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

被引:6
作者
Krueger, Kimberly [1 ]
Mayer, Zsuzsanna [1 ]
Gerckens, Miriam [2 ]
Boeck, Stefan [2 ]
Luppa, Peter [3 ]
Holdenrieder, Stefan [1 ]
机构
[1] Tech Univ Munich, German Heart Ctr Munich, Munich Biomarker Res Ctr, Inst Lab Med, D-80636 Munich, Germany
[2] Univ Hosp Munich Grosshadern, Dept Internal Med 3, D-81377 Munich, Germany
[3] Tech Univ Munich, Dept Clin Chem, Univ Hosp, D-81675 Munich, Germany
关键词
PD-1; PD-L1; PD-L2; analytical validation; ELISA; EXPRESSION; BIOMARKER; TOLERANCE; MECHANISM; RECEPTOR; LIGANDS; MEMBER; CELLS;
D O I
10.3390/biomedicines10102405
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
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页数:13
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