Establishment And Preliminary Clinical Application Of A Quantitative Detection Method For IgG-specific Antibodies Targeting Different Domains Of PLA2R

BackgroundIn 2009,M-Type Phospholipase A2 Receptor(PLA2R)was identified as the main target antigen in adult idiopathic membranous nephropathy(IMN).This finding is a major breakthrough in the study of IMN.PLA2R is expressed on the plasma membrane of human podocytes,and has a large extracellular region.The extracellular region is highly glycosylated and consists of multiple individual domains,which can directly bind to circulating autoantibodies.In this case,the immune response produced is polyclonal.In several important studies,reactive epitopes have been identified in the CysR,CTLD1,CTLD7 and CTLD8 domains of PLA2R.Moreover,there are differences in affinity of autoantibodies with different epitopes,the immunodominant epitope can be recognized by autoantibodies in most patients with PLA2R-associated MN,and the epitopes in C-terminal region of PLA2R(CTLD7 and CTLD8)are close to the cell membrane,which are difficult to be exposed to produce corresponding autoantibodies.IMN is an autoimmune disease,it is necessary to understand the antigenic epitopes for exploring pathological mechanism,because anti-PLA2R antibody cannot yet fully evaluate disease activity and kidney prognosis.However,researches on epitopes of PLA2R is few,and mainly rely on Western Blot(WB)and ELISA(Enzyme-linked immunosorbent assay)for qualitative or semi-quantitative analysis.Therefore,seeking a more sensitive quantitative assay method to detect the level of autoantibodies against different domains of PLA2R is essential to analyze the link between epitopes and disease activity.Method&ObjectiveThe purified recombinant human PLA2R protein corresponding to the entire extracellular domain and soluble domains of PLA2R(CysR,CTLD1 and CTLD678)were used as antigen to coated respectively,goat anti-human IgG antibody was labeled with rare earth ion Eu3+,and a highly sensitive quantitative assay method for detecting the levels of PLA2R domain-specific IgG antibodies by time resolved fluoroisnmunoassay(TRFIA).The TRFIAs established were used to detect the level of autoantibodies against CysR,CTLD1 and CTLD678 domains in the serum of patients with PLA2R-associated MN,and to analyze preliminarily on the clinical relevance of PLA2R domain-specific antibody levels and PLA2R domain recognition patterns.ResultsWe screened 78 serum samples through established PLA2R-TRFIA,and the normal range was defined by 20 serum samples from healthy volunteers.Anti-PLA2R-IgG antibody levels did not exceed the cut-off value in 9 patients with IgA nephropathy,4 patients with Henoch-Schonlein Purpura(HSP)and 5 patients with other kidney diseases.And anti-PLA2R-IgG antibodies were positive in 46 of 59 patients with IMN.Moreover,anti-PLA2R-IgG4 antibodies were positive in 54 patients with IMN.In a cohort of 54 patients with PLA2R-associated MN(patients with MN positive for PLA2R-specific antibodies),the positive rates of IgG-specific antibodies targeting CysR,CTLD1 and CTLD678 were 75.9%,46.3%,and 44.4%,respectively.And for IgG4-specific antibody,the positive rates were 94.4%,96.3%,respectively.In addition,there were significant differences in anti-CysR-IgG antibody levels and anti-CTLD678-IgG4 antibody levels between patients with different proteinuria.According to the reactivity of serum to CysR,CTLD1 and CTLD678 at initial diagnosis,patients were stratified:no domain reactivity or only 1 domain was recognized(pattern 1),2 domains were recognized(pattern 2),more than 3 domains were recognized(pattern 3).There were significant differences in anti-PLA2R-IgG antibody and anti-PLA2R-IgG4 antibody between patients in three groups(P=0.003,P=0.007),and patients whose autoantibodies recognized two or more domains at initial diagnosis had more proteinuria(P=0.04),lower serum albumin(P=0.01),higher serum creatinine(P=0.03),and lower estimated glomerular filtration rate(eGFR)(P=0.02).ConclusionThe PLA2R domain-specific TRFIA was established firstly,and has been preliminarily applied in patients with PLA2R-associated MN.We find that anti-CysR-IgG antibody levels and anti-CTLD678-IgG4 antibody levels may be used as biomarkers to monitor disease activity in the early stage of IMN.In addition,the more domains are recognized by anti-PLA2R-IgG antibodies,the more serious the kidney damage,and the significantly higher levels of anti-PLA2R antibodies.Therefore,the domain recognition pattern has the potential become a new tool to evaluate the disease severity,and to provide certain guidance for treatment plan.