High Quality Performance of Novel Immunoassays for the Sensitive Quantification of Soluble PD-1, PD-L1 and PD-L2 in Blood

Abstract

Programmed death-1 receptor PD-1(CD279) and its corresponding ligands PD-L1(CD274, B7-H1) and PD-L2(CD273, B7-DC) play important roles in physiological immune tolerance and for immune escape in cancer disease. Hence, the establishment and analytical validation of a novel enzyme-linked immunosorbent assay (ELISA) to measure soluble PD-1, PD-L1 and PD-L2 in blood samples according to high quality standards is required. Antibody pairs were used to establish novel highly sensitive ELISAs for all three markers on an open electrochemiluminescence Quickplex platform. Analytical validation comprised intra- and interassay imprecision, limit of quantification, dilution linearity, material comparison and analytical selectivity testing. The methods demonstrated a broad dynamic range and precise measurements down to the pg/mL range. The coefficient of variation (CV) during the intra-assay imprecision measurements with three patient pools did not exceed 10% for all three assays (PD-1: 6.4%, 6.5%, 7.8%, PD-L1: 7.1%, 4.2%, 6.8%; PD-L2: 4.5%, 10.0%, 9.9%). Dilution linearity experiments in both buffer and heparin plasma displayed good linearity. Selectivity was shown for each marker in titration cross-reactivity experiments up to concentrations of at least 15 ng/mL of these, possibly confounding other markers. Soluble PD-1, PD-L1 and PD-L2 can be measured highly sensitively in serum and plasma and can safely be applied to clinical study settings.

Cytokine and soluble programmed death-ligand 1 levels in serum and plasma of cancer patients treated with immunotherapy: Preanalytical and analytical considerations

“…Our results are consistent with several studies in healthy subjects showing levels of different markers including IL-8, sPD-L1, and MIP-1β higher in serum than plasma. [21][22][23] These differences between the two matrices were not surprising considering previous evidence that different levels of some cytokines can be found between serum and plasma samples collected from the same subjects at the same time. [24][25][26][27] Serum preparation includes the formation of a cross-linked fibrin clot, which causes removal of circulating proteins (e.g., fibrinogen, prothrombin, thrombin, and coagulation factors).…”

“…Our results are consistent with several studies in healthy subjects showing levels of different markers including IL-8, sPD-L1, and MIP-1β higher in serum than plasma. 21–23…”

Refining PD-1/PD-L1 assessment for biomarker-guided immunotherapy - a review

“…Soluble PD-1, PD-L1, and PD-L2 in plasma can be measured by enzyme-linked immunosorbent assay (ELISA) [ 62 ]. Intra-assay imprecision measurements showed that the coefficient of variation did not exceed 10% for all three assays, while other analyses estimated good dilution linearity and selectivity of this method.…”

Robust Preanalytical Performance of Soluble PD-1, PD-L1 and PD-L2 Assessed by Sensitive ELISAs in Blood

“…Currently, immunohistochemistry (IHC) methods based on anti-PD-L1 antibodies are mostly applied; however, they show inconsistent results depending on the specificity of the antibodies used. Recently, blood-based immunoassays were developed to sensitively detect and quantify soluble forms of PD-1, PD-L1, and PD-L2 [ 10 ]. This approach offers the advantage of quick, inexpensive, robust, and quantitative measurements in easily accessible patient blood that can be used to improve future oncoimmunology research as well as for the individual serial monitoring of the response to immune therapies in cancer patients [ 11 , 12 , 13 ].…”

“…Besides a thorough analytical validation [ 10 ], preanalytical handling conditions must be specified and standardized before the assays can be applied in clinical studies. It is well known that some protein profiles are affected by a variety of influencing factors occurring during collection and sample preparation processes [ 16 , 17 ].…”