Establishment And Application Of Detection Method For Anti-PLA2R Antibody In Membranous Nephropathy

Objective:The PLA2R eukaryotic recombinant plasmid was constructed and PLA2R was expressed in vitro. Indirect immunofluorescence test （IIFT） of anti-PLA2R antibody was established by the double antibody sandwich principle. The correlation between anti-PLA2R antibody and the disease severity of the patients and histopathology change was observed by detecting the anti-PLA2R antibodies in serum and urine of MN patients.Method:1. The construction of PLA2R eukaryotic recombinant plasmid and protein expression.We found the full-length PLA2R mRNA sequence in Genebank and synth esized the full-length gene, constructed pcDNA3.1/Hygro-PLA2R-IRES2-eGFP and pcDNA3.1/Hygro-PLA2R-His eukaryotic recombinant plasmid in laboratory. We expressed the interest protein by HEK293T cells which were transiently tr ansfected recombinant plasmid with lipofectamine2000. The target protein was determinated by flow cytometry and immunofluorescence technology, immunoh istochemistry and western blot.2. The establishment and validation of indirect immunofluorescence test for anti-PLA2R antibody.We used the HEK293T cells which express the target protein as antigen capture to bind the anti-PLA2R antibodies in serum of MN patients, established IIFT of anti-PLA2R antibody by the anti-anti fluorescent tags. We deteced the serum of150patients with glomerulopathy and30healthy adults using self-built IIFT for anti-PLA2R antibody, the foreign test kit as a control, to evaluate the effectiveness of our self-built detection method. And furtherly clarify the specificity for diagnosis of anti-PLA2R antibody in primary MN patients.3. The clinical research of anti-PLA2R antibody in membranous nephropathyWe collected serum and urine of114primary MN and18MN patients associated with secondary factor in Shanghai Changzheng Hospital, detected antibodies in serum and urine of these patients qualitatively by using the self-built IIFT for anti-PLA2R antibodies. In the meantime, we recorded the patient’s clinical data and pathological report and detect IgG and IgG4in serum of these patients quantificationally by the aggregation immune scattering method. The difference of clinical data and histopathology change between the primary MN patients with positive serum antibody and those with negative serum antibody was observed; The primary MN patients with positive serum antibody were divided in two groups:urine antibody positive group and negative group, to observe the difference of clinical data and histopathology change between two groups. The positive rate of antibody in MN patients associated with secondary factor was also observed by detecting anti-PLA2R antibodies in serum and urine.Results:1. We selected the full-length mRNA of PLA2R transcript variant1in G enebank（NCBI,NM₀07366.4） as our interest gene, synthesized the full-length base sequence, constructed PLA2R eukaryotic expression plasmid （pcDNA3.1/Hygro-PLA2R-IRES2-eGFP and pcDNA3.1/Hygro-PLA2R-His） by enzyme dige stion method. We expressed the PLA2R protein by HEK293T cells which wer e transiently transfected expression plasmid with lipofectamine2000. The PLA2R protein was determinated by flow cytometry and immunofluorescence tech nology, immunohistochemistry and western blot.2. We used the HEK293T cells which express the target protein as antige ncapture to bind the anti-PLA2R antibodies in serum of patients, established I IFT of anti-PLA2R antibody by the anti-anti fluorescent tags. We deteced the serum of150patients with glomerulopathy and30healthy adults using self-built IIFT for anti-PLA2R antibody, the antibody was only found in serum of primary MN patients group.but do not appear in other glomerular disease an d healthy people. The test results of Self-built detection method and the forei gn kit were consistent。3.84cases（73.68%） in114primary MN patients presented serum antibody positive. The positive group showed a significant difference compared with the negative group in serum albumin, triglycerides, creatinine, IgG,24-h proteinuria, urine TRF, IGU, eGFR and histopathological stage（p<0.05）, while no significant difference was showed in serum cholesterol, IgG4and IgG4/IgG in these two groups.6cases in18MN patients associated with secondary factor presented serum antibody positive. Patients with serum antibody negative presented urine antibody negative.60cases （71.42%）in84serum antibody positive patients presented urine antibody positive, the positive rate is52.63%in all primary MN, others presented urine antibody negative. The urine antibody positive group showed a significant difference compared with the negative group in serum albumin, creatinine, urine TRF, IGU, and eGFR（p<0.05）., while no statistical significance was showed in blood lipid,24-h proteinuria, serum IgG, IgG4, IgG4/IgG and histopathological stage.4cases in6MN patients associated with secondary factor and serum antibody positive presented urine antibody positive.Conclusion:The PLA2R eukaryotic recombinant plasmid which we constructed can express the PLA2R protein, the self-built IIFT for anti-PLA2R antibodies can detect anti-PLA2R antibody effectively in MN patients.The test of anti-PLA2R antibody can be used as a means of diagnosis of primary MN, the positive rate is73.68%in serum of primary MN, and52.63%in urine. Anti-PLA2R antibodies in MN patients associated with secondary factor had certain positive, but do not appear in other glomerular disease and healthy people. Serum antibody significantly associates with the disease severity of patients, and urine antibody may be related to renal damage degree, the antibody positive indicates that the disease severity and renal damage degree were getting worse.