Microbeta 1450 jet luminometer

To prepare VSVΔGluc pseudotypes, HEK293T cells were transfected with the H and F expression vectors from the respective viruses, followed by superinfection with VSVΔGluc (VSVG) as described previously (53 (link), 54 (link)). Supernatants were harvested 48 h postinfection, divided into aliquots, and frozen at −80°C. The titer of each viral pseudotype stock was estimated by preparing serial dilutions in triplicate and plating onto 293 canine SLAM cells followed by incubation for 48 to 72 h at 37°C, at which time luciferase substrate was added (steadylite plus; Perkin Elmer) and the signal analyzed on a MicroBeta 1450 Jet luminometer (Perkin Elmer). Canine SLAM was used because neither MeV nor PPRV appears to be restricted by this receptor (data not shown). The viral titer (50% tissue culture infectious dose [TCID₅₀]) was calculated using the Spearman-Kärber formula. To measure virus neutralization, 4-fold serum dilutions ranging from 1:8 to 1:32,768 were prepared in triplicate and added to 293 canine SLAM cells in 96-well white flat-bottomed plates, followed by 2.5 × 10³ TCID₅₀ of VSVΔG(F&H) pseudotype. Plates were incubated for 48 h at 37°C, at which time luciferase assays were performed. Antibody titers were calculated by interpolating the point at which there was a 90% reduction in luciferase activity (90% neutralization or 90% inhibitory concentration [EC₉₀]).

2 × 10⁴ 293-dogSLAM cells were plated into each well of a 96-well white flat-bottomed plate (Culturplate-96, Perkin Elmer, Coventry, UK). Four-fold serum dilutions were prepared in triplicate in complete medium ranging from 1:8 to 1:32768. The diluted serum samples were then added to the 293-dogSLAM cells followed by 2.5 × 10³ TCID50 of VSVΔG(CDV) pseudotype. Plates were incubated for 48–72 h at 37 °C, at which time luciferase substrate was added (Steadylite plus™, Perkin Elmer) and the signal analysed on a Microbeta 1450 Jet luminometer (Perkin Elmer). Antibody titres were calculated by interpolating the point at which there was a 90% reduction in luciferase activity (90% neutralisation, inhibitory concentration 90 or IC₉₀) [52] (link).

293T cells were transfected with the H and F expression vectors from the respective morbillivirus, followed by super-infection with VSVΔGluc (VSVG) as described [24] (link), [25] (link). Supernatants were harvested and titred on 293dogSLAM cells [24] (link), [25] (link). Luciferase activity was measured by the addition of Steadylite plus™ (Perkin Elmer) and a Microbeta 1450 Jet luminometer (Perkin Elmer). The viral titre [50% tissue culture infectious dose (TCID₅₀)] was calculated using the Spearman-Kärber formula [29] .