Novel ultrasensitive automated kinetic exclusion assay for measurement of plasma levels of soluble PD-L1, the predictive and prognostic biomarker in cancer patients treated with immune checkpoint inhibitors.

Recently, the blood plasma or serum levels of soluble programmed death protein 1 (PD-L1), but not tissue PD-L1 expression level, have been proposed as an effective predictive and prognostic biomarker in patients treated with immune checkpoint inhibitors for different types of cancers. The quantification of soluble PD-L1 in blood will provide a quick evaluation of patients' immune status; however, the available assays have limitations in their sensitivity, reproducibility, and accuracy for use in clinical settings. To overcome these problems, this study was dedicated to developing an ultrasensitive automated flow-based kinetic exclusion assay (KinExA) for the accurate and precise measurement of soluble PD-L1 in plasma. The assay was developed with the assistance of KinExA™ 3200 biosensor. In this assay, PD-L1 in its calibrator or plasma sample solution was pre-equilibrated with anti-PD-L1 monoclonal antibody. The equilibrated mixture solution was then passed rapidly over PD-L1 protein that has been coated onto polymethylmethacrylate beads consolidated as a microcolumn in the observation cell of the KinExA™ biosensor. The free anti- PD-L1 antibody was bound to the immobilized PD-L1, however, the unbound molecules were removed from the beads microcolumn by flushing the system with phosphate-buffered saline. Fluorescein-labeled secondary antibody was passed rapidly over the beads, and the fluorescence signals were monitored during the flow of the labeled antibody through the beads. The calibration curve was generated by plotting the binding percentages as a function of PD-L1 concentrations in its sample solution. The working range of the assay with very a good correlation coefficient on a 4-parameter equation (r = 0.9992) was 0.5 - 100 pg mL. The assay limit of detection and quantitation were 0.15 and 0.5 pg mL, respectively. The recovery values of plasma-spiked PD-L1 were in the range of 96.4-104.3 % (±3.7-6.2 %). The precision of the assay was satisfactory; the values of the coefficient of variations did not exceed 6.2 % for both intra- and inter-day precision. The automated analysis by the proposed KinExA facilitates the processing of many specimens in clinical settings. The overall performance of the proposed KinExA is superior to the available assays for plasma levels of soluble PD-L1. The proposed assay is anticipated to have a great value in the measurement of PD-L1 where a more confident result is needed.