Development of a method to detect anti-BP180NC16A IgG subclasses and the significance of antiBP180NC16A IgG in bullous pemphigoid / 中华皮肤科杂志

ABSTRACT

ObjectiveTo develop an assay to quantitatively detect anti-BP180NC16A IgG subclasses and to assess the significance of anti-BP180NC16A lgG in bullous pemphigoid (BP).MethodsThe Glutathione S-transferase(GST)-BPI80NC16A fusion protein was expressed in E.coli system and purified by affinity chromatography.An improved enzyme-linked immunoabsorbent assay (ELISA) was developed and used to detect anti-BP180NCI6A IgG subclasses in serum samples from 10 patients with BP,5 patients with pemphigoid gestationis (PG),1 patient with linear IgA bullous dermatosis (LIBD) and 2 patients with pemphigus.Results The optimal condition for the ELISA was determined by cross assay as followsthe concentration of GSTBP180NC16A fusion protein for coating,500 μg/L; the condition for coating,4 ℃ for 12 hours; the dilution ratio of sera and secondary antibody,1∶ 100 and 1 ∶ 2000 respectively; the condition for incubation,37 ℃ for 1 hour;,the condition for the enzyme-substrate reaction,37 ℃ for 20 minutes.Of the 10 patients with BP,all were positive for anti-BP180NC16A IgG1,9 for IgG2,5 for IgG3,and 9 for IgG4.Anti-BP180NC16A IgG was undetected in any of the serum samples from 2 patients with pemphigus vulgaris or 1 patient with adult LIBD.All the 5 sera from patients with PG; were positive for all the anti-BP180NC16A IgG subclasses,which were predominated by IgGl and IgG3.ConclusionThe developed ELISA is a highly specific and reproducible semiquantitative method for the detection of anti-BP180NCI6A IgG subclasses in patients with BP and PG.