Soluble programmed death-1 (sPD-1) plays an essential role in the pathogenesis and progression of various diseases, including chronic hepatitis B (CHB) and hepatocellular carcinoma (HCC). Currently, there is no Food and Drug Administration–approved sPD-1 immunoassay available for routine clinical testing. Most sPD-1 detections employed enzyme-linked immunosorbent assay (ELISA) method for research purpose, which is complicated by intensive manual operation and cannot achieve automatic detection. Therefore, we aimed to develop an automated, rapid immunoassay for sPD-1 measurement based on testing-on-a-probe (TOP) biosensors and evaluate its performance in patients with hepatic diseases.
We developed an automatic fluorescent immunoassay using TOP biosensors using a pair of mouse anti-PD-1 monoclonal antibodies (mAbs), which were evaluated by biolayer interferometry. The sensitivity, linearity, and repeatability of the novel immunoassay were analyzed, and its compatibility with an established ELISA kit was evaluated. Further, we quantified sPD-1 level in healthy individuals as well as patients with CHB, hepatic cirrhosis, and HCC.
The TOP assay to quantify sPD-1 was developed and performed on an automatic fluorescent analyzer within 20 min, which showed good precision with coefficients of variation less than 10% and good linearity ranging from 2 to 3,000 pg/mL. The results tested by our TOP assay correlated well with the established ELISA assay (r=0.92, p<0.0001). Using our TOP assay, sPD-1 was significantly elevated in patients with chronic hepatitis, hepatic cirrhosis and hepatocarcinoma if compared to healthy control, respectively (p<0.0001).
An automated, rapid fluorescent immunoassay to quantify serological sPD-1 protein using TOP biosensors was developed and showed acceptable analytical performance including precision, linearity, and good correlation with the established ELISA assay, with the great potential in clinical practice.
Funding source: Nanjing Medical Science and Technique Developmenthttp://dx.doi.org/10.13039/501100019349
Award Identifier / Grant number: QRX17141
Award Identifier / Grant number: YKK19056
Award Identifier / Grant number: YKK20058
Award Identifier / Grant number: YKK20076
Research funding: This study was supported by Nanjing Medical Science and Technique Development Foundation (https://dx.doi.org/10.13039/501100019349, QRX17141, YKK19056, YKK20058, and YKK20076).
Author contributions: 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: This study was approved by the Ethics Committee of Nanjing Drum Tower Hospital.
1. Zamani, MR, Aslani, S, Salmaninejad, A, Javan, MR, Rezaei, N. PD-1/PD-L and autoimmunity: a growing relationship. Cell Immunol 2016;310:27–41. https://doi.org/10.1016/j.cellimm.2016.09.009.Search in Google Scholar
2. Nielsen, C, Ohm-Laursen, L, Barington, T, Husby, S, Lillevang, ST. Alternative splice variants of the human PD-1 gene. Cell Immunol 2005;235:109–16. https://doi.org/10.1016/j.cellimm.2005.07.007.Search in Google Scholar
3. Frigola, X, Inman, BA, Krco, CJ, Liu, X, Harrington, SM, Bulur, PA, et al.. Soluble B7-H1: differences in production between dendritic cells and T cells. Immunol Lett 2012;142:78–82. https://doi.org/10.1016/j.imlet.2011.11.001.Search in Google Scholar
4. Chen, Y, Wang, Q, Shi, B, Xu, P, Hu, Z, Bai, L, et al.. Development of a sandwich ELISA for evaluating soluble PD-L1 (CD274) in human sera of different ages as well as supernatants of PD-L1+ cell lines. Cytokine 2011;56:231–8. https://doi.org/10.1016/j.cyto.2011.06.004.Search in Google Scholar
5. Wu, H, Miao, M, Zhang, G, Hu, Y, Ming, Z, Zhang, X. Soluble PD-1 is associated with aberrant regulation of T cells activation in aplastic anemia. Immunol Invest 2009;38:408–21. https://doi.org/10.1080/08820130902912332.Search in Google Scholar
6. Wan, B, Nie, H, Liu, A, Feng, G, He, D, Xu, R, et al.. Aberrant regulation of synovial T cell activation by soluble costimulatory molecules in rheumatoid arthritis. J Immunol 2006;177:8844–50. https://doi.org/10.4049/jimmunol.177.12.8844.Search in Google Scholar
7. Rossille, D, Gressier, M, Damotte, D, Maucort-Boulch, D, Pangault, C, Semana, G, et al.. Groupe Ouest-Est des Leucémies et Autres Maladies du Sang; Groupe Ouest-Est des Leucémies et Autres Maladies du Sang. High level of soluble programmed cell death ligand 1 in blood impacts overall survival in aggressive diffuse large B-Cell lymphoma: results from a French multicenter clinical trial. Leukemia 2014;28:2367–75. https://doi.org/10.1038/leu.2014.137.Search in Google Scholar
8. Cheng, HY, Kang, PJ, Chuang, YH, Wang, YH, Jan, MC, Wu, CF, et al.. Circulating programmed death-1 as a marker for sustained high hepatitis B viral load and risk of hepatocellular carcinoma. PLoS One 2014;9: e95870. https://doi.org/10.1371/journal.pone.0095870.Search in Google Scholar
9. Merli, M, Aprile, F. Le linee guida sulla nutrizione della Società europea per lo studio delle malattie epatiche (EASL) [The European Association for the Study of Liver (EASL) nutrition guidelines.]. Recenti Prog Med 2021;112:103–9. Italian. https://doi.org/10.1701/3559.35370.Search in Google Scholar
10. Polaris Observatory Collaborators. Global prevalence, treatment, and prevention of hepatitis B virus infection in 2016: a modelling study. Lancet Gastroenterol Hepatol 2018;3:383–403. https://doi.org/10.1016/S2468-1253(18)30056-6.Search in Google Scholar
11. Xia, J, Huang, R, Chen, Y, Liu, Y, Wang, J, Yan, X, et al.. Profiles of serum soluble programmed death-1 and programmed death-ligand 1 levels in chronic hepatitis B virus-infected patients with different disease phases and after anti-viral treatment. Aliment Pharmacol Ther 2020;51:1180–7. https://doi.org/10.1111/apt.15732.Search in Google Scholar PubMed
12. Li, P, Enea, NS, Zuk, R, Tan, H, Wu, AHB, Jaffe, AS. Performance characteristics of a high-sensitivity cardiac troponin assay using plasma and whole blood samples. Clin Biochem 2017;50:1249–52. https://doi.org/10.1016/j.clinbiochem.2017.08.002.Search in Google Scholar PubMed
13. Ye, Z, Dong, N, Liang, K, Hu, H, Xu, J. Analytical and clinical evaluation of a novel assay for measurement of interleukin 6 in human whole blood samples. J Clin Lab Anal 2021;35:e24011. https://doi.org/10.1002/jcla.24011.Search in Google Scholar PubMed PubMed Central
14. Yang, HS, Racine-Brzostek, SE, Karbaschi, M, Yee, J, Dillard, A, Steel, PAD, et al.. Testing-on-a-probe biosensors reveal association of early SARS-CoV-2 total antibodies and surrogate neutralizing antibodies with mortality in COVID-19 patients. medRxiv [Preprint] 2020:2020.11.19.20235044. https://doi.org/10.1101/2020.11.19.20235044.Search in Google Scholar PubMed PubMed Central
15. Wang, J, Fei, K, Jing, H, Wu, Z, Wu, W, Zhou, S, et al.. Durable blockade of PD-1 signaling links preclinical efficacy of sintilimab to its clinical benefit. mAbs 2019;11:1443–51. https://doi.org/10.1080/19420862.2019.1654303.Search in Google Scholar PubMed PubMed Central
16. Tan, S, Zhang, H, Chai, Y, Song, H, Tong, Z, Wang, Q, et al.. An unexpected N-terminal loop in PD-1 dominates binding by nivolumab. Nat Commun 2017;8:14369. https://doi.org/10.1038/ncomms14369.Search in Google Scholar PubMed PubMed Central
18. Keir, ME, Butte, MJ, Freeman, GJ, Sharpe, AH. PD-1 and its ligands in tolerance and immunity. Annu Rev Immunol 2008;26:677–704. https://doi.org/10.1146/annurev.immunol.26.021607.090331.Search in Google Scholar PubMed
19. Lee, J, Zhuang, Y, Wei, X, Shang, F, Wang, J, Zhang, Y, et al.. Contributions of PD-1/PD-L1 pathway to interactions of myeloid DCs with T cells in atherosclerosis. J Mol Cell Cardiol 2009;46:169–76. https://doi.org/10.1016/j.yjmcc.2008.10.028.Search in Google Scholar PubMed
20. Freeman, GJ, Long, AJ, Iwai, Y, Bourque, K, Chernova, T, Nishimura, H, et al.. Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J Exp Med 2000;192:1027–34. https://doi.org/10.1084/jem.192.7.1027.Search in Google Scholar PubMed PubMed Central
21. Tseng, SY, Otsuji, M, Gorski, K, Huang, X, Slansky, JE, Pai, SI, et al.. B7-DC, a new dendritic cell molecule with potent costimulatory properties for T cells. J Exp Med 2001;193:839–46. https://doi.org/10.1084/jem.193.7.839.Search in Google Scholar PubMed PubMed Central
22. Okazaki, T, Maeda, A, Nishimura, H, Kurosaki, T, Honjo, T. PD-1 immunoreceptor inhibits B cell receptor-mediated signaling by recruiting src homology 2-domain-containing tyrosine phosphatase 2 to phosphotyrosine. Proc Natl Acad Sci U S A 2001;98:13866–71. https://doi.org/10.1073/pnas.231486598.Search in Google Scholar PubMed PubMed Central
23. Muenst, S, Soysal, SD, Tzankov, A, Hoeller, S. The PD-1/PD-L1 pathway: biological background and clinical relevance of an emerging treatment target in immunotherapy. Expert Opin Ther Targets 2015;19:201–11. https://doi.org/10.1517/14728222.2014.980235.Search in Google Scholar PubMed
24. Pedoeem, A, Azoulay-Alfaguter, I, Strazza, M, Silverman, GJ, Mor, A. Programmed death-1 pathway in cancer and autoimmunity. Clin Immunol 2014;153:145–52. https://doi.org/10.1016/j.clim.2014.04.010.Search in Google Scholar PubMed
25. Hu, HH, Jeng, WJ, Pan, MH, Luo, WS, Chang, CL, Huang, YT, et al.. Serum soluble programmed cell death 1 levels predict spontaneous functional cure in inactive carriers with chronic hepatitis B. Aliment Pharmacol Ther 2022;55:558–67. https://doi.org/10.1111/apt.16752.Search in Google Scholar PubMed
26. Khan, M, Zhao, Z, Arooj, S, Fu, Y, Liao, G. Soluble PD-1: predictive, prognostic, and therapeutic value for cancer immunotherapy. Front Immunol 2020;11:587460. https://doi.org/10.3389/fimmu.2020.587460.Search in Google Scholar PubMed PubMed Central
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