Study Of The Comparison And Variation Trend Of BP180NC16A IgG, IgE And BP23O IgG In Serum And Blisters In Bullous Pemphigoid Patients

Part1ObjectiveTo establish a method to detect circulating IgE autoantibodies that recognize BP180-NC16A and to evaluate its significance in bullous pemphigoid (BP). Methods GST-NC16A fusion proteins were expressed in E. coli using the pGEX-2T expression system and were purified by glutathione affinity chromatography.To determine the optimal working conditions of the Enzyme-linked immunoabsorbent assay (ELISA), performed we checkerboard titrations with serial dilutions of antigen. In the same fashion, optimized dilution of secondary antibody was confirmed. Sera samples from56patients with BP,24healthy controls,18with pemphigus and1with SJS were examined by the modified ELISA approach. The optimum cut-off point for a positive result was selected using ROC analysis. Results The optimized ELISA was run using plates coated with500μg/ml GST-NC16A and the optimal dilution of sera samples and secondary antibody were1:10and1:1000respectively. According to the established cut-off value,40of56BP patients and none of controls had detectable levels of BP180NC16A IgE. Conclusion The established ELISA provides a highly specific tool for detection of IgE anti-BP180NC16A in BP patients. Part20bjective:To evaluate the correlation between antibodies against the immunodominant BP180NC16A domain in sera and blister fluid in BP patients and disease activity, and to analyze their titers changes during treatment. Methods:We evaluated IgE and IgG levels of anti-BP180autoantibodies and IgG levels of BP230in sera and blister fluid of BP patients using ELISA we established. We also observed the changes of antibodies in sera and blister fluids at different time points in2BP patients. Results:BP180NC16A IgG/IgE and BP230IgG Abs were detected in87.18%(34/39),46.15%(18/39) and72.97%(27/37) in peripheral blood of BP patients respectively. There was significant correlation between BP180NC16A IgG and disease activity scores but not between BP180NC16A IgE or BP230IgG titers and this scores. While in bister fluid, positive rates of the three autoantibodies were71.79%(28/39),33.33%(13/39) and72.97%(27/37) respectively. No autoantibodies paralleled with disease activity except anti-BP180NC16A IgG in blister(r=0.380, p=0.038). It could reflect disease activity generally but not in the early stage of treatment. Conclusion:BP180NC16A IgG antibodies can reflect disease activity either in sera or in blister but other antibodies can’t. Antibodies in BP patients are higher in peripheral blood. They could generally reflect disease severity in the whole disease course, but not in the early stage of medication.