Purification and identification of anti-BP180 NC16A antibodies from the sera of patients with bullous pemphigoid or herpes gestationis / 中华皮肤科杂志

Objective To develop a method to purify and identify anti-BP180 NC16A antibodies from the sera of patients with bullous pemphigoid (BP) or herpes gestationis.Methods The GST/NC16A fusion protein was expressed in a prokaryotic expression vector pGEX-2TBP180NC16A,and then crosslinked to glutathione sepharose beads.Anti-BP180 NC16A antibodies were isolated from the sera of 3 patients with BP and 2 patients with herpes gestationis by affinity chromatography,and analyzed by immunofluorescence,Western blot and enzyme linked immunosorbent assay (ELISA).Results The GST/NC16A fusion protein with a relative molecular mass of 37 000 was successfully expressed by the prokaryotic vector pGEX-2TBP180NC16A.Purified anti-BP180 NC16A antibodies were obtained from the sera of patients by the affinity chromatography,and ELISA revealed that the concentration of anti-BP180 NC16A was 2.4 mg/ml.The purified antibody could bind to the basement membrane zone of human skin,suggesting a strong biological activity of the antibodies.Western blot showed a single band corresponding to the expected molecular mass for anti-BP180 NC16A antibodies,indicating a high purity of the isolated antibodies.Conclusion The anti-BP180 NC16A antibodies purified by microbead-based affinity chromatography from the sera of patients with BP or herpes gestationis are highly active and specific.

Immunoblot Analysis Using Epidermal Extract and 180kD Bullous Pemphigoid Antigen NC16a Domain in the Diagnosis of Subepidermal Bullous Diseases / 대한피부과학회지

BACKGROUND: The 180kD bullous pemphigoid antigen (BP180) is known to be recognized by sera from patients with bullous pemphigoid (BP), herpes gestationis (HG), and cicatricial pemphigoid (CP). A series of previous studies using BP180 recombinant proteins has shown that most sera from patients with BP and HG react with the NC16a domain of BP180. OBJECTIVE: This study was performed to compare the immunoblotting results by using human epidermal extracts and by using recombinant protein of BP180 NC16a domain in the diagnosis of subepidermal bullous skin diseases. METHOD: We observed the reactivity with the epidermal extract of normal human foreskin and recombinant protein of BP180 NC16a domain in immunoblotting assay. RESULTS: In immunoblotting with epidermal extract, 7(78%) and 6(67%) of the 9 BP sera reacted with BP230 and BP180, respectively. 1(50%) of the 2 HG and 2(100%) of the 2 CP sera reacted with BP180. In immunoblotting with recombinant protein of BP180 NC16a domain, 8(89%) of the 9 BP sera and 2(100%) of the 2 HG sera were immunoreactive. But, none of the CP and pemphigus sera reacted with the recombinant protein. CONCLUSION: The immunoblotting analysis using BP180 NC16a domain is a highly sensitive method without ambiguity in the diagnosis of subepidermal bullous skin diseases, and also is useful for the differential diagnosis of BP and HG from CP.