Establishment Of Quantitative Detection Method For Phospholipase A2 Receptor Domain-specific IgG4 Antibody And Prognostic Analysis

Background:M-type phospholipase A2 receptor（PLA2R）is the major autoantigen of adult idiopathic membranous nephropathy（IMN）.Studies have demonstrated that there are four domain reactive epitopes（Cys R,CTLD1,CTLD7 and CTLD8）,and proposed that there is a close relationship between epitope spreading and disease activity,and epitope spreading at baseline is an independent risk factor for lack of clinical remission.However,some studies have put forward the opposite view,that is,epitope spreading is not supported as an indicator of the prognosis and treatment of patients with IMN.At present,there are still some controversies about PLA2R domains reactive epitopes for prognostic evaluation of IMN.Detection of anti-PLA2R antibodies is an important way to monitor IMN.The main subtype of autoantibodies in most patients with IMN is IgG4,and there is no quantitative detection method for PLA2R domains.Therefore,this study established a highly sensitive quantitative detection method for PLA2R domain-specific IgG4 antibodies levels,and by anti-PLA2R antibodies levels,domain-specific PLA2R antibody levels and the epitopes profiles in the follow-up patients to explore clinical value in efficacy evaluation and prognostic analysis.Method:A time-resolved fluorescence immunoassay（TRFIA）with high sensitivity and wide detection range was established to quantitatively detect anti-PLA2R-IgG4 and IgG4-specific anti-PLA2R antibodies levels targeting Cys R,CTLD1 and CTLD678 domains.Firstly,human recombinant antigens（PLA2R,Cys R,CTLD1 and CTLD678）were coated,and the mouse anti-human IgG4 monoclonal antibody labeled with rare earth ion Eu³⁺was used for tracking.Quantitatively detect anti-PLA2R-IgG4 and domain-specific IgG4 antibodies levels in25 follow-up patients with positive anti-PLA2R antibodies,and also to detect anti-PLA2R-IgG and domain-specific IgG antibodies levels,the relevant clinical data of follow-up patients at the initial diagnosis and 6-month follow-up were analyzed.Result:TRFIA was used to quantitatively detect anti-PLA2R antibody levels and domain-specific PLA2R antibody levels in the follow-up patients.The following results were obtained in this study:1.Anti-PLA2R antibody levels and domain-specific PLA2R antibody levels in the remission group（n=13）at initial diagnosis was significantly lower than that of the non-remission group（n=12）（P<0.05）.The anti-PLA2R-IgG antibody level（median）of the non-remission group at initial diagnosis was 5.6 times that of the remission group,and was also5.6 times that of the remission group at the end of follow-up.Compared with the remission group,the level of anti-CTLD678-IgG specific antibody level was not much different before and after treatment（4.2 times/1.5 times）,and the analysis of IgG-specific antibody levels alone cannot reveal a significant difference between the two groups.The anti-PLA2R-IgG4 antibody level（median）of the non-remission group at initial diagnosis was 6.2 times that of the remission,but the difference in antibody levels between the two groups at the end of follow-up was 85.2times;Compared with the remission group,anti-CTLD678-IgG4 specific antibody level was significantly different before and after treatment（2.6 times/22.3 times）.The IgG4-specific can more sensitively detect the degree of changes in specific anti-PLA2R antibodies levels in patients with IMN before and after treatment.2.After 6 months of immunosuppressive treatment,the reduction in anti-PLA2R-IgG antibody level of the remission group（77.9%）was higher than that of the non-remission group（65.9%）,but the reduction of domain-specific IgG antibodies levels was not significant.Whereas,the reduction of anti-PLA2R-IgG4 antibody and domain-specific IgG4 antibodies levels in the remission group（89%and above）were higher than those of the non-remission group（70%-85%）.And the decreased rate of anti-PLA2R-IgG4,anti-Cys R-IgG4,anti-CTLD1-IgG4 and anti-CTLD678-IgG4 antibody levels in the remission group were 34.3 times,6.6 times,2.1 times and 13.4 times that of the non-remission group,respectively.3.At the end of the follow-up,according to IgG-specific revealed that 5（38.5%）patients in the remission group and 5（41.7%）patients in the non-remission group were in pattern 1 epitope recognition mode.In contrast,specific-IgG4 revealed that 8 patients（61.5%）in the remission group but 0 patient（0%）in the non-remission group were in pattern 1 epitope recognition mode.At the initial diagnosis,there were 24（96.0%）follow-up patients who identified the three domains Cys R,CTLD1 and CTLD678,and among them,12 patients（50%）had remission.The results of the study found that anti-PLA2R-IgG4 antibody level and domain-specific IgG4antibody level in remission patients were significantly lower than those of patients who were not remission（P<0.05）.Conclusion:1.A TRFIA with high sensitivity is successfully established to quantitatively detect IgG4-specific anti-PLA2R antibodies levels targeting Cys R,CTLD1 and CTLD678 domains.2.Compared with the IgG-specific,anti-PLA2R-IgG4 and domain-specific IgG4 antibody levels can better evaluate the efficacy of patients with IMN and predict the degree of stubbornness of the disease.3.The use of IgG4-specific can more sensitively detect the changes in the epitope profiles of patients with IMN before and after treatment,and can more sensitively monitor the progress of the disease.In the middle and late stages of IMN,for patients whose PLA2R autoantibodies have targeted multiple domains at the initial diagnosis,quantitative detection of anti-PLA2R-IgG4 antibody levels and domain-specific IgG4 antibody levels is more helpful to assess the treatment effect and prognosis.