Spectrafluor plate reader

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 4

In order to test the potency of the antibodies in the blockade of GM-CSF binding to the GM-CSF receptor alpha chain, recombinant human GM-CSF receptor alpha protein (CD116) was coated at 2 μg/ml in PBS onto microtiter plates for over-night at 4° C. 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 in the presence of biotinylated human GM-CSF protein (0.05 μg/ml) and incubated for 1 hr at RT.

For detection of the binding of biotinylated GM-CSF to the coated receptor, the HRP-conjugated Streptavidin was 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. 5, both antibodies showed dose-dependent inhibition of GM-CSF binding to the GM-CSF receptor alpha.

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.