Wu Juan, Zhang Qingjuan, Du Yuanyuan, Zheng Tianyu, Jin Juan, Kao Shangbin, Zhou Xiumei, Qin Yuan, Zhao Xueqin, He Qiang, Yang Fuzhou, Huang Biao
College of Life Sciences and Medicine, Zhejiang Sci-Tech University, Hangzhou, Zhejiang, China.
Department of Nephrology, The Affiliated Jiangning Hospital with Nanjing Medicine University, Nanjing, Jiangsu, China.
Clin Kidney J. 2025 Jan 13;18(3):sfaf010. doi: 10.1093/ckj/sfaf010. eCollection 2025 Mar.
This study aimed to develop a multiplex suspension assay for the simultaneous quantitative detection of anti-cysteine-rich domain (anti-CysR)/C-type lectin domain 1 (CTLD1)/C-type lectin domain 6-8 (CTLD678)-immunoglobulin G4 (IgG4) antibodies of M-type phospholipase A2 receptor (PLA2R) in the serum samples of patients to evaluate the clinical application value of PLA2R epitope spreading in disease prognosis.
CysR, CTLD1 and CTLD678 antigen domains were coupled to three types of microspheres. The optimal dilution ratio of biotinylated anti-human IgG4 was identified, and a multiplex suspension assay was evaluated in terms of linearity, sensitivity, precision, specificity and recovery rate. Lastly, the relationship between epitope spreading and the severity of idiopathic membranous nephropathy was evaluated.
The content of all three epitopes could be detected simultaneously within 2 h. The intra-assay precision ranged from 4.74% to 9.48%, and the inter-assay precision was between 5.13% and 13.92%. Specific experiments showed that the human IgA antibody did not cause a cross-reaction. The recovery rates ranged between 90% and 100%. The cut-off values of epitopes CysR, CTLD1 and CTLD678 between healthy individuals and patients were 6.30, 12.38 and 10.06 Ru/mL, respectively. Among them, the positive rates of epitopes CysR, CTLD1 and CTLD678 were 100%, 34% and 75%, respectively. In addition, Group 3 (CTLD1 response) accounted for 73% of the 12 patients who were not in remission. Meanwhile, when the concentration of CTLD1 exceeds 42.76 Ru/mL, the patient's prognosis may be poorer.
A multiplex suspension assay was developed for the simultaneous quantitative detection of anti-CysR/CTLD1/CTLD678-IgG4 antibodies. The epitope migration sequence of PLA2R molecules during disease progression may not follow a simple linear rule. Among them, the epitope CTLD1 is likely to exert the most significant influence on patient remission.
本研究旨在开发一种多重悬浮分析法，用于同时定量检测患者血清样本中M型磷脂酶A2受体（PLA2R）的抗富含半胱氨酸结构域（抗CysR）/C型凝集素结构域1（CTLD1）/C型凝集素结构域6 - 8（CTLD678）-免疫球蛋白G4（IgG4）抗体，以评估PLA2R表位扩展在疾病预后中的临床应用价值。
将CysR、CTLD1和CTLD678抗原结构域偶联到三种类型的微球上。确定生物素化抗人IgG4的最佳稀释比例，并从线性、灵敏度、精密度、特异性和回收率方面评估多重悬浮分析法。最后，评估表位扩展与特发性膜性肾病严重程度之间的关系。
所有三个表位的含量均可在2小时内同时检测到。批内精密度范围为4.74%至9.48%，批间精密度在5.13%至13.92%之间。特异性实验表明人IgA抗体未引起交叉反应。回收率在90%至100%之间。健康个体与患者之间表位CysR、CTLD1和CTLD678的临界值分别为6.30、12.38和10.06 Ru/mL。其中，表位CysR、CTLD1和CTLD678的阳性率分别为100%、34%和75%。此外，在未缓解的12例患者中，第3组（CTLD1反应）占73%。同时当CTLD1浓度超过42.76 Ru/mL时，患者预后可能较差。
开发了一种多重悬浮分析法用于同时定量检测抗CysR/CTLD1/CTLD678 - IgG4抗体。疾病进展过程中PLA2R分子的表位迁移序列可能不遵循简单的线性规律。其中，表位CTLD1可能对患者缓解产生最显著影响。
