Modeling of main characteristics of bullous pemphigoid antigen-2 (BPAG2) peptide structure in serological recognition by autoantibodies.
The serum level of autoantibodies against autoantigens of the bullous pemphigoid peptides 1 and 2 (BPAG1 and BPAG2) is a relevant diagnostic marker. Twelve representative sera of BP were tested against the RSILPYGDSMDRIEKDRLQMAP amino acid sequence that is an epitope fragment of the NC16A domain of BPAG2 (AC Q02802; 507-528) to find the most suitable antigenic form for specific detection of autoantibodies of BP patients' sera by quantitative ELISA system. The antigenic epitope sequence was presented as an antigen in a carrier free form of dimeric peptide (BP22), dimeric peptide fused to glutathione S-transferase (GST-BP22) or dimeric peptide chemically conjugated to polyLys(Ser-DL-Alam) (SAK-BP22). The intensity of ELISA reaction was highest against the recombinant fusion antigen GSTBP22; the chemically conjugated SAK-BP22 performed less well than the free dimeric form of the peptide. In the case of the GST-BP22 antigen, the (GST-BP22)-(GST)492nm optical density values were determined. There was no significant difference between the mean ODs of the GST-BP22 and the SAK-BP22 (0.888 vs. 0.892, p= 0.9726). Conjugating the epitope peptide with the synthetic carrier SAK was advantageous, as it abrogated cross-reactivity with GST carrier protein. Consequently, the SAKBP22 conjugate appears to be the most reliable assay component, avoiding cross-reactivity with GST and simplifying the detection and evaluation of BP autoantibodies in routine ELISA diagnostic system.
Pál
József
J
Department of Immunology and Biotechnology, University of Pécs, Faculty of Medicine, Pécs, Hungary.
Marczinovits
Ilona
I
Hudecz
Ferenc
F
Tóth
Gábor K
GK
Mezõ
Gábor
G
Molnár
János
J
Németh
Péter
P
D001323:Autoantibodies / Q000097:blood*; D001324:Autoantigens / Q000276:immunology*; D002352:Carrier Proteins / Q000276:immunology; D016022:Case-Control Studies; D003598:Cytoskeletal Proteins / Q000276:immunology; D000071821:Dystonin; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D009419:Nerve Tissue Proteins / Q000276:immunology; D024041:Non-Fibrillar Collagens / Q000276:immunology*; D010391:Pemphigoid, Bullous / Q000097:blood / Q000276:immunology*; D010446:Peptide Fragments / Q000276:immunology; D000097805:Collagen Type XVII
D001323:Autoantibodies; D001324:Autoantigens; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D000071821:Dystonin; D000939:Epitopes; D005982:Glutathione Transferase; D009419:Nerve Tissue Proteins; D024041:Non-Fibrillar Collagens; D010446:Peptide Fragments; D000097805:Collagen Type XVII; C486268:DST protein, human