【摘要】 目的建立抗PLA2R抗体的超灵敏定量检测方法,改进抗PLA2R抗体检测在膜性肾病诊治中的应用。方法筛选出一个含高浓度抗PLA2R抗体的血清,采用尿素解离结合在PLA2R包被孔上的抗PLA2R抗体,并进行IgG抗体定量,推算出原血清中抗PLA2R抗体的浓度,将此已知抗PLA2R抗体浓度的样品稀释到不同浓度作为抗PLA2R抗体的标准品。建立抗PLA2R抗体TRFIA检测方法,从批内批间精密度、灵敏度、回收率、测量范围、临床应用等方面对建立的TRFIA方法进行评价。结果制备了抗PLA2R抗体标准品,设置的抗PLA2R抗体的标准品浓度分别为0μg/ml、0.17μg/ml、0.68μg/ml、3.42μg/ml、17.1μg/ml、68.4μg/ml、343μg/ml。有效检测范围为0.02μg/mL-343μg/ml,3份标本的回收率均值为98.96%,低、中、高浓度批内CV%(n=20)分别为6.38%、5.55%、4.88%,低、中、高浓度批间(n=8)CV%分别为7.69%、6.23%、6%,均<10%。正常参考范围为0-0.89μg/ml,29%的IgA肾病、44.4%的红斑狼疮肾病、88.5%的IMN抗PLA2R抗体阳性。红斑狼疮肾病患者、IgA肾病患者和IMN患者的血清PLA2R抗体含量均显著高于健康体检者,对应的P值分别为0.036、0.034和0.000,P均<0.05。以正常健康人作为对照组,血清抗PLA2R-IgG抗体在红斑狼疮肾病、IgA肾病和IMN的灵敏度和特异度曲线下面积AUCROC分别为:0.738、0.607、0.968,其中对应肾病的检测准确性程度由高到低分别为:IMN、红斑狼疮、IgA肾病。结论为膜性肾病患者提供一个除穿刺以外的可临床实际应用的血清学诊断指标,为该病的诊断、活动性的判断、治疗时机的把握、药物的选择及疗效判断提供了一个理想的血清学标志物。更多还原

【Abstract】 Objective To establish an ultrasensitive detection method of PLA2 Rantibodies.Improve the application of PLA2 Rantibodies in membranous nephropathy.Methods Select a serum with high-concentration PLA2 Rantibodies.Dissociated PLA2 Rantibodies based on urea from PLA2 R.Calculated the original anti PLA2 Rantibodies concentration in serum with IgG antibody quantitative.The concentration of a known PLA2 Rantibodies of the sample diluted to different concentrations as a standard of anti PLA2 Rantibodies.To establish a detection method of PLA2 Rantibodies by TRFIA.To evaluate TRFIA method from the intra-and inter-batch precision,sensitivity,recovery rate,measurement range and clinical application.Results Recombinant PLA2 Rof the specific immune reactivity was generated and it can be used to detect PLA2 Rantibodies.The standard of PLA2 Rantibodies were prepared.Quantitative detection of PLA2 Rantibodies was performed on the basis of the PLA2 Rantibodies standards.We developed a highly sensitive and quantitative assay for PLA2 Rusing a time-resolved fluoroimmunoassay(TRFIA)for the detection of PLA2 Rantibodies.This method detects PLA2 Rantibodies to a lower limit of 0.02μg/L,with a broad measurement range of 0.02-343μg/L.The average recovery rate was 98.96%.The intra-assay and inter-assay coefficients of variation(CVs)were both< 10%.According to our results,when the cut-off for normal anti-PLA2 R-IgG concentration was defined as<0.89μg/mL,the positive rate of detection was 88.5%in IMN,29%in IgA nephropathy cases,44.4%in lupus nephropathy cases.Lupus erythematosus(sle)patients with kidney disease patients,patients with IgA nephropathy and IMN of PLA2 Rserum antibody levels were significantly higher than that of healthy physical examination,the corresponding P values were 0.036,0.034 and 0.000.In normal healthy people as control group,serum anti PLA2 R-IgG antibody in lupus erythematosus(sle)and kidney disease,IgA nephropathy,IMN sensitivity and specific AUCROC offline area are:0.738、0.607、0.968.The corresponding kidney disease detection accuracy degree from high to low are:IMN,lupus erythematosus,IgA nephropathy.Conclusion 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.更多还原