Anti-PDCD1 Recombinant Antibody (TAB-770)

ELISA analysis of TAB-770 was performed by coating with PD-1 Protein, Human, Recombinant (His Tag).

WB analysis of TAB-770 was performed by with PD-1 Protein, Human, Recombinant (His Tag).
Lane 1: TAB-770 antibody
Lane 2: Negative control
Lane 3: 100ng PDCD1 antigen
Lane 4: 200ng PDCD1 antigen
Lane 5: 400ng PDCD1 antigen

The purified anti-PD-1 antibody (Cat# TAB-770) was loaded in SDS-PAGE gels in β-mercaptoethanol-reduced (Lane 1) and non-reduced (Lane 2) conditions. Gel stained for 30 minutes with Coomassie Blue. As a result of different β-mercaptoethanol-reduced proteins (Heavy chain and Light chain) migrate as about 50 kDa and 25 kDa, respectively.

Size exclusion chromatography (SEC-HPLC) is a technique of purity analysis and macromolecular separation based on molecule size. The purity of anti-PD-1 antibody (Cat# TAB-770) was determined by this method with Agilent 1200 Series.

ELISA analysis of anti-PD-1 antibody (Cat# TAB-770) was performed by coating with recombinant human PD-1 protein (His tag). Then blocked with BSA and incubated with anti-PD-1 antibodies. The HRP-conjugated goat anti-human IgG was used as a secondary antibody. Detection was performed using TMB substrate and stopped with sulfuric acid. The absorbances were read on a spectrophotometer at 450 nm.

Western blot analysis of anti-PD-1 antibody (Cat# TAB-770) was performed by loading human PD-1 protein (His tag) onto a 12% Tris-HCl polyacrylamide gel. Proteins were transferred to a NC membrane and blocked with 5% skim milk for at least one hour. Membranes were probed with anti-PD-1 antibodies and HRP goat anti-human IgG was used as a secondary antibody (1/2000).
Lane 1: Reduced antigen (0.1 μg)
Lane 2: Reduced antigen (0.3 μg)
Lane 3: Reduced antigen (0.6 μg)

Dot blot analysis of anti-PD-1 antibody (Cat# TAB-770) was performed by coating with human PD-1 protein (His tag).
The anti-PD-1 antibody incubation concentration: 2 μg/mL.
HRP-conjugated goat anti-human IgG was used as a secondary antibody (1/2000).

Dilute the antibody ligand (5 μg/mL) and antigen analyte with running buffer. The ligand was injected to sample channel (Fc2) at a flow rate of 10 μL/min to reach a capture level of about 400 RU. The analyte was injected to Fc1-Fc2 of the channel at a flow rate of 30 μL/min for an association phase of the corresponding time. The association and dissociation process were all handling in the running buffer. Repeat 6 cycles of analyte according to analyte concentrations in ascending order.

In a functional ELISA, the recombinant human PD‑1 protein (His tag) was coated at 2 µg/mL, 100 µL/well. Anti-PD-1 antibody (Cat# TAB-770) were 3-fold serial diluted from 20 µg/mL, 100 µL/well. Coated PD-1 protein was incubated with the human PD-L1 mFc chimera (5 µg/mL) and anti-PD-1 antibody (Cat# TAB-770). The PD-L1 binding to PD-1 was detected by HRP-goat anti-mouse IgG secondary antibody. Human IgG4 antibody (Cat# MOB-0408ZL, Creative Biolabs) was used as an isotype control.

PBMCs were stimulated with 2 μg/mL anti-CD3 antibody (Cat# TAB-0416CL, Creative Biolabs) and cultured for 144 hours with PD-1 antibody (Cat# TAB-770, Creative Biolabs). Human IgG4 antibody (Cat# MOB-0408ZL, Creative Biolabs) was used as an isotype control. After cultivation, the supernatant was collected and the IFN-γ secretion level was analyzed using a human IFN gamma ELISA kit. The assay was performed in three parallel wells.