Hrp conjugated goat anti human igg fc

Enzyme-linked immunosorbent assay for VEGF-trap was developed as has been previously described (Koh et al, 2010 (link)). A 96-well plate was coated overnight at 4°C with 200 ng of VEGF in 100 μl of PBS. The plate was washed four times with 400 μl wash buffer [PBS, 10% SBlock (Pierce, cat. # 37538), 0.05% Tween-20] and then coated for 2 min with SBlock at RT. Fifty microlitres of trap standards, samples and controls were added along with 50 μl of incubation buffer (100% SBlock, 0.1% Tween-20) and incubated on the bench, at RT for 2 h. After washing four times with 400 μl of wash buffer, 50 μl of HRP-conjugated goat anti-human IgG-Fc (1:10,000 dilution in PBS; Jackson Immunoresearch, cat. # 109-035-098) along with 50 μl of incubation buffer was added in each well and the plate was incubated at RT for 2 h. The plate was washed four more times with 400 μl of wash buffer, before addition of 50 μl of 3,3′,5,5′-tetramethylbenzidine (TMB) solution (Thermo Scientific, cat. # 34028) and incubation at RT for 30 min. The reaction was stopped with stopping solution (Sigma, cat. # S5814), and the absorbance at 450 nm was measured using an ELISA reader (Bio-Rad).
Human VEGF was measured using a commercially available sandwich ELISA (R&D systems, cat. # DVE00) following the manufacturer's protocol. One hundred microlitres of tissue supernatant was used for each sample.

To investigate whether IMB-R1 prevented heparin/HS binding to FGFR1, 50 μg/ml heparin was coated onto GAG-binding plates in the standard assay buffer provided (Iduron). After blocked with 0.5 % BSA, the plate was incubated with 2 μg/ml FGFR-Fc in PBS for 2 h at 37 °C. FGFR-Fc bound to heparin was then detected using 0.5–1 μg/ml HRP-conjugated goat anti-human IgG-Fc (Jackson ImmunoResearch Laboratories) for 1 h and visualized as described above.
To examine whether IMB-R1 affected the interaction between FGFRs and FGF2, 5 μg/ml heparin was coated onto the plate (using the standard assay buffer) as a substrate to bind FGF2. The heparin-coated surface was blocked with 0.5 % fish gelatin before 200 ng/ml FGF2 (R&D systems) in PBS was added for 2 h at 37 °C. Next, FGFR-Fc (500 ng/ml) was pre-complexed in PBS with increasing amounts of IMB-R1 (on ice for 2 h). The complex was then applied to the FGF2 surface described above for 1.5 h. The amount of FGFR-Fc bound to FGF2 was determined as described above.

The binding of HK010 to PD-L1 or 4-1BB proteins (human or cynomolgus monkey) and B7 or TNF receptor superfamily proteins was analyzed by enzyme-linked immunosorbent assay (ELISA). B7 or TNF receptor superfamily proteins (human B7-1: B71-HM480; human B7-2: B72-HM486; human B7-H2: BH7-HM472; human B7-H4: BH7-HM174; human B7-H3: BH7-HM173; human CD40: CD4-HM140; human OX40: P43489; human CD27: CD2-HM127) were purchased from Kactus Biosystems (Shanghai). The Nunc Maxisorp plate was coated with 1 µg/ml of proteins in carbonate buffer at 4 °C overnight. After blocking with 1% bovine serum albumin (BSA, Gibco) at 37 °C for 2 h, serially diluted test antibodies were added to each well and incubated at room temperature for 2 h. The wells were washed and incubated for 1 h at 37 °C with horseradish peroxidase (HRP)-conjugated goat anti-human IgG Fc (146460, Jackson). After washing with a phosphate buffered solution containing 0.05% Tween-20 (PBST) solution three times, tetramethylbenzidine (TMB, Invitrogen) was added as a substrate, and the absorbance was detected at 450 nm by VersaMax (Molecular Devices). For the dual-antigen capture ELISA, human PD-L1-His was coated, and HK010 binding was detected using human 4-1BB and HRP-conjugated anti-mouse antibody (115-035-062, Jackson).