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Licensed Unlicensed Requires Authentication Published by De Gruyter May 11, 2022

An automated, rapid fluorescent immunoassay to quantify serum soluble programmed death-1 (PD-1) protein using testing-on-a-probe biosensors

  • Jun Zhang , Lin Chen , Qin Xu , Yue Tao , Jie Pan , Jianmin Guo , Jing Su , Hui Xie EMAIL logo and Yuxin Chen EMAIL logo



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.

Corresponding authors: Yuxin Chen and Hui Xie, Department of Laboratory Medicine, Nanjing Drum Tower Hospital Clinical College of Nanjing Medical University, Nanjing, Jiangsu, P.R. China, E-mail: (Y. Chen), (H. Xie)
Jun Zhang and Lin Chen contributed equally to this work.

Funding source: Nanjing Medical Science and Technique Development

Award Identifier / Grant number: QRX17141

Award Identifier / Grant number: YKK19056

Award Identifier / Grant number: YKK20058

Award Identifier / Grant number: YKK20076

  1. Research funding: This study was supported by Nanjing Medical Science and Technique Development Foundation (, QRX17141, YKK19056, YKK20058, and YKK20076).

  2. Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.

  3. Competing interests: Authors state no conflict of interest.

  4. Informed consent: Informed consent was obtained from all individuals included in this study.

  5. Ethical approval: This study was approved by the Ethics Committee of Nanjing Drum Tower Hospital.


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Received: 2022-02-23
Accepted: 2022-04-25
Published Online: 2022-05-11
Published in Print: 2022-06-27

© 2022 Walter de Gruyter GmbH, Berlin/Boston

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