The neutralization activity of antibodies was determined as previously described [43 (link), 58 ]. In brief, 293T cells were transfected with pSG3ΔEnv and Env expression vector, and the supernatant after 48 h of transfection was stored at ˗80 °C. The median tissue culture infectious dose (TCID50) of each pseudovirus was determined using TZM-bl cells. Serially diluted antibody and virus (400 TCID50) were incubated for 1 h, and TZM-bl cells were added. After incubation for 48 h, the galactosidase activity was measured using galactosidase substrate (Tropix Gal-Screen substrate, Applied Biosystems) and an EnSpire Multimode Plate Reader (PerkinElmer, MA, USA). The relative light units (RLU) were compared to calculate the reduction in infectivity and 50% of the maximal inhibitory concentration (IC50) was calculated using nonlinear regression.
Tropix gal screen substrate
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Tropix Gal-Screen substrate is a chemiluminescent detection reagent used for the quantitative analysis of β-galactosidase activity in various biological applications, such as reporter gene assays and enzyme-linked immunosorbent assays (ELISAs).
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Lab products found in correlation
2 protocols using tropix gal screen substrate
1
Antibody Neutralization Assay for Pseudoviruses
2
Neutralization Assay for Antibodies
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The neutralization activity of antibodies was determined as previously described (45, 61) . In brief, 293T cells were transfected with pSG3ΔEnv and Env expression vector, and the supernatant after 48 h of transfection was stored at 80 °C. The median tissue culture infectious dose (TCID 50 ) of each pseudovirus was determined using TZM-bl cells. Serially diluted antibody and virus (400 TCID 50 ) were incubated for 1 h, and TZM-bl cells were added. After incubation for 48 h, the galactosidase activity was measured using galactosidase substrate (Tropix Gal-Screen substrate, Applied Biosystems) and an EnSpire Multimode Plate Reader (PerkinElmer, MA, USA). The relative light units (RLU) were compared to calculate the reduction in infectivity and 50% of the maximal inhibitory concentration (IC 50 ) was calculated using nonlinear regression.
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