最近，血浆或血清中的可溶性程序性死亡蛋白 1 (PD-L1) 水平（而非组织 PD-L1 表达水平）被提议作为针对不同类型的免疫检查点抑制剂治疗的患者的有效预测和预后生物标志物。癌症。血液中可溶性PD-L1的定量可以快速评估患者的免疫状态；然而，现有的检测方法在临床环境中的灵敏度、重现性和准确性方面存在局限性。为了克服这些问题，本研究致力于开发一种超灵敏的自动流式动力学排除测定 (KinExA)，用于精确测量血浆中可溶性 PD-L1。该检测是在 KinExA™ 3200 生物传感器的协助下开发的。在此测定中，校准品或血浆样品溶液中的 PD-L1 用抗 PD-L1 单克隆抗体预平衡。然后将平衡后的混合溶液快速通过 PD-L1 蛋白，该蛋白已涂覆在聚甲基丙烯酸甲酯珠上，并在 KinExA™ 生物传感器的观察池中固结为微柱。游离的抗PD-L1抗体与固定的PD-L1结合，然而，通过用磷酸盐缓冲盐水冲洗系统，将未结合的分子从珠微柱中除去。荧光素标记的二抗快速通过珠子，并在标记抗体流过珠子的过程中监测荧光信号。通过绘制结合百分比作为样品溶液中 PD-L1 浓度的函数来生成校准曲线。该测定的工作范围在 4 参数方程 (r = 0.9992) 上具有非常好的相关系数，为 0.5 - 100 pg mL─1。检测和定量的检测限分别为 0.15 和 0.5 pg mL─1。血浆掺加的 PD-L1 的回收率范围为 96.4-104.3% (±3.7-6.2%)。测定的精密度令人满意；日内和日间精度的变异系数值均不超过 6.2%。所提议的 KinExA 的自动分析有助于在临床环境中处理许多样本。所提出的 KinExA 的整体性能优于可溶性 PD-L1 血浆水平的现有检测方法。预计所提出的检测方法对于需要更可信结果的 PD-L1 测量具有巨大价值。© 2024 作者。

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─1. The assay limit of detection and quantitation were 0.15 and 0.5 pg mL─1, 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.© 2024 The Authors.