Enzyme-linked immunosorbent assay using bacterial recombinant proteins of human BP230 as a diagnostic tool for bullous pemphigoid.
BACKGROUND
By immunoblot analyses of normal human epidermal extracts, the 230kDa bullous pemphigoid antigen (BP230) is recognized by most bullous pemphigoid (BP) sera. We produced different recombinant glutathione-S-transferase-fusion proteins, which roughly presented N-terminal domain, central rod domain and C-terminal domain of human BP230.


OBJECTIVE
In the present study, we developed an enzyme-linked immunosorbent assay (ELISA) using the recombinant proteins for detection of anti-BP230 IgG antibodies and assessed the usefulness of this assay in conjunction with an anti-BP180 ELISA to establish the diagnosis of BP.


METHODS
Using the bacterial recombinant proteins of N-terminal and C-terminal domains, we developed an ELISA. A receiver-operating-characteristic (ROC) analysis was performed to determine a cut-off value for the BP230 ELISA.


RESULTS
By this BP230 ELISA, 173 (72.4%) of 239 BP sera were positive, while only one (1.1%) of 94 sera from pemphigus vulgaris and pemphigus foliaceus patients was positive and all the 109 normal control sera were negative. Thus, the sensitivity and specificity of the BP230 ELISA were 72.4 and 99.5%, respectively. Interestingly, while 54 (84.4%) of 64 BP sera in active stage and 113 (64.6%) of 175 BP sera in remission were positive in BP180 ELISA, 37 (57.8%) of 64 BP sera in active stage and 136 (77.7%) of 175 BP sera in remission were positive in BP230 ELISA. These results indicate that the titer of anti-BP230 antibodies is not related with disease activity in some BP cases. Most significantly, by combining the results of BP230 ELISA and BP180 ELISA, 232 (97.1%) of 239 BP sera were positive.


CONCLUSIONS
The combination of BP230 ELISA and BP180 ELISA is the highly sensitive method for the diagnosis of BP.
Yoshida
Mariko
M
Department of Dermatology, Kurume University School of Medicine, 67 Asahi-machi, Kurume, Fukuoka 830-0011, Japan.
Hamada
Takahiro
T
Amagai
Masayuki
M
Hashimoto
Koji
K
Uehara
Rie
R
Yamaguchi
Kentaro
K
Imamura
Koji
K
Okamoto
Eiji
E
Yasumoto
Shinichiro
S
Hashimoto
Takashi
T
D001324:Autoantigens / Q000097:blood / Q000276:immunology*; D001419:Bacteria; D015415:Biomarkers; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D000071821:Dystonin; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004817:Epidermis / Q000276:immunology; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D009419:Nerve Tissue Proteins; D024041:Non-Fibrillar Collagens / Q000097:blood / Q000276:immunology*; D010391:Pemphigoid, Bullous / Q000097:blood / Q000175:diagnosis* / Q000276:immunology; D011994:Recombinant Proteins / Q000276:immunology; D012680:Sensitivity and Specificity; D000097805:Collagen Type XVII
D001324:Autoantigens; D015415:Biomarkers; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D000071821:Dystonin; D009419:Nerve Tissue Proteins; D024041:Non-Fibrillar Collagens; D011994:Recombinant Proteins; D000097805:Collagen Type XVII; C486268:DST protein, human