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.
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
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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).
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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.
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Competing interests: Authors state no conflict of interest.
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Informed consent: Informed consent was obtained from all individuals included in this study.
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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).
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