Hrp conjugated secondary antibodies against mouse fc

Example 2

This example tests the dose response of ELISA binding of mouse anti-GM-CSF mAb to recombinant human or rhesus GM-CSF protein (1 μg/ml@100 μl).

Recombinant human or rhesus GM-CSF protein (Genscript) was coated at 1 μg/ml in PBS onto microtiter plates for 2 h at room temperature (RT). After coating of antigen the wells were blocked with PBS/0.05% Tween (PBST) with 1% BSA for 1 h at RT. After washing of the wells with PBST, different concentrations of anti-GM-CSF antibodies were added to the well and incubated for 1 at RT. For detection of the binding antibodies, the HRP-conjugated secondary antibodies against mouse Fc (Jackson Immuno Research) were added, followed by the addition of fluorogenic substrates (Roche). Between all incubation steps, the wells of the plate were washed with PBST three times. Fluorescence was measured in a TECAN Spectrafluor plate reader.

As shown in FIG. 3, both 23F4 and 50C5 antibodies showed dose-dependent binding to human GM-CSF with EC50s of 11.8 ng/ml and 14.6 ng/ml, respectively, and rhesus GM-CSF with EC50s of 10.2 ng/ml and 21.7 ng/ml, respectively.

Example 8

The humanized variants were tested for the binding to recombinant human GM-CSF as previously described. Recombinant human GM-CSF protein (Genscript) was coated at 1 ug/ml in PBS onto microtiter plates for 2 h at room temperature (RT). After coating of antigen the wells were blocked with PBS/0.05% Tween (PBST) with 1% BSA for 1 h at RT. After washing of the wells with PBST, different concentrations of anti-GM-CSF humanized antibodies were added to the well and incubated for 1 at RT.

For detection of the binding antibodies, the HRP-conjugated secondary antibodies against mouse Fc (Jackson Immuno Research) were added, followed by the addition of fluorogenic substrates (Roche). Between all incubation steps, the wells of the plate were washed with PBST three times. Fluorescence was measured in a TECAN Spectrafluor plate reader. As shown in FIG. 8, all the humanized variants demonstrated a similar binding potency against human GM-CSF as compared with chimeric antibody.

Example 12

Recombinant rhesus GM-CSF protein (Genscript) was coated at 1 ug/ml in PBS onto microtiter plates for 2 h at room temperature (RT). After coating of antigen, the wells were blocked with PBS/0.05% Tween (PBST) with 1% BSA for 1 h at RT. After washing of the wells with PBST, different concentrations of humanized anti-GM-CSF antibodies were added to the well and incubated for 1 at RT. For detection of the binding antibodies, the HRP-conjugated secondary antibodies against mouse Fc (Jackson Immuno Research) were added, followed by the addition of fluorogenic substrates (Roche). Between all incubation steps, the wells of the plate were washed with PBST three times. Fluorescence was measured in a TECAN Spectrafluor plate reader. As shown in FIG. 11, Hu23F4-13, Hu23F4-27 and Hu23F4-36 showed a dose-dependent binding to rhesus GM-CSF with an EC50 of 7.44 ng/ml, 6.25 ng/ml and 7.75 ng/ml, respectively; Hu50C5-17 and Hu50C5-23 showed a dose-dependent binding to rhesus GM-CSF with an EC50 of 18.86 ng/ml and 21.63 ng/ml, respectively.