College of Life Sciences and Medicine, Zhejiang Sci-Tech University, Hangzhou, China.
The Affiliated Wuxi Children's Hospital of Nanjing Medical University, Wuxi People's Hospital affiliated to Nanjing Medical University, Wuxi, China.
J Clin Lab Anal. 2020 Dec;34(12):e23508. doi: 10.1002/jcla.23508. Epub 2020 Aug 6.
To detect phospholipase A2 receptor (PLA2R) antibody by established time-resolved fluorescent bead immunochromatographic assay.
The reaction time of coupling, pH of the reaction, and coupling ratio of the label to PLA2R were determined. The EDC method was used to covalently couple PLA2R to time-resolved fluorescent beads, which were sprayed onto a bonding pad. PLA2R and rabbit anti-PLA2R antibody sprayed onto a nitrocellulose membrane were used as detection and quality control lines, respectively. Immunochromatographic test strips were prepared to enable rapid detection of PLA2R antibodies. Various technical indicators were evaluated, and the correlation among this method, enzyme-linked immunosorbent assay (ELISA), and serum analysis was examined.
The pH suitable for labeling was 6.5. The optimal mass ratio of PLA2R protein to fluorescent beads was 0.08:1, and the reaction time of coupling was at least 1.5 hours. The appropriate spray film size of the coupled fluorescent bead was 5 μL/cm, and the appropriate staining concentration of the test line was 0.28 mg/mL. Further, 80 µL of sample was required for the test, and the result was obtained in only 15 minutes. The measurable range of this method was 5-1500 RU/mL. Intra- and inter-assay coefficients of variation were 7.61% and 11.07%, respectively, with an average recovery rate of 93.77%. The method showed a good correlation with ELISA, with a correlation coefficient of 0.936.
This method could better meet the clinical demand for idiopathic membranous nephropathy (IMN) detection.
通过建立时间分辨荧光珠免疫层析法检测磷脂酶 A2 受体（PLA2R）抗体。
确定偶联反应时间、反应 pH 值和标记物与 PLA2R 的偶联比。采用 EDC 法将 PLA2R 共价偶联到时间分辨荧光珠上，然后将其喷涂到结合垫上。将 PLA2R 和兔抗 PLA2R 抗体喷涂到硝酸纤维素膜上，分别作为检测线和质控线。制备免疫层析测试条，以实现 PLA2R 抗体的快速检测。评估各种技术指标，并检查该方法与酶联免疫吸附测定（ELISA）和血清分析之间的相关性。
适合标记的 pH 值为 6.5。PLA2R 蛋白与荧光珠的最佳质量比为 0.08:1，偶联反应时间至少为 1.5 小时。偶联荧光珠的适当喷涂膜尺寸为 5 μL/cm，测试线的适当染色浓度为 0.28 mg/mL。此外，该试验需要 80 μL 样本，15 分钟即可获得结果。该方法的测量范围为 5-1500 RU/mL。内和间试验的变异系数分别为 7.61%和 11.07%，平均回收率为 93.77%。该方法与 ELISA 相关性良好，相关系数为 0.936。
该方法可以更好地满足特发性膜性肾病（IMN）检测的临床需求。
