As in the high-throughput in vitro neutralization assay, pHIV–ZGP–Fluc was incubated with each mAb for 1 h at 37 °C and then mixed with HEK293T cells in a 96-well plate and incubated for 48 h. The infectivity of pHIV–ZGP–Fluc was determined by measuring the bioluminescence, as described previously36 (link).
The ADCC activities of the mAbs were determined with a new method based on the pseudotyped virus, pHIV–ZGP–Fluc. First, 5,000 CEM cells/well were combined with the pseudotyped virus (MOI = 1), mixed well, and centrifuged at 1,200 × g for 2 h at room temperature. After incubation for 4–6 h, the infected CEM cells were washed thoroughly to remove both dead cells and uninfected virus, and then used as the target cells. MAbs against GP were serially diluted from 100 μg/mL to 10 ng/mL and were then added into each well along with 50,000 Jurkat cells, followed by a 4–6 h killing incubation. Jurkat cells can be stimulated by mAb-recognized target cells, 4–6 h after which they express Fluc. The ADCC activities of the mAbs were measured with the Promega ADCC Reporter Bioassay (Promega, G7102, Madison, WI, USA).
The ADCC activities of the mAbs were determined with a new method based on the pseudotyped virus, pHIV–ZGP–Fluc. First, 5,000 CEM cells/well were combined with the pseudotyped virus (MOI = 1), mixed well, and centrifuged at 1,200 × g for 2 h at room temperature. After incubation for 4–6 h, the infected CEM cells were washed thoroughly to remove both dead cells and uninfected virus, and then used as the target cells. MAbs against GP were serially diluted from 100 μg/mL to 10 ng/mL and were then added into each well along with 50,000 Jurkat cells, followed by a 4–6 h killing incubation. Jurkat cells can be stimulated by mAb-recognized target cells, 4–6 h after which they express Fluc. The ADCC activities of the mAbs were measured with the Promega ADCC Reporter Bioassay (Promega, G7102, Madison, WI, USA).
Free full text: Click here
