Hrp conjugated goat anti human fc antibody

Example 7

The CD47-SIRPα interaction blocking activities of recombinant anti-CD47 antibodies were tested using ELISA assay. ELISA wells (Nunc, Denmark) were coated with 1 μg/ml of recombinant human His-tagged SIRP-α protein (R&D Systems, USA) for 16 hr at 4° C. After blocking, 1 μg/ml of recombinant CD47-Fc protein and 3-fold serially diluted of anti-CD47 antibody starting from 30 nM were added and incubated at room temperature for 1 hr. After incubation, wells were washed and incubated with HRP-conjugated goat anti-human Fc antibody (1:2500 dilution, Jackson ImmunoResearch, USA) for 1 hr. After final washing, the bound CD47-Fc proteins were detected with TMB substrate. The reaction was stopped by adding 1M HCl and OD450 readings were obtained. IC₅₀, the antibody concentration required to inhibit half of max absorbance, was calculated by GraphPad Software for each tested CD47 antibody.

The anti-CD47 antibodies as shown in FIG. 5 were able to block the interaction between recombinant human CD47 and recombinant human SIRP-α. The calculated IC₅₀s for Hu5F9-G4, CwP1A1, CwP1C4, CwP2E8, CwP2F8 and CwP2F12 is 0.063 nM, 0.053 nM, 0.155 nM, 0.556 nM, 0.068 nM and 0.063 nM, respectively. Results from this study indicated CwP1A1, CwP2F8 and CwP2F12 have similar or even better blocking activities than that of Hu5F9-G4.