Optimal multiplicity of infection (MOI) determination for each pseudotype virus TIMING 1.0 h, then 48-54 hours
1| Select the two strains that are most potently neutralized by the donor’s serum, or alternatively easy-to-neutralize strains, such as HIV-1-BaL and HIV-1-MN.
2| Titer pseudovirus stock in a mock neutralization assay, using medium as a substitute for antibody. In a 384-well plate prepare 11 2-fold and 11 5-fold serial dilutions of each virus in the presence or absence of DEAE-dextran which may facilitate viral entry. Each lot should be optimized typically approximately 7.5 mgs ml-1. Choose a dilution that gives a 40,000 to 100,000 RLU signal that is 50-100 fold above background. See steps 3-13 below.
Incubation of B cell culture supernatant with virus● TIMING 1-2 h
3| Thaw supernatants and dilute virus in complete DMEM using the concentration titered in step 2. A total of 2 viruses are tested one on each of the 2 harvested plates.
4| Robotics: transfer 20 μl virus to each of the 2 plates harvested at step 48 each of which contains 20 μl of supernatant. (one of each pair of plates for each virus).
CRITICAL STEP: when designing the plate plan, set up at least 8 wells with medium instead of antibody (virus-only) and 8 wells with medium instead of antibody and medium instead of virus (cells-only). When using supernatants from B cell culture, all of these wells should use supernatants from culture plate wells that received feeders and cytokines but no B cells.
5| Incubate at 37oC for 45-90 minutes.
Incubation with TZM-bl cells ● TIMING 48-54 h
6| During the incubation, discard the TZM-bl culture media and remove the adherent cells by slowly adding 5 ml of 0.25% Trypsin-EDTA.
7| Incubate at 37°C for 10 minutes.
8| Add 10 ml of complete DMEM and suspend the cells by gentle pipet action.
9| Transfer the suspension to a 50 ml conical tube.
10| Spin, resuspend in complete DMEM, and count the cells.
11| Dilute TZM-bl cells in complete DMEM to a concentration of 1.5 x 105. Add DEAE–dextran to the cell suspension at a lot optimized concentration of approximately 7.5 µg ml-1. Dispense cells to each well of the assay plate at a total of 3x103 cells to each well.
12| Incubate for 48-54 hours at 37°C .
Plate Harvest and Result Evaluation● TIMING 0.5 h
13| Remove 30 μl from each assay well. Lyse the cells with the addition of 30 µl of Britelite Plus substrate.
CRITICAL STEP: Read in a luminometer that is capable of accurately reading 384- well plates. Some models will read 384-well plates but have an unacceptably high level of cross-talk between wells due to the detector configuration. We use a Molecular Devices Paradigm luminometer.
14| To calculate neutralization, first subtract the average signal from the cells-only wells from all wells. Next, average the signal from wells with feeders but no B cells. For each well with antibodies, % neutralization = 100*(Vo-Vn)/Vo where Vn is the RLU in the virus plus antibody wells and V0 is the RLU in the virus only wells.