Human pd l1 elisa kit

HEK293T, Jurkat, B-3T3, EL4, MCA-38, CHO, B3, Vero, CV-1 and R9ab cells were obtained from and verified as mycoplasma free by the Cell Services facility at The Francis Crick Institute. Human cell lines were also validated by DNA fingerprinting. All cells were grown in Iscove’s Modified Dulbecco’s Medium (Sigma-Aldrich) supplemented with 5% fetal bovine serum (Thermo Fisher Scientific), L-glutamine (2 mmol/L, Thermo Fisher Scientific), penicillin (100 U/mL, Thermo Fisher Scientific), and streptomycin (0.1 mg/mL, Thermo Fisher Scientific). HEK293T, B-3T3, MCA-38, B3 and Jurkat sublines transduced with CD274v1, CD274-L2A, or PDCD1 were generated by adding viral stocks (as described above) to target cells in the presence of polybrene (4 μg/mL) and sorted based on GFP expression to >98% purity on a S3e Cell Sorter (Propel Labs Inc), 72 hr after transduction. Supernatant PD-L1 levels were measured 48 hr after seeding 5 × 10⁵ sorted cells/ml, using the human PD-L1 ELISA Kit (clone 28–8, Abcam) or the murine PD-L1 ELISA Kit (EKU06803, Biomatik), according to manufacturers’ instructions.

Protein levels of PD-L1 were measured using a human PD-L1 ELISA kit (Abcam, clone: 28-8) according to the recommendations of the manufacturer. All concentrations are expressed as means ± SEM of triplicates.

Blood samples were obtained from all patients before biopsy or treatment. To remove remaining cells, serum tubes were centrifuged at 1500 g for 10 min at 4 °C. The serum samples were aliquoted and stored at −80 °C.
Serum PD-L1 levels were measured quantitatively by enzyme immunoassay. On the measurement day, stored serum samples were thawed, and 100 µL of serum were used for further analysis. Levels of PD-L1 were measured using a commercially available sandwich enzyme-linked immunosorbent assay (Human PD-L1 ELISA Kit, ab214565, Abcam, Cambridge, MA) according to the manufacturer’s recommendations. The minimum detectable level of sPD-L1 was 2.91 pg/mL; values under the detectable level were assigned a value of 0 pg/mL.

Resected fresh tumor tissue(s) and tumor-adjacent normal tissue(s) were obtained from the pathology department. Samples were collected in DMEM/F-12 + Glutamax 500 mL (Base) supplemented with HyClone Penicillin-Streptomycin 100 × 100 mL (1%). Surgically resected tumor and tumor-associated normal tissues were obtained per the pathologist’s recommendation, and the pathology reports for the respective patients were included in the study. Blood was collected in cell-save tubes (CellSave Preservative Tubes, Menarini Silicon Biosystems, BrynAthyn, PA, USA catalog #7900005), and plasma was stored at −80 °C for sPD-L1, sPD-L2, and sPD-1 determination by ELISA (Abcam, Boston, MA, USA; Human PD-L1 ELISA kit, Product #ab277712; Abcam Human PD-L2 ELISA kit, Product #ab231928; Abcam Human PD-1 ELISA kit, Product #ab252360) as per the manufacturer’s protocol. For PD-L1/PD-1, plasma samples were used undiluted (50 μL sample) and diluted 1:1 in a diluent buffer. For PD-L2, plasma samples were first diluted at 1:10. From this 1:10 dilution, final dilutions of 1:25 and 1:50 were made for the assay. Samples were run in triplicate as desired/necessary, parallel to the standard curves for each batch.