Skip to main content
SpringerLink
Log in

ELISA Testing and Immunoblotting

  • Chapter
  • First Online:
Book cover Blistering Diseases

Abstract

Diagnoses of autoimmune bullous diseases are made by clinical findings, histopathological findings, and immunological findings. Among immunological tests, enzyme-linked immunosorbent assay (ELISA) and immunoblotting are now two major methods to diagnose autoimmune bullous diseases. Two major groups of autoimmune bullous disease are pemphigus and pemphigoid. The major autoantigens of pemphigus are desmogleins, and those of pemphigoid are BP180 and BP230. Diagnosis and assessment of the disease activity of both diseases are made mainly by desmoglein or BP180 ELISA. Moreover, diagnoses of other rare autoimmune bullous disease subtypes, including mucous membrane pemphigoid, herpes gestationis, epidermolysis bullosa acquisita, anti-laminin γ[gamma]1 pemphigoid, and linear IgA bullous dermatosis, are made by a combination of ELISA and immunoblotting using various substrates, including normal human epidermal and dermal extracts, recombinant proteins of NC16a and C-terminal domains of BP180, purified human laminin-332, and concentrated HaCaT cell culture supernatant.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Chapter
USD 29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 249.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Abbreviations

Dsg:

Desmoglein

ELISA:

Enzyme-linked immunosorbent assay

References

  1. Lleo A, Invernizzi P, Gao B, Podda M, Gershwin ME Definition of human autoimmunity—autoantibodies versus autoimmune disease. Autoimmun Rev. 2010;​9(5):A259–66.

    Article  CAS  PubMed  Google Scholar 

  2. Cunha PR, Barraviera SR. Autoimmune bullous dermatoses. An Bras Dermatol. 2009;84(2):111–24.

    Article  PubMed  Google Scholar 

  3. Vaughan FL, Bernstein IA. Molecular aspects of control in epidermal differentiation. Mol Cell Biochem. 1976;12(3):171–9.

    Article  CAS  PubMed  Google Scholar 

  4. Brooke MA, Nitoiu D, Kelsell DP. Cell-cell connectivity: desmosomes and disease. J Pathol. 2012;226(2):158–71.

    Article  CAS  PubMed  Google Scholar 

  5. Green KJ, Simpson CL. Desmosomes: new perspectives on a classic. J Invest Dermatol. 2007;127(11):2499–515.

    Article  CAS  PubMed  Google Scholar 

  6. Hatsell S, Cowin P. Deconstructing desmoplakin. Nat Cell Biol. 2001;3(12):E270–2.

    Article  CAS  PubMed  Google Scholar 

  7. Sterk LM, Geuijen CA, Oomen LC, Calafat J, Janssen H, Sonnenberg A. The tetraspan molecule CD151, a novel constituent of hemidesmosomes, associates with the integrin alpha6beta4 and may regulate the spatial organization of hemidesmosomes. J Cell Biol. 2000;149(4):969–82.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  8. Tsuruta D, Kobayashi H, Imanishi H, Sugawara K, Ishii M, Jones JC. Laminin-332-integrin interaction: a target for cancer therapy? Curr Med Chem. 2008;15(20):1968–75.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  9. Koster J, van Wilpe S, Kuikman I, Litjens SH, Sonnenberg A. Role of binding of plectin to the integrin beta4 subunit in the assembly of hemidesmosomes. Mol Biol Cell. 2004;15(3):1211–23.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  10. Koster J, Geerts D, Favre B, Borradori L, Sonnenberg A. Analysis of the interactions between BP180, BP230, plectin and the integrin alpha6beta4 important for hemidesmosome assembly. J Cell Sci. 2003;116(Pt 2):387–99.

    Article  CAS  PubMed  Google Scholar 

  11. Schmidt E, Brocker EB, Zillikens D. Pemphigus. Loss of desmosomal cell-cell contact. Hautarzt. 2000;51(5):309–18. Pemphigus. Verlust des desmosomalen Zell-Zell-Kontaktes.

    Article  CAS  PubMed  Google Scholar 

  12. Fontao L, Favre B, Riou S, Geerts D, Jaunin F, Saurat JH, et al. Interaction of the bullous pemphigoid antigen 1 (BP230) and desmoplakin with intermediate filaments is mediated by distinct sequences within their COOH terminus. Mol Biol Cell. 2003;14(5):1978–92.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  13. Karashima T, Tsuruta D, Hamada T, Ishii N, Ono F, Hashikawa K, et al. Interaction of plectin and intermediate filaments. J Dermatol Sci. 2012;66(1):44–50.

    Article  CAS  PubMed  Google Scholar 

  14. Amagai M, Stanley JR. Desmoglein as a target in skin disease and beyond. J Invest Dermatol. 2012;132(3 Pt 2):776–84.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  15. Arya SR, Valand AG, Krishna K. A clinico-pathological study of 70 cases of pemphigus. Indian J Dermatol Venereol Leprology. 1999;65(4):168–71.

    CAS  Google Scholar 

  16. Monshi B, Marker M, Feichtinger H, Schmid G, Kriehuber E, Fodinger D, et al. Pemphigus vegetans—immunopathological findings in a rare variant of pemphigus vulgaris. J Dtsch Dermatol Ges. 2010;8(3):179–83.

    Article  PubMed  Google Scholar 

  17. Tsuruta D, Ishii M, Hashimoto T. The diagnosis and treatment of pemphigus. Immunotherapy. 2012;4(7):735–45.

    Article  CAS  PubMed  Google Scholar 

  18. Tsuruta D, Kobayashi H. Recent patents in pemphigus research, prophylaxis, diagnosis and treatment in USA (1988–2006). Recent Pat Inflamm Allergy Drug Discov. 2007;1(1):77–81.

    Article  CAS  PubMed  Google Scholar 

  19. Stanley JR, Amagai M. Pemphigus, bullous impetigo, and the staphylococcal scalded-skin syndrome. N Engl J Med. 2006;355(17):1800–10.

    Article  CAS  PubMed  Google Scholar 

  20. Tsuruta D, Ishii N, Hamada T, Ohyama B, Fukuda S, Koga H, et al. IgA pemphigus. Clin Dermatol. 2011;29(4):437–42.

    Article  PubMed  Google Scholar 

  21. Tateishi C, Tsuruta D, Nakanishi T, Uehara S, Kobayashi H, Ishii M, et al. Antidesmocollin-1 antibody-positive, antidesmoglein antibody-negative pemphigus herpetiformis. J Am Acad Dermatol. 2010;63(1):e8–10.

    Article  PubMed  Google Scholar 

  22. Ishii N, Hamada T, Koga H, Sogame R, Ohyama B, Fukuda S, et al. Decline of disease activity and autoantibodies to desmoglein 3 and envoplakin by oral prednisolone in paraneoplastic pemphigus with benign thymoma. Eur J Dermatol. 2012;22(4):547–9.

    PubMed  Google Scholar 

  23. Amagai M. The molecular logic of pemphigus and impetigo: the desmoglein story. Vet Dermatol. 2009;20(5–6):308–12.

    Article  PubMed  Google Scholar 

  24. Bernard P, Vaillant L, Labeille B, Bedane C, Arbeille B, Denoeux JP, et al. Incidence and distribution of subepidermal autoimmune bullous skin diseases in three French regions. Bullous Diseases French Study Group. Arch Dermatol. 1995;131(1):48–52.

    Article  CAS  PubMed  Google Scholar 

  25. Marazza G, Pham HC, Scharer L, Pedrazzetti PP, Hunziker T, Trueb RM, et al. Incidence of bullous pemphigoid and pemphigus in Switzerland: a 2-year prospective study. Br J Dermatol. 2009;161(4):861–8.

    Article  CAS  PubMed  Google Scholar 

  26. Ujiie H, Shibaki A, Nishie W, Shimizu H. What’s new in bullous pemphigoid. J Dermatol. 2010;37(3):194–204.

    Article  CAS  PubMed  Google Scholar 

  27. Liu Z, Giudice GJ, Swartz SJ, Fairley JA, Till GO, Troy JL, et al. The role of complement in experimental bullous pemphigoid. J Clin Invest. 1995;95(4):1539–44.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  28. Liu Z, Giudice GJ, Zhou X, Swartz SJ, Troy JL, Fairley JA, et al. A major role for neutrophils in experimental bullous pemphigoid. J Clin Invest. 1997;100(5):1256–63.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  29. Verraes S, Hornebeck W, Polette M, Borradori L, Bernard P. Respective contribution of neutrophil elastase and matrix metalloproteinase 9 in the degradation of BP180 (type XVII collagen) in human bullous pemphigoid. J Invest Dermatol. 2001;117(5):1091–6.

    Article  CAS  PubMed  Google Scholar 

  30. Kitajima Y, Hirako Y, Owaribe K, Mori S, Yaoita H. Antibody-binding to the 180-kD bullous pemphigoid antigens at the lateral cell surface causes their internalization and inhibits their assembly at the basal cell surface in cultured keratinocytes. J Dermatol. 1994;21(11):838–46.

    CAS  PubMed  Google Scholar 

  31. Iwata H, Kamio N, Aoyama Y, Yamamoto Y, Hirako Y, Owaribe K, et al. IgG from patients with bullous pemphigoid depletes cultured keratinocytes of the 180-kDa bullous pemphigoid antigen (type XVII collagen) and weakens cell attachment. J Invest Dermatol. 2009;129(4):919–26.

    Article  CAS  PubMed  Google Scholar 

  32. Hall 3rd RP, Murray JC, McCord MM, Rico MJ, Streilein RD. Rabbits immunized with a peptide encoded for by the 230-kD bullous pemphigoid antigen cDNA develop an enhanced inflammatory response to UVB irradiation: a potential animal model for bullous pemphigoid. J Invest Dermatol. 1993;101(1):9–14.

    Article  CAS  PubMed  Google Scholar 

  33. Kiss M, Husz S, Janossy T, Marczinovits I, Molnar J, Korom I, et al. Experimental bullous pemphigoid generated in mice with an antigenic epitope of the human hemidesmosomal protein BP230. J Autoimmun. 2005;24(1):1–10.

    Article  CAS  PubMed  Google Scholar 

  34. Endo H, Rees TD, Kuyama K, Kono Y, Yamamoto H. Clinical and diagnostic features of mucous membrane pemphigoid. Compend Contin Educ Dent. 2006;27(9):512–6; quiz 7–8.

    PubMed  Google Scholar 

  35. Dainichi T, Kurono S, Ohyama B, Ishii N, Sanzen N, Hayashi M, et al. Anti-laminin gamma-1 pemphigoid. Proc Natl Acad Sci U S A. 2009;106(8):2800–5.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  36. Shimanovich I, Skrobek C, Rose C, Nie Z, Hashimoto T, Brocker EB, et al. Pemphigoid gestationis with predominant involvement of oral mucous membranes and IgA autoantibodies targeting the C-terminus of BP180. J Am Acad Dermatol. 2002;47(5):780–4.

    Article  PubMed  Google Scholar 

  37. Kurashige Y, Mitsuhashi Y, Saito M, Fukuda S, Hashimoto T, Tsuboi R. Herpetiform pemphigus with anti-Dsg 1 and full-length BP180 autoantibodies. Eur J Dermatol. 2012;22(2):269–70.

    PubMed  Google Scholar 

  38. Osawa M, Demitsu T, Toda S, Yokokura H, Umemoto N, Yamada T, et al. A case of mixed bullous disease of epidermolysis bullosa acquisita and linear IgA bullous dermatosis. Dermatology. 2005;211(2):146–8.

    Article  PubMed  Google Scholar 

  39. Harman KE. New laboratory techniques for the assessment of acquired immunobullous disorders. Clin Exp Dermatol. 2002;27(1):40–6.

    Article  CAS  PubMed  Google Scholar 

  40. Ishii K, Amagai M, Hall RP, Hashimoto T, Takayanagi A, Gamou S, et al. Characterization of autoantibodies in pemphigus using antigen-specific enzyme-linked immunosorbent assays with baculovirus-expressed recombinant desmogleins. J Immunol. 1997;159(4):2010–7.

    CAS  PubMed  Google Scholar 

  41. Kobayashi M, Amagai M, Kuroda-Kinoshita K, Hashimoto T, Shirakata Y, Hashimoto K, et al. BP180 ELISA using bacterial recombinant NC16a protein as a diagnostic and monitoring tool for bullous pemphigoid. J Dermatol Sci. 2002;30(3):224–32.

    Article  CAS  PubMed  Google Scholar 

  42. Yoshida M, Hamada T, Amagai M, Hashimoto K, Uehara R, Yamaguchi K, et al. Enzyme-linked immunosorbent assay using bacterial recombinant proteins of human BP230 as a diagnostic tool for bullous pemphigoid. J Dermatol Sci. 2006;41(1):21–30.

    Article  CAS  PubMed  Google Scholar 

  43. Amagai M, Tsunoda K, Zillikens D, Nagai T, Nishikawa T. The clinical phenotype of pemphigus is defined by the anti-desmoglein autoantibody profile. J Am Acad Dermatol. 1999;40(2 Pt 1):167–70.

    Article  CAS  PubMed  Google Scholar 

  44. Miida H, Fujiwara H, Ito M. Association between effective dose of prednisolone, alone or in conjunction with other immunosuppressants, and titre of anti-bullous pemphigoid 180 antibody: a retrospective study of 42 cases. Clin Exp Dermatol. 2011;36(5):485–8.

    Article  CAS  PubMed  Google Scholar 

  45. Lee JB, Fumimori T, Kurose K, Mori O, Hashimoto T. A case of bullous pemphigoid successfully treated by plasmapheresis: assessment of the change in titers of circulating antibodies by immunoblotting and enzyme-linked immunosorbent assay. J Dermatol. 2003;30(4):326–31.

    Article  CAS  PubMed  Google Scholar 

  46. Kim JH, Kim YH, Kim S, Noh EB, Kim SE, Vorobyev A, et al. Serum levels of anti-type VII collagen antibodies detected by enzyme-linked immunosorbent assay in patients with epidermolysis bullosa acquisita are correlated with the severity of skin lesions. J Eur Acad Dermatol Venereol. 2012;27(2):e224–30.

    Article  PubMed  Google Scholar 

  47. Eslami A, Lujan J. Western blotting: sample preparation to detection. J Vis Exp. 2010;44:2359.

    PubMed  Google Scholar 

  48. Natsuga K, Nishie W, Shinkuma S, Moriuchi R, Shibata M, Nishimura M, et al. Circulating IgA and IgE autoantibodies in antilaminin-332 mucous membrane pemphigoid. Br J Dermatol. 2010;162(3):513–7.

    Article  CAS  PubMed  Google Scholar 

  49. Dmochowski M, Hashimoto T, Bhogal BS, Black MM, Zone JJ, Nishikawa T. Immunoblotting studies of linear IgA disease. J Dermatol Sci. 1993;6(3):194–200.

    Article  CAS  PubMed  Google Scholar 

  50. Ohata Y, Hashimoto T, Nishikawa T. Comparative study of autoantigens for various bullous skin diseases by immunoblotting using different dermo-epidermal separation techniques. Clin Exp Dermatol. 1995;20(6):454–8.

    Article  CAS  PubMed  Google Scholar 

  51. Kowalczyk AP, Anderson JE, Borgwardt JE, Hashimoto T, Stanley JR, Green KJ. Pemphigus sera recognize conformationally sensitive epitopes in the amino-terminal region of desmoglein-1. J Invest Dermatol. 1995;105(2):147–52.

    Article  CAS  PubMed  Google Scholar 

  52. Nagata Y, Karashima T, Watt FM, Salmhofer W, Kanzaki T, Hashimoto T. Paraneoplastic pemphigus sera react strongly with multiple epitopes on the various regions of envoplakin and periplakin, except for the c-terminal homologous domain of periplakin. J Invest Dermatol. 2001;116(4):556–63.

    Article  CAS  PubMed  Google Scholar 

  53. Giudice GJ, Emery DJ, Zelickson BD, Anhalt GJ, Liu Z, Diaz LA. Bullous pemphigoid and herpes gestationis autoantibodies recognize a common non-collagenous site on the BP180 ectodomain. J Immunol. 1993;151(10):5742–50.

    CAS  PubMed  Google Scholar 

  54. Matsuzaki T, Mashima Y, Idei T, Hashimoto T, Mashima Y. Unilateral ocular cicatricial pemphigoid with circulating IgA and IgG autoantibodies reactive with the 180 kD bullous pemphigoid antigen. Br J Ophthalmol. 1996;80(8):769.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  55. Matsumura K, Amagai M, Nishikawa T, Hashimoto T. The majority of bullous pemphigoid and herpes gestationis serum samples react with the NC16a domain of the 180-kDa bullous pemphigoid antigen. Arch Dermatol Res. 1996;288(9):507–9.

    Article  CAS  PubMed  Google Scholar 

  56. Lee JB, Liu Y, Hashimoto T. Cicatricial pemphigoid sera specifically react with the most C-terminal portion of BP180. J Dermatol Sci. 2003;32(1):59–64.

    Article  CAS  PubMed  Google Scholar 

  57. Dainichi T, Hirakawa Y, Ishii N, Ohyama B, Kohda F, Takahara M, et al. Mucous membrane pemphigoid with autoantibodies to all the laminin 332 subunits and fatal outcome resulting from liver cirrhosis and hepatocellular carcinoma. J Am Acad Dermatol. 2011;64(6):1199–200.

    Article  PubMed  Google Scholar 

  58. Zone JJ, Taylor TB, Meyer LJ, Petersen MJ. The 97 kDa linear IgA bullous disease antigen is identical to a portion of the extracellular domain of the 180 kDa bullous pemphigoid antigen, BPAg2. J Invest Dermatol. 1998;110(3):207–10.

    Article  CAS  PubMed  Google Scholar 

  59. Ishii N, Ohyama B, Yamaguchi Z, Hashimoto T. IgA autoantibodies against the NC16a domain of BP180 but not 120-kDa LAD-1 detected in a patient with linear IgA disease. Br J Dermatol. 2008;158(5):1151–3.

    Article  CAS  PubMed  Google Scholar 

  60. Hashimoto T, Ishiko A, Shimizu H, Tanaka T, Dodd HJ, Bhogal BS, et al. A case of linear IgA bullous dermatosis with IgA anti-type VII collagen autoantibodies. Br J Dermatol. 1996;134(2):336–9.

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Dermatology, Kurume University School of Medicine, and Kurume University Institute of Cutaneous Cell Biology, Kurume, Fukuoka, Japan

    Daisuke Tsuruta MD, PhD & Takashi Hashimoto MD

Authors
  1. Daisuke Tsuruta MD, PhD
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Takashi Hashimoto MD
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Takashi Hashimoto MD .

Editor information

Editors and Affiliations

  1. Department of Dermatology, St George Hospital, University of New South Wales, Sydney, New South Wales, Australia

    Dédée F. Murrell

Rights and permissions

Reprints and permissions

Copyright information

© 2015 Springer-Verlag Berlin Heidelberg

About this chapter

Cite this chapter

Tsuruta, D., Hashimoto, T. (2015). ELISA Testing and Immunoblotting. In: Murrell, D. (eds) Blistering Diseases. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-662-45698-9_17

Download citation

  • DOI: https://doi.org/10.1007/978-3-662-45698-9_17

  • Published:

  • Publisher Name: Springer, Berlin, Heidelberg

  • Print ISBN: 978-3-662-45697-2

  • Online ISBN: 978-3-662-45698-9

  • eBook Packages: MedicineMedicine (R0)

Publish with us

Policies and ethics

Search

Navigation