Quantitative detection of anti-PLA2R antibodies targeting different epitopes and its clinical application in primary membranous nephropathy

Yuan Qin; Qingqing Wu; Huiming Sheng; Ting Li; Xiaobin Liu; Xue Yang; Bo Lin; Xiumei Zhou; Juan Jin; Liang Wang; Biao Huang; Qiang He; Zhigang Hu

doi:10.1515/cclm-2022-0720

Article

Quantitative detection of anti-PLA2R antibodies targeting different epitopes and its clinical application in primary membranous nephropathy

Yuan Qin , Qingqing Wu , Huiming Sheng , Ting Li , Xiaobin Liu , Xue Yang , Bo Lin , Xiumei Zhou , Juan Jin , Liang Wang , Biao Huang , Qiang He and Zhigang Hu

Published/Copyright: November 10, 2022

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From the journal Clinical Chemistry and Laboratory Medicine (CCLM) Volume 61 Issue 2

Abstract

Objectives

This study aimed to establish time-resolved fluorescence immunoassays to quantitatively detect the autoantibodies targeting different epitopes of M-type phospholipase A2 receptor (PLA2R) and evaluate its clinical application in primary membranous nephropathy (PMN).

Methods

PLA2R and its reactive epitope-specific IgG/IgG4 time-resolved fluorescence immunoassays (TRFIAs) were established using europium-labeled anti-human IgG/IgG4 antibodies, recombinant proteins, and patient serum. The levels of IgG/IgG4 targeting PLA2R and its epitopes in PMN patient serum were detected, and the relationship between epitope spreading of PLA2R and the severity of patients with PMN was evaluated.

Results

The TRFIAs established in this study could quantitatively detect PLA2R and its epitope-specific IgG and IgG4. Sera from 59 patients with PMN were subjected to detection using anti-PLA2R IgG and anti-PLA2R IgG4. Among them, 46 and 54 patients were found positive for PLA2R antibodies, respectively. Moreover, the levels of PLA2R antibodies were strongly correlated with the severity of patients with PMN. Patients who were detected to have two or more epitopes had more serious renal injury.

Conclusions

PLA2R domain-specific IgG/IgG4 TRFIAs were established in this study, and detection with anti-PLA2R IgG4 could more sensitively screen the reactivity of patients to the PLA2R domain. Moreover, detection epitope spreading of PLA2R was confirmed which is related to the severity of patients with PMN.

Keywords: epitope; IgG4; M-type phospholipase A2 receptor; membranous nephropathy; time-resolved fluorescence immunoassay

Corresponding authors: Biao Huang, Immunoassay laboratory, College of Life Sciences and Medicine, Zhejiang Sci-Tech University, Hangzhou, Zhejiang, 310016, P.R. China, E-mail: jswxhb@163.com; Qiang He, The First Affiliated Hospital of Zhejiang Chinese Medical University (Zhejiang Provincial Hospital of Traditional Chinese Medicine), Hangzhou, Zhejiang, 310000, P.R. China, E-mail: qianghe1973@126.com; and Zhigang Hu, Wuxi People’s Hospital affiliated to Nanjing Medical University, Wuxi, P.R. China; and Wuxi Children’s Hospital, Wuxi, P.R. China, E-mail: jswxhzg@163.com

Yuan Qin, Qingqing Wu and Huiming Sheng contributed equally to this work.

Funding source: National Natural Science Foundation of China

Award Identifier / Grant number: 82172336 and 82070730

Funding source: the Social Development Fund of Zhejiang Province

Award Identifier / Grant number: No. LGF20H200008

Funding source: The precision medicine key project of Wuxi health commission

Award Identifier / Grant number: J202001

Funding source: Key Research and Development Project of Zhejiang Province

Award Identifier / Grant number: No. 2022C03118

Funding source: Key Research and Development Project of Hangzhou

Award Identifier / Grant number: No. 202004A23

Research funding: This study was supported by the Chinese National Natural Science Foundation (No. 82172336 and 82070730), the Social Development Fund of Zhejiang Province (No. LGF20H200008), The precision medicine key project of Wuxi health commission (J202001), Key Research and Development Project of Zhejiang Province (No. 2022C03118), Key Research and Development Project of Hangzhou (No. 202004A23).
Author contributions: B.H., Q.H. and Z.H. designed the experiments; Y.Q., Q.W. and H.S. performed most of the experiments; Q.W.T.L., X.L., H.S., and B.H. analyzed experimental data; X.Y., Y.L., B.L., Q.Z., X.Z., Y.W., L.Z., Y.Q., Z.H., and Y.S. revised the manuscript; L.W., T.L., Q.H., and Z.H. performed the clinical analysis. All authors contributed to the manuscript. All authors have accepted responsibility for the entire content of this manuscript and approved its submission.
Competing interests: Authors state no conflict of interest.
Informed consent: Informed consent was obtained from all individuals included in this study.
Ethical approval: The study protocol was approved by the Institutional Review Board of the Affiliated Wuxi People’s Hospital of Nanjing Medical University (KL12016001), and conducted in accordance with ethical principles stated by the Declaration of Helsinki.

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Supplementary Material

The online version of this article offers supplementary material (https://doi.org/10.1515/cclm-2022-0720).

Received: 2022-07-26

Accepted: 2022-10-27

Published Online: 2022-11-10

Published in Print: 2023-01-27

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Supplementary Material

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Keywords for this article

epitope; IgG4; M-type phospholipase A2 receptor; membranous nephropathy; time-resolved fluorescence immunoassay