Cell-based binding assay
Vero E6 cells were grown to a confluent layer in wells of 48-well plate (NUNC). The CHO transfected cells were labeled with Hoechst 33258 at 2 μM for 5 min, rinsed three times with PBS, and then suspended after incubation with 0.025% trypsin in 0.02% EDTA. 1 × 105 cells suspended in 500 μL were gently laid onto the Vero cell layer in at least triplicate wells. After 2-h incubation at 37°C, wells were gently rinsed with a culture medium three times, and cells were fixed by a 10-min incubation with 2% formaldehyde. Adherent CHO cells were counted under an epifluorescence microscope (Zeiss, Jena, Germany) with a 10× objective. Three to six fields/well were counted and results expressed as the mean number of cells per field. In the antibody blocking experiments, monoclonal antibodies or human plasma samples were added to the transfected CHO cells suspension before seeding on the Vero cell layer.