4.5. ELISA

Using sandwich enzyme-linked immunosorbent assays (ELISAs, R&D Systems, Bio-Techne, DY666-05, Ancillary Reagent kit 2: R&D Systems, Bio-Techne, DY008, Specificity: no cross-reactivity or interference with 50 ng/mL recombinant human CD155/Fc Chimera CD96/Fc Chimera Integrin β2 Nectin-2/Fc Chimera PVRIG/Fc Chimera and recombinant mouse DNAM-1/Fc Chimera; Sensitivity: 62.5–4000 pg/mL), the CD226 concentrations were measured following previously published methods [34]. A 96-well flat-bottom microplate was coated overnight with 100 μL/well anti-human CD226 antibody at RT. Subsequently, the plate was washed with 3 × 400 μL/well of wash buffer and blocked with 400 μL/well of reagent diluent for an hour at RT. After another three-washing step, 100 μL of serially diluted standards and thawed serum samples were added to the plate and incubated for two hours at RT. Following incubation, the wells were washed again, and 100 μL of detection antibody was added to the wells for two hours at RT. After another three-washing step, 100 μL/well of streptavidin HRP was pipetted for 20 min. Following the final three washes, the plate was developed for 20 min with a 1:1 mixture of substrate reagents A (H₂O₂) + B (tetramethylbenzidine) in darkness. The reaction was then terminated with a Stop solution. The absorbance of the test plates was read immediately at 450 nm or 450 nm with a reference filter of 540 nm using a BMG SPECTOstar Nano spectrophotometer (BMG Labtech, Ortenberg, Germany). Following background subtraction, standard curves were generated using 4-parametric logistic analysis, and the CD226 concentrations were calculated using MARS Data Analysis Software version 3.32 (BMG Labtech, Ortenberg, Germany).