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

Abstract Objectives 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. Methods 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. Results 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). Conclusions 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.