INTENDED USE

This human PD-L1 ELISA kit is to be used for the in vitro quantitative determination of human Programmed Death-Ligand 1 (PD-L1) concentrations in cell culture supernatant and other biological fluids. This kit is intended for LABORATORY RESEARCH USE ONLY

INTRODUCTION

Programmed Death-Ligand 1 (PD-L1) is a 40KD type I transmembrane protein found on various hematopoietic and non-hematopoietic cells. PD-L1 and its receptor Programmed Death-1 (PD-1) are members in the extended B7/CD28 co-stimulator superfamily of T cell immunity regulators. PD-1 is expressed by activated T-cell, pro B-cell and macrophage. When the T-cell receptor (TCR) on activated T-cell engages with antigen presented by MHC, the binding of PD-1 on activated T-cell with PD-L1 on the antigen presenting cells occurs simultaneously. The ligation triggers the recruitment of Src homology region 2 domain containing phosphatase 1 (SHP) and SHP-2 (Chemnitz et al., 2004) through the immunereceptor tyrosine-based switch motif (ITSM) on the PD-1 cytoplasmic tail. These events can inhibit CD28-mediated up-regulation of IL-2, IL-10, IL-13, IFN-γ, and Bcl-xL, and suppress T-cell activation. In immune-privileged organs such as placenta, testes and eyes, PD-L1 is constitutively expressed and plays a role in suppressing autoimmunity and maintaining maternal-fetal tolerance. The PD-L1/PD-1 pathway is thought to have a self-protective function, which is exploited by tumor cells and chronic infectious agents to escape immunity (Riley, 2009). Butte et al. discovered B7-1 (CD80) as another specific binding partner for PD-L1 in 2007 and observed the inhibitory effect of B7-1 on in vitro proliferation of CD28/CTLA4 -/- T cells and the inhibitory effect PD-L1 on PD-1 -/- T cells comparing with Wild Type T cells. The study revealed a possible inhibitory pathway of T-cell associated immunity through B7-1 and PD-L1 binding. Upregulation of PD-L1 has been found to be associated with many cancer types, such as bladder cancer, non-small cell lung cancer (NSCLC), renal cancer, melanoma, ovarian cancer, and breast cancer. High PD-L1 levels are related to tumor aggressiveness and higher mortality in patients with renal cell carcinoma, and related to significantly poorer prognosis and lower intraepithelial CD8+ T-lymphocyte count in patience with ovarian cancer. The development of PD-L1/PD-1 pathway has been shown to be associated with transformed tumor microenvironment (He et al., 2015). During persistent viral infection, motility arrest of antigen specific T-cells has been observed. Blockage of PD-1/PD-L1 pathway with antibody has been shown to restore the mobility and function of exhausted T-cells, indicating that the pathway is implicated with infection-associated T-cell exhaustion (Kaufmann et al., 2009). In the study with HIV patients and normal individual, Said et al. demonstrated that PD-1 expression on macrophage from HIV patients was increased and ligation with PD-L1 induced IL-10 production by monocytes. The upregulation of IL-10 impaired CD4+ T cell activation and resulted in a reversible CD4+ T-cell dysfunction during HIV infection. PD-L1 is also believed to play a role in the generation of regulatory T cells (Tregs), and in the enhancement of Treg cell function. PD-L1 co-stimulation can down-regulate the ligand induced T-cell receptor on CD8+ T-cells (Karwacz et al., 2011)

PRINCIPLE OF THE ASSAY

This enzyme-linked immunosorbent assay (ELISA) applies a technique called a quantitative sandwich immunoassay. The microtiter plate provided in this kit has been pre-coated with a monoclonal antibody specific for human PD-L1. When standards or samples are added to the appropriate microtiter plate wells, human PD-L1 in the standards or samples will be immobilized by the pre-coated antibody during incubation. Then, a biotin-conjugated antibody preparation specific for human PD-L1 is added to each well and incubated. The biotin labelled antibody attaches to the wells by binding to human PD-L1. After plate washing, other proteins, components and unattached biotin labelled antibody is removed. Then, avidin-horseradish peroxidase (HRP) conjugate is added to each well. Avidin has a very high affinity for biotin, thus, it links the tracer (HRP) sturdily to the biotin labelled antibody. The wells are thoroughly washed to remove all unbound avidin-HRP conjugate and a TMB (3,3’,5,5" tetramethyl-benzidine) substrate solution is added to each well. The enzyme (HRP) and substrate are allowed to react over a short incubation period. Only wells that contain human PD-L1 will exhibit a change in colour. The extent of colour change is proportional to the quantity of human PD-L1 presented in the standards/samples. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the colour change is measured spectrophotometrically at a wave length of 450 nm ± 2 nm. According to the testing system, the provided standard is diluted (2-fold) with the appropriate diluent and assayed at the same time as the samples. This allows the operator to produce a curve of Optical Density (O.D.) versus human PD-L1 concentration (pg/mL) in standards. The concentration of human PD-L1 in the samples is then determined by comparing the O.D. of the samples to the standard curve and multiplying with sample dilution factor.