Application Of Membranous Nephropathy Diagnosis With Ultrasensitive Quantitative Detection Of Anti-Phospholipase A2 Receptor

Research purposes:To establish an ultrasensitive detection method of PLA2R antibodies.Improve the application of PLA2R antibodies in idiopathic membranous nephropathy,the secondary membranous nephropathy and other kidney disease.Research content:(1)Expression,purification and identification of recombinant PLA2R.(2)The preparation of quantitative standard of PLA2R antibodies.(3)Eu3+-labeled anti human IgG antibody preparation.(4)Establishing of detection method of anti PLA2R antibodies by time-resolved fluorescence immunoassay(TRFIA).The optimization of reaction conditions and the methodological evaluation of the assay.(5)This technique was applied to study of membranous nephropathy.Is it can accurate assessment PLA2R antibodies in idiopathic membranous nephropathy,secondary membranous nephropathy and other kidney disease?Is it possible to through the different threshold value to distinguish the degree and type of membranous nephropathy?Research methods:(1)Recombinant PLA2R was generated by the PLA2R plasmid with flag labeled.Recombinant PLA2R was purified on a Sephadex G100 column with coupling a flag antibody.Identification of recombinant PLA2R by the molecular weight and the activity of immune response.(2)Select a serum with high-concentration PLA2R antibodies.Dissociated PLA2R antibodies based on urea from PLA2R.Calculated the original anti PLA2R antibodies concentration in serum with IgG antibody quantitative.The concentration of a known PLA2R antibodies of the sample diluted to different concentrations as a standard of anti PLA2R antibodies.(3)To establish a detection method of PLA2R antibodies by TRFIA.To optimize reaction conditions of the coating concentration,the labelled antibody dilution degree,the serum dilution degrees,the reaction time and so on.To evaluate TRFIA method from the intra-and inter-batch precision,sensitivity,recovery rate and measurement range.(4)To detect concentration of PLA2R antibodies in serum of 167 kidney patients(including 77 cases of idiopathic model,53 ases of IgA nephropathy,16 cases of lupus nephropathy,21 cases of other kidney disease)and 286 cases of healthy volunteers.To establish the normal reference range and diagnostic value of PLA2R antibodies in serum.Innovative achievements:(1)Recombinant PLA2R of the specific immune reactivity was generated and it can be used to detect PLA2R antibodies.(2)The standard of PLA2R antibodies were prepared.Quantitative detection of PLA2R antibodies was performed on the basis of the PLA2R antibodies standards.(3)PLA2R antibodies serological detection is still in the stage of theoretical research currently,mainly adopts the western blot method,a few researchers using indirect immunofluorescence method and ELISA method,but these methods are limited by their sensitivity,and these methods are mainly used for the qualitative or half quantitative analysis,and the operation are complex,there is no other commercial PLA2R antibodies detection kits.Significance of this study is to provide patients with membranous nephropathy a clinical serological diagnosis index of practical application except puncture.We developed a highly sensitive and quantitative assay for PLA2R using a time-resolved fluoroimmunoassay(TRFIA)for the detection of PLA2R antibodies.This method detects PLA2R antibodies to a lower limit of 0.02 ?g/L,with a broad measurement range of 0.02?343 ?g/L.The average recovery rate was 98.96%.The intra-assay and inter-assay coefficients of variation(CVs)were both<10%.This research provides an ideal serological marker for the diagnosis of the disease,active judgment,treatment timing,selection of drugs and curative effect judgment.This method will make the diagnosis of the disease and monitoring is more convenient,and it is easy to be accepted by patients,it is helpful to early diagnosis and treatment of idiopathic membranous nephropathy.(4)According to our results,when the cut-off for normal anti-PLA2R-IgG concentration was defined as<0.91 ?g/mL,the positive rate of detection was 88.3%in IMN,35.8%in IgA nephropathy cases,50%in lupus nephropathy cases and 14.3%in other kidney cases.The specificity was 90.9%.To improve the anti-PLA2R clinical diagnosis utility,we adjusted the cut-off concentration to 2.025 ?g/mL according to the ROC curves.All cases of IgA nephropathy,lupus erythematosus,and other kidney diseases in our study were under the threshold;however,57/77 cases of IMN were positive.Thus,the positive rate was 74%for that group.The specificity was 100%.