith CM, Steitz JA. 1999. The growing world of small nuclear ribonucleoprotein In: Gesteland RF, Cech TR, Atkins JF, editors. The RNA world. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; p 487–524. [Google Scholar]
  • 10. Nilsen TW. RNA‐RNA interactions in the spliceosome: unraveling the ties that bind. Cell 1994;78:1–4. [DOI] [PubMed] [Google Scholar]
  • 11. Smith C, Steitz JA. Sno storm in the nucleolus: new roles for myriad small RNPs. Cell 1997;89:669–672. [DOI] [PubMed] [Google Scholar]
  • 12. Fatica A, Gallardi S, Altieri F, Bozzoni I. Fibrillarin binds directly and specifically to U16 C/D snoRNA. RNA 2000;6:88–95. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13. Ni J, Fournier MJ. 1997. Small nucleolar RNAs direct site‐specific synthesis of pseurouridine in ribosomal RNA. Cell 1997;89:565–573. [DOI] [PubMed] [Google Scholar]
  • 14. Ganot P, Bortolin ML, Kiss T. Site specific pseudouridine formation in preribosomal RNA is guided by small nucleolar RNAs. Cell 1997;89:799–809. [DOI] [PubMed] [Google Scholar]
  • 15. Yang Y, Isaac C, Wang C, Dragon F, Pogai V, Meier T. Conserved composition of mammalian box H/ACA and Box C/D small nucleolar ribonucleoprotein particles and their interaction with the common factor Nopp140. Mol Biol Cell 2000;11:567–577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16. Thiry M. Ultrastructural detection of DNA within the nucleolus by sensitive molecular immunocytochemistry. Exp Cell Res 1992;20:135–144. [DOI] [PubMed] [Google Scholar]
  • 17. Thiry M. New data concerning the functional organization of the mammalian cell mucleolus: detection of RNA and rRNA by in situ molecular imunocytochemistry. Nucl Acid Res 1992;20:6195–6200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18. Aris JP, Blobel G. cDNA cloning and sequencing of human fibrillarin, a conserved nucleolar protein recognized by autoimmune antisera. Proc Natl Acad Sci U S A 1991;88:931–935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19. Jensen RP, Hurt EC, Kern H, et al. Evolutionary conservation of the human nucleolar protein fibrillarin and its functional expression in yeast. J Cell Biol 1991;113:715–729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20. Tyc K, Steitz JA. U3, U8, and U13 comprise a new class of mammalin snRNPs localized to the cell nucleolus. EMBO J 1989;8:3113–3119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21. Baserga SJ, Yang X, Steitz JA. An intact box C sequence in the U3 snoRNA is required for binding of fibrillarin, the protein common to the major family of nucleolar snRNPs. EMBO J 1991;10:2645–2651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22. Peculis B, Steitz JA. Sequence and structural elements critical for U8 snRNP function in Xenopus oocytes are evolutionary conserved. Genes Dev 1994;8:2241–2255. [DOI] [PubMed] [Google Scholar]
  • 23. Tycowski KT, Shu MD, Steitz JA. Requirement for intron‐encoded U22 small nucleolar RNA in 18S ribosomal RNA maturation. Science 1994;266:1558–1561. [DOI] [PubMed] [Google Scholar]
  • 24. Tycowski K, Shu MD, Steitz JA. A mammalian gene whose introns instead of exons generate stable RNA products. Nature 1996;379:464–466. [DOI] [PubMed] [Google Scholar]
  • 25. Tycowski K, Smith C, Shu MD, Steitz JA. A small nucleolar RNA requirement for site specific ribose methylation of rRNA in Xenopus . Proc Natl Acad Sci U S A 1996;93:14480–14485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26. Caffarelli E, Fatica A, Prislei S, De Gregorio E, Fragapane P, Bozzoni I. Processing of the intron‐encoded U16 and U18 snoRNAs: the conserved C and D boxes control both the processing reaction and the stability of the mature snoRNA. EMBO J 1996;15:1121–1131. [PMC free article] [PubMed] [Google Scholar]
  • 27. Balakin AG, Smith M, Fournier J. The RNA world of the nucleolus: two major families of small RNAs defined by different box elements related functions. Cell 1996;86:823–834. [DOI] [PubMed] [Google Scholar]
  • 28. Nicoloso M, Qu LH, Michot B, Bachellerie JP. Intron‐encoded, antisense small nucleolar RNAs: the characterization of nine novel species points to their direct role as guides for the 2′‐O‐ribose methylation of rRNAs. J Mol Biol 1996;260:178–195. [DOI] [PubMed] [Google Scholar]
  • 29. Ochs RL, Lischwe MA, Spohn WH, Busch H. Fibrillarin: a new protein of the nucleolus identified by autoimmune sera. Biol Cell 1985;54:123–134. [DOI] [PubMed] [Google Scholar]
  • 30. Lischwe MA, Ochs RL, Reddy R, et al. Purification and partial characterization of a nucleolar scleroderma antigen (Mr = 34.000; pI, 8.5) rich in NGdimethylarginine. J Biol Chem 1985;260:14304–14310. [PubMed] [Google Scholar]
  • 31. LeRoy EC, Black CM, Fleischmajer R, Jablonska S. Scleroderma (systemic sclerosis), classification, subsets and pathogenesis. J Rheumatol 1988;15:202–205. [PubMed] [Google Scholar]
  • 32. Matera AG, Ward DC. Nucleoplasmic organization of small nuclear ribonucleoproteins in cultured human cells. J Cell Biol 1993;121:715–727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33. Fritzler MJ. Fluorescent antinuclear antibody test In: Rose NR, Friedman H, editors. Manual of clinical immunology. Washington, DC: American Society for Microbiology; 1980. p 852. [Google Scholar]
  • 34. Matera AG, Frey MR, Margelot K, Wolin SL. A perinucleolar compartment contains several RNA polymerase III transcripts as well as the polypyrimidine tract‐binding protein, hnRNP I. J Cell Biol 1995;129:1181–1193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35. Steitz JA. Immunoprecipitation of ribonucleoproteins using autoantibodies. Methods Enzymol 1989;180:468–481. [DOI] [PubMed] [Google Scholar]
  • 36. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680–685. [DOI] [PubMed] [Google Scholar]
  • 37. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and applications. Proc Natl Acad Sci U S A 1979;76:4350–4354. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38. England TE, Bruce AG, Uhlenbeck OC. Specific labelling of 3′ end termini of RNA with T4 RNA ligase. Methods Enzymol 1980;65:65–74. [DOI] [PubMed] [Google Scholar]
  • 39. Chaconas G, Van de Sande JH. 5′32P labelling of RNA and DNA fragments. Methods Enzymol 1980;65:75–88. [DOI] [PubMed] [Google Scholar]
  • 40. Fritzler MJ. Autoantibodies: diagnostic fingerprints and etiologic perplexities. Clin Invest Med 1997;20:50–66. [PubMed] [Google Scholar]
  • 41. Evans J. Antinuclear antibody testing in systemic autoimmune disease. Clin Chest Med 1998;19:613–625. [DOI] [PubMed] [Google Scholar]
  • 42. Bauerfeind S, Heide KG. Determination of autoimmune antibodies using Western blot technique in the routine laboratory. Problems with determining and comparing three test kits. Z Rheumatol 1994;53:66–71. [PubMed] [Google Scholar]
  • 43. Tan EM, Smolen JS, McDougal JS, et al. A critical evaluation of enzyme immunoassays for detection of antinuclear autoantibodies of defined specificities. I. Precision, sensitivity, and specificity. Arthritis Rheum 1999;42:455–464. [DOI] [PubMed] [Google Scholar]
  • 44. Villalta D, Bizarro N, Tonnuti E, et al. Detection of anti‐ENA autoantibodies in patients with systemic connective tissue diseases. Analytical variability and diagnostic sensitivity of 4 methods. Recent Prog Med 1999;90:579–584. [PubMed] [Google Scholar]
  • 45. Zampieri S, Ghirardello A, Doria A, et al. The use of tween 20 in immunoblotting assays for the detection of autoantibodies in connective tissue diseases. J Immunol Methods 2000;239:1–11. [DOI] [PubMed] [Google Scholar]
  • 46. Okano Y, Steen VD, Medsger TAJ. Autoantibody to U3 nucleolar ribonucleoprotein (fibrillarin) in patients with systemic sclerosis. Arthritis Rheum 1992;35:95–100. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Clinical Laboratory Analysis are provided here courtesy of Wiley

RESOURCES