| Objective To establish indirect ELISA methods for detection of anti-human Ig E and anti-human FcεRⅠ autoantibodies,and to analyze the serum levels and clinical significance of these two autoantibodies and other mast cell activation related molecules in chronic urticarial(CU)and inflammatory bowel disease(IBD).Methods(1)Using Omazumab as standard,human Ig E as capture antigen and HRPanti-human Ig G as detection antibody,an indirect quantitative ELISA method for anti-Ig E autoantibody was established.Using recombinant human FcεRⅠα as capture antigen and HRP-anti human Ig G as detection antibody,a qualitative and indirect elisa method for the detection of anti-FcεRⅠ autoantibody was established.The conditions of the method were optimized and the performance of the method was evaluated.(2)Serum from CU(n=43),IBD(n=87)patients,age-sex matched healthy controls and their clinical data were collected.(3)The self-made indirect ELISA methods were used to detect the serum level of anti-Ig E,anti-FcεRⅠ in patients with CU to verify the correctness of the method.The serum Ig E,s FcεRⅠ levels of CU patients was detected by immunoturbidimetry and ELISA respectively.The correlation of four molecules and its diagnostic significance for CU were analyzed.(4)Serum Ig E,anti-FcεRⅠ levels were measured in patients with IBD by ELISA.Serum anti-nuclear antibody(ANA),anti-neutrophil cytoplasmic antibody(ANCA),anti-intestinal goblet cell antibody(GAB),anti-panereatic antibody(PAB)and anti-saceharomyces cerevisiae antibody(ASCA)were detected by indirect immunofluorescence assay.Evaluate the diagnosticefficacy of each index alone or jointly and its correlation with clinical features.Results(1)The anti-Ig E quantitative ELISA methods were established successfully.The methods exhibited good specificity and anti-interference ability.The limit of quantitation was 2.0μg/m L.The intra-and inter-CV<10%.The recovery rate80%～110%.A semi-quantitative ELISA method for ant-FcεRI was successfully established.The intra-and inter-CV<10% and the sensitivity was <1:4800.The performance verification results of the two methods met the basic requirements of clinical application.(2)The results showed that the levels of serum Ig E,anti-Ig E,anti-FcεRⅠ in CU patients were significantly higher than that in healthy controls(P<0.001),and s FcεRⅠα also increased,but there was no statistical significance(P>0.05).The diagnostic performances of Ig E,anti-Ig E and anti-FcεRⅠ for CU were similar(AUC=0.72),which were better than s FcεRⅠα(AUC=0.61).The levels of Ig E and s FcεRⅠα,anti-Ig E and anti-FcεRⅠ were positively correlated.(3)The serum total Ig E in IBD patients showed an increasing trend,but there was no significant difference compared with the healthy controls(P=0.072>0.05).Anti-FcεRⅠ increased significantly in ulcerative colitis(P<0.001).However,there was no significant difference in anti-Ig E level between CD,UC and HC group,P=0.170>0.05.CD patients with anti-FcεRI positive were more likely to have higher disease activity,OR:1.312(1.085,1.588),P=0.005.The combined detection of anti-FcεRⅠ,ANA,ANCA and ASCA improved the diagnostic efficiency of IBD,AUC=0.908(0.870～0.947).Conclusions The indirect ELISA methods of anti-Ig E,and anti-FcεRⅠ were successfully established,which provided reliable means for the detection and study of mast cell related molecules.The serum levels of Ig E,anti-Ig E and anti-FcεRⅠ in patients with CU were significantly higher than those in healthy controls.Anti-Ig E,anti-FcεRI combined with IBD related autoantibodies can better analyze the autoimmune factors of disease.Mast cell activation related molecules have the potential to be diagnostic markers of CU and IBD. |
