Detection of IgG autoantibodies in the sera of patients with bullous and gestational pemphigoid: ELISA studies utilizing a baculovirus-encoded form of bullous pemphigoid antigen 2

Claudia Haase, Lioba Büdinger, Luca Borradori, Carole Yee, Hans F. Merk, Kim Yancey, Michael Hertl

Research output: Contribution to journalArticle

93 Citations (Scopus)

Abstract

Autoantibodies against the extracellular domain of bullous pemphigoid antigen 2 (BPAG2) are thought to play a key role in the pathogenesis of bullous pemphigoid and their detection may thus be of diagnostic and prognostic value. The aim of this study was to develop a standardized enzyme- linked immunosorbent assay utilizing the baculovirus-derived protein BV13 (extracellular domain of BPAG2 devoid of 68 amino acids at the C terminus linked to glutathione-S-transferase and 6x His tag) to detect BPAG2-specific autoantibodies. For the enzyme-linked immunosorbent assay, nickel agarose affinity-purified BV13 protein was incubated with sera from patients with bullous pemphigoid (n = 39), gestational pemphigoid (n = 10), and pemphigus vulgaris/pemphigus foliaceus (PV/PF; n = 15), or normal human sera (NHS; n = 18). Nickel affinity-purified proteins from wild-type baculovirus-infected insect cells served as a control. A positive enzyme-linked immunosorbent assay value was defined as reactivity (OD(BV13) -OD(WT)) > mean reactivity + 1 SD of the negative control sera (PV/PF; NHS). Thirty-five of 39 bullous pemphigoid sera and 10 of 10 gestational pemphigoid sera were reactive to BPAG2 compared with none of 15 PV/PF sera and one of 18 NHS (sensitivity, 91.8%; specificity, 97%). Of 16 BPAG2-reactive sera in the enzyme-linked immunosorbent assay, only six were BPAG2-reactive in the western blot, whereas 14 sera immunoprecipitated BPAG2 from extracts of epidermal keratinocytes. The enzyme-linked immunosorbent assay utilizing an eukaryotic BPAG2 protein thus seems to be highly sensitive and specific in the detection of BPAG2-specific antibodies and, hence, may be useful in the diagnosis of bullous autoimmune diseases, such, as bullous pemphigoid and gestational pemphigoid.

Original languageEnglish (US)
Pages (from-to)282-286
Number of pages5
JournalJournal of Investigative Dermatology
Volume110
Issue number3
DOIs
StatePublished - 1998

Fingerprint

Pemphigoid Gestationis
Bullous Pemphigoid
Baculoviridae
Autoantibodies
Immunoglobulin G
Enzyme-Linked Immunosorbent Assay
Immunosorbents
Antigens
Serum
Assays
Enzymes
Nickel
Pemphigus
Proteins
Glutathione Transferase
Sepharose

Keywords

  • Autoimmunity
  • B lymphocytes
  • Hemidesmosome

ASJC Scopus subject areas

  • Dermatology

Cite this

Detection of IgG autoantibodies in the sera of patients with bullous and gestational pemphigoid : ELISA studies utilizing a baculovirus-encoded form of bullous pemphigoid antigen 2. / Haase, Claudia; Büdinger, Lioba; Borradori, Luca; Yee, Carole; Merk, Hans F.; Yancey, Kim; Hertl, Michael.

In: Journal of Investigative Dermatology, Vol. 110, No. 3, 1998, p. 282-286.

Research output: Contribution to journalArticle

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abstract = "Autoantibodies against the extracellular domain of bullous pemphigoid antigen 2 (BPAG2) are thought to play a key role in the pathogenesis of bullous pemphigoid and their detection may thus be of diagnostic and prognostic value. The aim of this study was to develop a standardized enzyme- linked immunosorbent assay utilizing the baculovirus-derived protein BV13 (extracellular domain of BPAG2 devoid of 68 amino acids at the C terminus linked to glutathione-S-transferase and 6x His tag) to detect BPAG2-specific autoantibodies. For the enzyme-linked immunosorbent assay, nickel agarose affinity-purified BV13 protein was incubated with sera from patients with bullous pemphigoid (n = 39), gestational pemphigoid (n = 10), and pemphigus vulgaris/pemphigus foliaceus (PV/PF; n = 15), or normal human sera (NHS; n = 18). Nickel affinity-purified proteins from wild-type baculovirus-infected insect cells served as a control. A positive enzyme-linked immunosorbent assay value was defined as reactivity (OD(BV13) -OD(WT)) > mean reactivity + 1 SD of the negative control sera (PV/PF; NHS). Thirty-five of 39 bullous pemphigoid sera and 10 of 10 gestational pemphigoid sera were reactive to BPAG2 compared with none of 15 PV/PF sera and one of 18 NHS (sensitivity, 91.8{\%}; specificity, 97{\%}). Of 16 BPAG2-reactive sera in the enzyme-linked immunosorbent assay, only six were BPAG2-reactive in the western blot, whereas 14 sera immunoprecipitated BPAG2 from extracts of epidermal keratinocytes. The enzyme-linked immunosorbent assay utilizing an eukaryotic BPAG2 protein thus seems to be highly sensitive and specific in the detection of BPAG2-specific antibodies and, hence, may be useful in the diagnosis of bullous autoimmune diseases, such, as bullous pemphigoid and gestational pemphigoid.",
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