大疱性类天疱疮患者血清抗BP180分子不同表位自身抗体定量分析及亲和力分析

Quantification and affinity assessment of autoantibodies against different epitopes of BP180 from patients with bullous pemphigoid

目的探讨大疱性类天疱疮（13P）患者血清抗BP180分子不同表位的自身抗体量。方法制备BP180分子NC16A片段的不同抗原表位区NC16A-1、NC16A-2和NC16A-3，利用柱亲和层析的方法从10例BP患者血清中分别纯化抗不同表位区的自身抗体，定量，并用硫氰酸盐洗脱法测定抗NC16A-1、抗NC16A-2和抗NC16A-3自身抗体的相对亲和力。结果经亲和层析纯化获得针对BP180-NC16A不同表位区的自身抗体，从20nag总IgG中纯化抗Nc16A-1、NC16A-2和NC16A-3自身抗体的产量分别为（49．0±20．7）ug、（117．7±22．4）ug和（39．5±18．9）ug：抗NC16A-2自身抗体的量和亲和力水平均明显高于其他两个表位区的自身抗体？结论BP致病性自身抗体所识别的主要抗原表位可能位于BP180分子的NC16A-2表位区（aa507-aa520）。

Objective To purify and quantify the autoantibodies against various epitopes within BP180-NC 16A domain from the sera of patients with bullous pemphigoid （BP）. Methods Three epitopes within BPI80-NC16A domain, NC16A-1, NCI6A-2 and NC16A-3, were prepared. Blood samples were obtained from 10 patients who were diagnosed as active BP by clinical, pathological and immunofluorescence examination. Autoantibodies against these epitopes were purified with affinity chromatography column from the sera of these patients with active BP, and quantified separately. The relative binding affinity of autoantibodies to NC 16A-1, NC 16A-2 and NC16A-3 was measured using thiocyanate elution method. Results The autoantibodies against NC16A-1, NC16A-2 and NC16A-3 were successfully purified from the sera of patients. On average, the amount was 49.0 ± 20.7 ug, 117.7 ± 22.4 ug and 39.5 ± 18.9 ug respectively for autoantibodies to NC16A-1, NCI6A-2 and NC16A-3 purified from a portion of serum containing about 20 mg IgG. Both the amount and binding affinity of anti-NC16A-2 autoantibody were significantly higher than those of anti-NC16A-1 and anti-NC16A-3 autoantibodies. Conclusion BP180 NC16A-2 （aa507-aa520） may be the major epitope recognized by pathogenic autoantibodies in patients with BP.