mAb competition assay

mAb competition assay

1. Incubate 100 ul of 100 nM test IgG with 2 ul of goat anti-human IgG capture beads (Spherotech Goat anti-Human IgG beads, catalog # HUP-60-5) for 20 minutes at room temperature. Prepare in a 96-well v-bottom plate.
2. Centrifuge beads at 2400 x g for 3 min to pellet and remove supernatant.
3. Wash beads with 200 ul of PBS+0.1% BSA, centrifuge beads and remove supernatant.
4. Pre-incubate 50 nM biotinylated Gn H/Gc with 1 uM competitor F(ab) for 30 minutes at room temperature in a total volume of 50 ul and then combine with the IgG-loaded beads.
5. Incubate for 5 minutes at room temperature.
6. Wash beads by adding 200 ul of PBS+0.1% BSA, centrifuge, and remove supernatant.
7. Repeat wash.
8. Resuspend beads in 50 ul of streptavidin-Alexa Fluor 633 (Life Technologies) diluted 1:500 in PBS+0.1% BSA. Incubate for 20 minutes on ice. IgG loading on the bead can be detected by including anti-LC-FITC (Southern Biotech catalog #2062-02) diluted 1:100 into the staining mix.
9. Wash beads with 200 ul of PBS+0.1% BSA, centrifuge beads and remove supernatant.
10. Repeat wash.
11. Resuspend beads in 100 ul of PBS+0.1% BSA and analyze by flow cytometry using a FACSCanto II (BD Biosciences) or comparable instrument.
12. Assess competition by measuring the fold reduction in PUUV Gn H/Gc binding in the presence of competitor F(ab) relative to an antigen-only control. Antibodies were considered competitors when a greater than five-fold reduction was observed in the presence of control F(ab) relative to an antigen-only control.

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