Skip to main content
NCBI home page
Clinical and Experimental Immunology logoLink to Clinical and Experimental Immunology
. 1995 Jul;101(1):45–54. doi: 10.1111/j.1365-2249.1995.tb02275.x

Characterization of murine monoclonal antibodies against 60-kD Ro/SS-A and La/SS-B autoantigens.

C H Veldhoven 1, G J Pruijn 1, J F Meilof 1, J P Thijssen 1, A W Van der Kemp 1, W J Van Venrooij 1, R J Smeenk 1
PMCID: PMC1553296  PMID: 7621591

Abstract

Small cytoplasmic ribonucleoproteins (scRNPs) are important autoantigens in patients with systemic lupus erythematosus and Sjögren's syndrome. MoAbs against these proteins were made by immunization of BALB/c mice with purified human recombinant 60-kD Ro/SS-A or 50-kD La/SS-B proteins. Five stable hybridoma cell lines were obtained, of which four secreted anti-Ro/SS-A antibodies (clones 1D8, 1D11, 2G10 and 6G8) and one produced anti-La/SS-B antibodies (clone 7F6). The MoAbs were further characterized using four different immunoassays: immunofluorescence, immunoblotting, RNA precipitation combined with Northern blotting, and recombinant protein precipitation. All four MoAbs against Ro/SS-A recognized the native protein and one of them (2G10) recognized also intact scRNP particles. Interestingly, hY3-RNA was reproducibly not efficiently precipitated by MoAb 2G10. Epitope mapping using deletion mutants of the 60-kD Ro/SS-A antigen showed that MoAb 1D8 recognized the C-terminal part of this protein, while 1D11 and 2G10 recognized distinct epitopes in the region between the RNP motif and the putative zinc finger domain. The epitopes recognized by these MoAbs are highly conserved among species, and the epitope recognized by MoAb 2G10 may be identical to an autoepitope recognized by sera of patients. This is the first report describing the isolation and characterization of MoAbs of the IgG class against the 60-kD Ro/SS-A and La/SS-B autoantigens obtained by immunization with purified human recombinant proteins. These MoAbs can be of great use in studying the cellular processes in which scRNPs are involved, and may help to determine why these scRNPs become autoantigenic in autoimmune diseases.

Full text

PDF
45

Images in this article

48Fig. 1
on p.48
49Fig. 2
on p.49
50Fig. 3
on p.50
51Fig. 4
on p.51
52Fig. 5
on p.52

Selected References

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

  1. Alspaugh M., Maddison P. Resolution of the identity of certain antigen-antibody systems in systemic lupus erythematosus and Sjögren's syndrome: an interlaboratory collaboration. Arthritis Rheum. 1979 Jul;22(7):796–798. doi: 10.1002/art.1780220719. [DOI] [PubMed] [Google Scholar]
  2. Bachmann M., Mayet W. J., Schröder H. C., Pfeifer K., Meyer zum Büschenfelde K. H., Müller W. E. Association of La and Ro antigens with intracellular structures in HEp-2 carcinoma cells. Proc Natl Acad Sci U S A. 1986 Oct;83(20):7770–7774. doi: 10.1073/pnas.83.20.7770. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bachmann M., Mayet W. J., Schröder H. C., Pfeifer K., Meyer zum Büschenfelde K. H., Müller W. E. Identification of the Ro and La antigens in the endoribonuclease VII--ribonucleoprotein complex. Biochem J. 1987 Apr 1;243(1):189–194. doi: 10.1042/bj2430189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bachmann M., Pfeifer K., Schröder H. C., Müller W. E. The La antigen shuttles between the nucleus and the cytoplasm in CV-1 cells. Mol Cell Biochem. 1989 Feb 21;85(2):103–114. doi: 10.1007/BF00577106. [DOI] [PubMed] [Google Scholar]
  5. Barakat S., Meyer O., Torterotot F., Youinou P., Briand J. P., Kahn M. F., Muller S. IgG antibodies from patients with primary Sjögren's syndrome and systemic lupus erythematosus recognize different epitopes in 60-kD SSA/Ro protein. Clin Exp Immunol. 1992 Jul;89(1):38–45. doi: 10.1111/j.1365-2249.1992.tb06874.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ben-Chetrit E., Chan E. K., Sullivan K. F., Tan E. M. A 52-kD protein is a novel component of the SS-A/Ro antigenic particle. J Exp Med. 1988 May 1;167(5):1560–1571. doi: 10.1084/jem.167.5.1560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Boire G., Lopez-Longo F. J., Lapointe S., Ménard H. A. Sera from patients with autoimmune disease recognize conformational determinants on the 60-kd Ro/SS-A protein. Arthritis Rheum. 1991 Jun;34(6):722–730. doi: 10.1002/art.1780340613. [DOI] [PubMed] [Google Scholar]
  8. Bozic B., Pruijn G. J., Rozman B., van Venrooij W. J. Sera from patients with rheumatic diseases recognize different epitope regions on the 52-kD Ro/SS-A protein. Clin Exp Immunol. 1993 Nov;94(2):227–235. doi: 10.1111/j.1365-2249.1993.tb03436.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Casciola-Rosen L. A., Anhalt G., Rosen A. Autoantigens targeted in systemic lupus erythematosus are clustered in two populations of surface structures on apoptotic keratinocytes. J Exp Med. 1994 Apr 1;179(4):1317–1330. doi: 10.1084/jem.179.4.1317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chan E. K., Francoeur A. M., Tan E. M. Epitopes, structural domains, and asymmetry of amino acid residues in SS-B/La nuclear protein. J Immunol. 1986 May 15;136(10):3744–3749. [PubMed] [Google Scholar]
  11. Chan E. K., Tan E. M. Human autoantibody-reactive epitopes of SS-B/La are highly conserved in comparison with epitopes recognized by murine monoclonal antibodies. J Exp Med. 1987 Dec 1;166(6):1627–1640. doi: 10.1084/jem.166.6.1627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Derksen R. H., Meilof J. F. Anti-Ro/SS-A and anti-La/SS-B autoantibody levels in relation to systemic lupus erythematosus disease activity and congenital heart block. A longitudinal study comprising two consecutive pregnancies in a patient with systemic lupus erythematosus. Arthritis Rheum. 1992 Aug;35(8):953–959. doi: 10.1002/art.1780350817. [DOI] [PubMed] [Google Scholar]
  13. Dunn S. D. Effects of the modification of transfer buffer composition and the renaturation of proteins in gels on the recognition of proteins on Western blots by monoclonal antibodies. Anal Biochem. 1986 Aug 15;157(1):144–153. doi: 10.1016/0003-2697(86)90207-1. [DOI] [PubMed] [Google Scholar]
  14. Gottlieb E., Steitz J. A. Function of the mammalian La protein: evidence for its action in transcription termination by RNA polymerase III. EMBO J. 1989 Mar;8(3):851–861. doi: 10.1002/j.1460-2075.1989.tb03446.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gottlieb E., Steitz J. A. The RNA binding protein La influences both the accuracy and the efficiency of RNA polymerase III transcription in vitro. EMBO J. 1989 Mar;8(3):841–850. doi: 10.1002/j.1460-2075.1989.tb03445.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Harmon C. E., Deng J. S., Peebles C. L., Tan E. M. The importance of tissue substrate in the SS-A/Ro antigen-antibody system. Arthritis Rheum. 1984 Feb;27(2):166–173. doi: 10.1002/art.1780270207. [DOI] [PubMed] [Google Scholar]
  17. Hendrick J. P., Wolin S. L., Rinke J., Lerner M. R., Steitz J. A. 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. 1981 Dec;1(12):1138–1149. doi: 10.1128/mcb.1.12.1138. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hopp T. P., Woods K. R. Prediction of protein antigenic determinants from amino acid sequences. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3824–3828. doi: 10.1073/pnas.78.6.3824. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Itoh Y., Kriet J. D., Reichlin M. Organ distribution of the Ro (SS-A) antigen in the guinea pig. Arthritis Rheum. 1990 Dec;33(12):1815–1821. doi: 10.1002/art.1780331209. [DOI] [PubMed] [Google Scholar]
  20. Itoh Y., Reichlin M. Autoantibodies to the Ro/SSA antigen are conformation dependent. I: Anti-60 kD antibodies are mainly directed to the native protein; anti-52 kD antibodies are mainly directed to the denatured protein. Autoimmunity. 1992;14(1):57–65. doi: 10.3109/08916939309077357. [DOI] [PubMed] [Google Scholar]
  21. Itoh Y., Reichlin M. Ro/SS-A antigen in human platelets. Different distributions of the isoforms of Ro/SS-A protein and the Ro/SS-A-binding RNA. Arthritis Rheum. 1991 Jul;34(7):888–893. doi: 10.1002/art.1780340715. [DOI] [PubMed] [Google Scholar]
  22. Lerner M. R., Boyle J. A., Hardin J. A., Steitz J. A. Two novel classes of small ribonucleoproteins detected by antibodies associated with lupus erythematosus. Science. 1981 Jan 23;211(4480):400–402. doi: 10.1126/science.6164096. [DOI] [PubMed] [Google Scholar]
  23. Lerner M. R., Steitz J. A. Snurps and scyrps. Cell. 1981 Aug;25(2):298–300. doi: 10.1016/0092-8674(81)90047-7. [DOI] [PubMed] [Google Scholar]
  24. Madore S. J., Wieben E. D., Pederson T. Eukaryotic small ribonucleoproteins. Anti-La human autoantibodies react with U1 RNA-protein complexes. J Biol Chem. 1984 Feb 10;259(3):1929–1933. [PubMed] [Google Scholar]
  25. Matter L., Schopfer K., Wilhelm J. A., Nyffenegger T., Parisot R. F., De Robertis E. M. Molecular characterization of ribonucleoprotein antigens bound by antinuclear antibodies. A diagnostic evaluation. Arthritis Rheum. 1982 Nov;25(11):1278–1283. doi: 10.1002/art.1780251102. [DOI] [PubMed] [Google Scholar]
  26. Mayet W. J., Bachmann M., Pfeifer K., Schröder H. C., Müller W. E., Gudat W., Korting G. W., Meyer zum Büschenfelde K. H. A monoclonal Ro-antibody and the serum of a Ro-positive patient with subacute cutaneous lupus erythematosus (SCLE) react with basal layers of human epidermis. Eur J Clin Invest. 1988 Oct;18(5):465–471. doi: 10.1111/j.1365-2362.1988.tb01041.x. [DOI] [PubMed] [Google Scholar]
  27. McCauliffe D. P., Zappi E., Lieu T. S., Michalak M., Sontheimer R. D., Capra J. D. A human Ro/SS-A autoantigen is the homologue of calreticulin and is highly homologous with onchocercal RAL-1 antigen and an aplysia "memory molecule". J Clin Invest. 1990 Jul;86(1):332–335. doi: 10.1172/JCI114704. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Meilof J. F. Autoantibodies against small cytoplasmic ribonucleoproteins: the anti-Ro/SS-A and anti-La/SS-B autoimmune response. A review of autoantibody detection, autoantigen composition, autoantibody-disease associations and possible etiologic mechanisms. Rheumatol Int. 1992;12(4):129–140. doi: 10.1007/BF00274932. [DOI] [PubMed] [Google Scholar]
  29. Meilof J. F., Bantjes I., De Jong J., Van Dam A. P., Smeenk R. J. 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 Methods. 1990 Oct 19;133(2):215–226. doi: 10.1016/0022-1759(90)90362-y. [DOI] [PubMed] [Google Scholar]
  30. Meilof J. F., Frohn-Mulder I. M., Stewart P. A., Szatmari A., Hess J., Veldhoven C. H., Smeenk R. J., Swaak A. J. Maternal autoantibodies and congenital heart block: no evidence for the existence of a unique heart block-associated anti-Ro/SS-A autoantibody profile. Lupus. 1993 Aug;2(4):239–246. doi: 10.1177/096120339300200406. [DOI] [PubMed] [Google Scholar]
  31. Meilof J. F., Van der Lelij A., Rokeach L. A., Hoch S. O., Smeenk R. J. Autoimmunity and filariasis. Autoantibodies against cytoplasmic cellular proteins in sera of patients with onchocerciasis. J Immunol. 1993 Nov 15;151(10):5800–5809. [PubMed] [Google Scholar]
  32. Peek R., Pruijn G. J., van der Kemp A. J., van Venrooij W. J. Subcellular distribution of Ro ribonucleoprotein complexes and their constituents. J Cell Sci. 1993 Nov;106(Pt 3):929–935. doi: 10.1242/jcs.106.3.929. [DOI] [PubMed] [Google Scholar]
  33. Pruijn G. J., Slobbe R. L., van Venrooij W. J. Analysis of protein--RNA interactions within Ro ribonucleoprotein complexes. Nucleic Acids Res. 1991 Oct 11;19(19):5173–5180. doi: 10.1093/nar/19.19.5173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rader M. D., O'Brien C., Liu Y. S., Harley J. B., Reichlin M. Heterogeneity of the Ro/SSA antigen. Different molecular forms in lymphocytes and red blood cells. J Clin Invest. 1989 Apr;83(4):1293–1298. doi: 10.1172/JCI114014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Rokeach L. A., Haselby J. A., Meilof J. F., Smeenk R. J., Unnasch T. R., Greene B. M., Hoch S. O. Characterization of the autoantigen calreticulin. J Immunol. 1991 Nov 1;147(9):3031–3039. [PubMed] [Google Scholar]
  36. Rosario M. O., Fox O. F., Koren E., Harley J. B. Anti-Ro (SS-A) antibodies from Ro (SS-A)-immunized mice. Arthritis Rheum. 1988 Feb;31(2):227–237. doi: 10.1002/art.1780310211. [DOI] [PubMed] [Google Scholar]
  37. Scofield R. H., Dickey W. D., Jackson K. W., James J. A., Harley J. B. A common autoepitope near the carboxyl terminus of the 60-kD Ro ribonucleoprotein: sequence similarity with a viral protein. J Clin Immunol. 1991 Nov;11(6):378–388. doi: 10.1007/BF00918804. [DOI] [PubMed] [Google Scholar]
  38. Scofield R. H., Harley J. B. Autoantigenicity of Ro/SSA antigen is related to a nucleocapsid protein of vesicular stomatitis virus. Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):3343–3347. doi: 10.1073/pnas.88.8.3343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Scott J. S., Maddison P. J., Taylor P. V., Esscher E., Scott O., Skinner R. P. Connective-tissue disease, antibodies to ribonucleoprotein, and congenital heart block. N Engl J Med. 1983 Jul 28;309(4):209–212. doi: 10.1056/NEJM198307283090403. [DOI] [PubMed] [Google Scholar]
  40. Silverman E., Mamula M., Hardin J. A., Laxer R. Importance of the immune response to the Ro/La particle in the development of congenital heart block and neonatal lupus erythematosus. J Rheumatol. 1991 Jan;18(1):120–124. [PubMed] [Google Scholar]
  41. Slobbe R. L., Pluk W., van Venrooij W. J., Pruijn G. J. Ro ribonucleoprotein assembly in vitro. Identification of RNA-protein and protein-protein interactions. J Mol Biol. 1992 Sep 20;227(2):361–366. doi: 10.1016/0022-2836(92)90890-v. [DOI] [PubMed] [Google Scholar]
  42. Slobbe R. L., Pruijn G. J., Damen W. G., van der Kemp J. W., van Venrooij W. J. Detection and occurrence of the 60- and 52-kD Ro (SS-A) antigens and of autoantibodies against these proteins. Clin Exp Immunol. 1991 Oct;86(1):99–105. doi: 10.1111/j.1365-2249.1991.tb05780.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Smeenk R. J., Brinkman K., van den Brink H. G., Westgeest A. A. Reaction patterns of monoclonal antibodies to DNA. J Immunol. 1988 Jun 1;140(11):3786–3792. [PubMed] [Google Scholar]
  44. Smith P. R., Williams D. G., Venables P. J., Maini R. N. Monoclonal antibodies to the Sjögren's syndrome associated antigen SS-B (La). J Immunol Methods. 1985 Feb 28;77(1):63–76. doi: 10.1016/0022-1759(85)90184-x. [DOI] [PubMed] [Google Scholar]
  45. Tan E. M. Antinuclear antibodies: diagnostic markers for autoimmune diseases and probes for cell biology. Adv Immunol. 1989;44:93–151. doi: 10.1016/s0065-2776(08)60641-0. [DOI] [PubMed] [Google Scholar]
  46. Theofilopoulos A. N., Dixon F. J. Murine models of systemic lupus erythematosus. Adv Immunol. 1985;37:269–390. doi: 10.1016/s0065-2776(08)60342-9. [DOI] [PubMed] [Google Scholar]
  47. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Veldhoven C. H., Meilof J. F., Huisman J. G., Smeenk R. J. The development of a quantitative assay for the detection of anti-Ro/SS-A and anti-LA/SS-B autoantibodies using purified recombinant proteins. J Immunol Methods. 1992 Jul 6;151(1-2):177–189. doi: 10.1016/0022-1759(92)90116-b. [DOI] [PubMed] [Google Scholar]
  49. Wahren M., Rudén U., Andersson B., Ringertz N. R., Pettersson I. Identification of antigenic regions of the human Ro 60 kDa protein using recombinant antigen and synthetic peptides. J Autoimmun. 1992 Jun;5(3):319–332. doi: 10.1016/0896-8411(92)90146-h. [DOI] [PubMed] [Google Scholar]
  50. Westgeest A. A., Bons J. C., Van den Brink H. G., Aarden L. A., Smeenk R. J. An improved incubation apparatus for Western blots used for the detection of antinuclear antibodies. J Immunol Methods. 1986 Dec 24;95(2):283–288. doi: 10.1016/0022-1759(86)90417-5. [DOI] [PubMed] [Google Scholar]
  51. Wolin S. L., Steitz J. A. 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. 1984 Apr;81(7):1996–2000. doi: 10.1073/pnas.81.7.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES