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. 1989 Oct;84(4):1050–1055. doi: 10.1172/JCI114266

Production of human monoclonal anti-basement membrane zone (BMZ) antibodies from a patient with bullous pemphigoid (BP) by Epstein-Barr virus transformation. Analyses of the heterogeneity of anti-BMZ antibodies in BP sera using them.

T Sugi 1, T Hashimoto 1, T Hibi 1, T Nishikawa 1
PMCID: PMC329759  PMID: 2551921

Abstract

We established three lymphoblastoid cell lines from a bullous pemphigoid (BP) patient's peripheral blood by means of EBV transformation, which produced human monoclonal anti-basement membrane zone (BMZ) IgG antibodies. A blocking immunofluorescence test using these MAbs, designated 5A, 5E, and 10D, revealed that 5A and 5E recognized the same or a closely associated epitope, but the epitope for 10D was completely different, 18 of 30 BP sera blocked the reactivity of 10D MAb and 17 sera blocked 5E, while 9 sera did not block the staining of either antibody. Immunoblot analysis demonstrated that both 5A and 5E MAbs reacted exclusively with a protein band of approximately 230 kD in normal human epidermal extracts. However, 10D did not show any protein band. 22 of 30 BP sera strongly reacted with the same 230-kD protein, while none of control sera showed such reactivity. These results clearly demonstrated the heterogeneity of anti-BMZ antibodies in terms of epitopes. These MAbs should be useful in future investigations concerning not only the immunopathology but also the biochemial and molecular analyses of the BP antigen.

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Selected References

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  1. Cote R. J., Morrissey D. M., Houghton A. N., Beattie E. J., Jr, Oettgen H. F., Old L. J. Generation of human monoclonal antibodies reactive with cellular antigens. Proc Natl Acad Sci U S A. 1983 Apr;80(7):2026–2030. doi: 10.1073/pnas.80.7.2026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Croce C. M., Linnenbach A., Hall W., Steplewski Z., Koprowski H. Production of human hybridomas secreting antibodies to measles virus. Nature. 1980 Dec 4;288(5790):488–489. doi: 10.1038/288488a0. [DOI] [PubMed] [Google Scholar]
  3. Diaz L. A., Calvanico N. J., Tomasi T. B., Jr, Jordon R. E. Bullous pemphigoid antigen: isolation from normal human skin. J Immunol. 1977 Feb;118(2):455–460. [PubMed] [Google Scholar]
  4. Diaz L. A., Patel H., Calvanico N. J. Bullous pemphigoid antigen. II. Isolation from the urine of a patient. J Immunol. 1979 Feb;122(2):605–608. [PubMed] [Google Scholar]
  5. Edwards P. A., Smith C. M., Neville A. M., O'Hare M. J. A human-hybridoma system based on a fast-growing mutant of the ARH-77 plasma cell leukemia-derived line. Eur J Immunol. 1982 Aug;12(8):641–648. doi: 10.1002/eji.1830120804. [DOI] [PubMed] [Google Scholar]
  6. Gammon W. R., Lewis D. M., Carlo J. R., Sams W. M., Jr, Wheeler C. E., Jr Pemphigoid antibody mediated attachment of peripheral blood leukocytes at the dermal-epidermal junction of human skin. J Invest Dermatol. 1980 Oct;75(4):334–339. doi: 10.1111/1523-1747.ep12531082. [DOI] [PubMed] [Google Scholar]
  7. Garzelli C., Taub F. E., Jenkins M. C., Drell D. W., Ginsberg-Fellner F., Notkins A. L. Human monoclonal autoantibodies that react with both pancreatic islets and thyroid. J Clin Invest. 1986 May;77(5):1627–1631. doi: 10.1172/JCI112479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Haustein U. F. The role of bullous pemphigoid antibodies in dermal-epidermal separation. J Invest Dermatol. 1989 Jan;92(1):133–133. doi: 10.1111/1523-1747.ep13071360. [DOI] [PubMed] [Google Scholar]
  9. Heggeness M. H., Ash J. F. Use of the avidin-biotin complex for the localization of actin and myosin with fluorescence microscopy. J Cell Biol. 1977 Jun;73(3):783–788. doi: 10.1083/jcb.73.3.783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hibi T., Chan M. A., Petsche D., Dosch H. M. Phenotype, frequency, and EBV responsiveness of human marrow B and pre-B cells. J Immunol. 1986 May 1;136(9):3211–3218. [PubMed] [Google Scholar]
  11. Hjelm H., Hjelm K., Sjöquist J. Protein A from Staphylococcus aureus. Its isolation by affinity chromatography and its use as an immunosorbent for isolation of immunoglobulins. FEBS Lett. 1972 Nov 15;28(1):73–76. doi: 10.1016/0014-5793(72)80680-x. [DOI] [PubMed] [Google Scholar]
  12. Ichimori Y., Harada K., Hitotsumachi S., Tsukamoto K. Establishment of hybridoma secreting human monoclonal antibody against hepatitis B virus surface antigen. Biochem Biophys Res Commun. 1987 Feb 13;142(3):805–812. doi: 10.1016/0006-291x(87)91485-9. [DOI] [PubMed] [Google Scholar]
  13. Irie R. F., Sze L. L., Saxton R. E. Human antibody to OFA-I, a tumor antigen, produced in vitro by Epstein-Barr virus-transformed human B-lymphoid cell lines. Proc Natl Acad Sci U S A. 1982 Sep;79(18):5666–5670. doi: 10.1073/pnas.79.18.5666. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Jordon R. E., Day N. K., Sams W. M., Jr, Good R. A. The complement system in bullous pemphigoid. I. Complement and component levels in sera and blister fluids. J Clin Invest. 1973 May;52(5):1207–1214. doi: 10.1172/JCI107288. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jordon R. E., Triftshauser C. T., Schroeter A. L. Direct immunofluorescent studies of pemphigus and bullous pemphigoid. Arch Dermatol. 1971 May;103(5):486–491. doi: 10.1001/archderm.103.5.486. [DOI] [PubMed] [Google Scholar]
  16. LEVER W. F. Pemphigus. Medicine (Baltimore) 1953 Feb;32(1):1–123. doi: 10.1097/00005792-195302000-00001. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Labib R. S., Anhalt G. J., Patel H. P., Mutasim D. F., Diaz L. A. Molecular heterogeneity of the bullous pemphigoid antigens as detected by immunoblotting. J Immunol. 1986 Feb 15;136(4):1231–1235. [PubMed] [Google Scholar]
  19. 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]
  20. Mueller S., Klaus-Kovtun V., Stanley J. R. A 230-kD basic protein is the major bullous pemphigoid antigen. J Invest Dermatol. 1989 Jan;92(1):33–38. doi: 10.1111/1523-1747.ep13070476. [DOI] [PubMed] [Google Scholar]
  21. Muramatsu T., Shirai T., Iida T., Yamashina Y., Sakamoto K. Antigen specificities of antibasement membrane zone antibodies: immunofluorescence and Western immunoblotting studies. Arch Dermatol Res. 1988;280(7):411–415. doi: 10.1007/BF00429979. [DOI] [PubMed] [Google Scholar]
  22. Naito K., Morioka S., Ikeda S., Ogawa H. Experimental bullous pemphigoid in guinea pigs: the role of pemphigoid antibodies, complement, and migrating cells. J Invest Dermatol. 1984 Mar;82(3):227–230. doi: 10.1111/1523-1747.ep12260057. [DOI] [PubMed] [Google Scholar]
  23. Naito K., Morioka S., Ogawa H. The pathogenic mechanisms of blister formation in bullous pemphigoid. J Invest Dermatol. 1982 Nov;79(5):303–306. doi: 10.1111/1523-1747.ep12500082. [DOI] [PubMed] [Google Scholar]
  24. Nishikawa T., Kurihara S., Harada T., Hatano H. Binding of bullous pemphigoid antibodies to basal cells. J Invest Dermatol. 1980 Jun;74(6):389–391. doi: 10.1111/1523-1747.ep12544466. [DOI] [PubMed] [Google Scholar]
  25. Nowinski R., Berglund C., Lane J., Lostrom M., Bernstein I., Young W., Hakomori S. I., Hill L., Cooney M. Human monoclonal antibody against Forssman antigen. Science. 1980 Oct 31;210(4469):537–539. doi: 10.1126/science.7423202. [DOI] [PubMed] [Google Scholar]
  26. Okada N., Kitano Y., Miyagawa S., Sakamoto K., Steinberg M. L. Expression of pemphigoid antigen by SV40-transformed human keratinocytes. J Invest Dermatol. 1986 Apr;86(4):399–401. doi: 10.1111/1523-1747.ep12285666. [DOI] [PubMed] [Google Scholar]
  27. Olsson L., Kaplan H. S. Human-human hybridomas producing monoclonal antibodies of predefined antigenic specificity. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5429–5431. doi: 10.1073/pnas.77.9.5429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Scaletta L. J., Occhino J. C., MacCallum D. K., Lillie J. H. Isolation and immunologic identification of basement membrane zone antigens from human skin. Lab Invest. 1978 Jul;39(1):1–9. [PubMed] [Google Scholar]
  29. Stanley J. R. A specific antigen-antibody interaction triggers the cellular pathophysiology of bullous pemphigoid. Br J Dermatol. 1985 Jul;113 (Suppl 28):67–73. doi: 10.1111/j.1365-2133.1985.tb15628.x. [DOI] [PubMed] [Google Scholar]
  30. Stanley J. R., Hawley-Nelson P., Yuspa S. H., Shevach E. M., Katz S. I. Characterization of bullous pemphigoid antigen: a unique basement membrane protein of stratified squamous epithelia. Cell. 1981 Jun;24(3):897–903. doi: 10.1016/0092-8674(81)90115-x. [DOI] [PubMed] [Google Scholar]
  31. Stanley J. R., Woodley D. T., Katz S. I. Identification and partial characterization of pemphigoid antigen extracted from normal human skin. J Invest Dermatol. 1984 Jan;82(1):108–111. doi: 10.1111/1523-1747.ep12259224. [DOI] [PubMed] [Google Scholar]
  32. Steinitz M., Klein G., Koskimies S., Makel O. EB virus-induced B lymphocyte cell lines producing specific antibody. Nature. 1977 Sep 29;269(5627):420–422. doi: 10.1038/269420a0. [DOI] [PubMed] [Google Scholar]
  33. Thivolet C. H., Hintner H. H., Stanley J. R. The effect of retinoic acid on the expression of pemphigus and pemphigoid antigens in cultured human keratinocytes. J Invest Dermatol. 1984 Apr;82(4):329–334. doi: 10.1111/1523-1747.ep12260634. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. Watson D. B., Burns G. F., Mackay I. R. In vitro growth of B lymphocytes infiltrating human melanoma tissue by transformation with EBV: evidence for secretion of anti-melanoma antibodies by some transformed cells. J Immunol. 1983 May;130(5):2442–2447. [PubMed] [Google Scholar]
  36. Wood G. W., Beutner E. H. Blocking-immunofluorescence studies on the specificity of pemphigus autoantibodies. Clin Immunol Immunopathol. 1977 Mar;7(2):168–175. doi: 10.1016/0090-1229(77)90045-9. [DOI] [PubMed] [Google Scholar]
  37. Yamada H., Hashimoto T., Nishikawa T. IgG subclasses of intercellular and basement membrane zone antibodies: the relationship to the capability of complement fixation. J Invest Dermatol. 1989 Apr;92(4):585–587. doi: 10.1111/1523-1747.ep12709613. [DOI] [PubMed] [Google Scholar]
  38. Yamasaki Y., Nishikawa T. Ultrastructural localization of in vitro binding sites of circulating anti-basement membrane zone antibodies in bullous pemphigoid. Acta Derm Venereol. 1983;63(6):501–506. doi: 10.2340/00015555463501506. [DOI] [PubMed] [Google Scholar]
  39. Zhu X. J., Bystryn J. C. Heterogeneity of pemphigoid antigens. J Invest Dermatol. 1983 Jan;80(1):16–20. doi: 10.1111/1523-1747.ep12530900. [DOI] [PubMed] [Google Scholar]

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