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.
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.
Haase
C
C
Hautklinik der RWTH Aachen, Germany.
Büdinger
L
L
Borradori
L
L
Yee
C
C
Merk
H F
HF
Yancey
K
K
Hertl
M
M
D000328:Adult; D001323:Autoantibodies / Q000032:analysis*; D001324:Autoantigens; D015153:Blotting, Western; D002352:Carrier Proteins; D003094:Collagen; D003598:Cytoskeletal Proteins; D000071821:Dystonin; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology*; D009419:Nerve Tissue Proteins; D024041:Non-Fibrillar Collagens; D010391:Pemphigoid, Bullous / Q000276:immunology*; D010446:Peptide Fragments; D011233:Precipitin Tests; D011247:Pregnancy; D011248:Pregnancy Complications / Q000276:immunology*; D012680:Sensitivity and Specificity; D000097805:Collagen Type XVII
D001323:Autoantibodies; D001324:Autoantigens; D002352:Carrier Proteins; D003094:Collagen; D003598:Cytoskeletal Proteins; D000071821:Dystonin; D007074:Immunoglobulin G; D009419:Nerve Tissue Proteins; D024041:Non-Fibrillar Collagens; D010446:Peptide Fragments; D000097805:Collagen Type XVII; C486268:DST protein, human