Hrp labeled goat anti human igg fab

Example 10

The binding of purified hAb21 (IgG4-kappa) to recombinant FcR receptor (such as FcγRI, CD64) protein coated on the 96-well plate can be detected by direct ELISA, and the results are compared with two other IgG4 type McAbs (Nivolumab and MK3475) and three IgG1 type McAbs (Avastin, hPV19 and Eribitux).

The basic procedures for this ELISA detection are as follows:

Add a serial diluted samples of IgG4 type McAbs (hAb21, Nivolumab and MK3475) or IgG1 type McAbs (Avastin, hPV19 and Eribitux) into the 96-well plate pre-coated with recombinant human FcγRI receptor proteins (CD64, a product from Sino Biological Inc.). After 2-hour incubation at 37° C. and washing, added HRP-labeled goat-anti-human-IgG-Fab (a product of Sigma) into each well. After another 1-hour incubation at 37° C. and washing, added the OPD substrate into each well for color development.

FIG. 7 is the representative ELISA results. As shown in the Figure, compared with three IgG1 McAbs, (Avastin, hPV19 and Eribitux), the binding activity of three IgG4 McAbs (hAb21, Nivolumab and MK3475) to the FcγRI acceptor (CD64) is significantly reduced, which is in line with the forecast.

Example 11

The binding of purified hAb21, Nivolumab and MK3475 to PD-1 protein and other related proteins can be detected by ELISA.

The basic procedures for the ELISA method are as follows:

Add a serial diluted samples of PD-1 McAbs (hAb21, Nivolumab and MK3475) or un-related hPV19 into 96-wells plate pre-coated with recombinant PD1-Fc or other immunity related gene-Fc fusion proteins (including CD28, B7, CTLA4, CD3, PD-L1, PD-L2, BTLA, etc). After 2-hour incubation at 37° C. and washing, added HRP-labeled goat-anti-human-IgG-Fab (a product of Sigma) into each well; after 1-hour incubation at 37° C. and washing, added the OPD substrate into each well for color development.

FIG. 8 is the representative ELISA results. As shown in the figure, similar to Nivolumab and MK3475, hAb21 only binds to human PD-1 protein and does not bind other immunity related proteins such as CD28, B7, CTLA4, CD3, PD-L1, PD-L2 and BTLA.