Abstract:

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.

Key words: identification