Alkaline phosphatase goat anti rabbit igg h l

For the plaque reduction neutralization test (PRNT), heat-inactivated serum was diluted 4-fold and incubated with RSV-A2 virus (diluted to yield 20–50 plaques/well) containing 10% guinea pig complement (Rockland Immunochemical; Philadelphia, PA, USA) and incubated for 1 h at 37°C. After incubation, 100 μl of the antibody-virus mixtures were inoculated in duplicate onto A549 monolayers in 48-well plates and incubated for 1 h at 37°C. Inoculum was removed prior to adding infection medium containing 0.8% methylcellulose. Plates were incubated for 5 to 7 days at which time the overlay medium was removed and cell monolayers fixed with 100% methanol; plaques were detected by immunostaining with rabbit RSV polyclonal anti-F sera (14), followed by addition of alkaline phosphatase goat anti-rabbit IgG (H+L) (Jackson) antibody. The reactions were developed by using Vector Black Alkaline Phosphatase (AP) substrate kit (Vector Labs, Burlingame, CA). Numbers of plaques were counted per well and the neutralization titers were calculated by adding a trend line to the neutralization curves and using the following formula to calculate 50% endpoints: antilog of [(50+y-intercept)/slope].

For the plaque reduction neutralization test (PRNT), heat-inactivated serum was diluted 4-fold and incubated with RSV-A2 virus (diluted to yield 20-50 plaques/well) containing 10% guinea pig complement (Rockland Immunochemical; Philadelphia, PA, USA) and incubated for 1 h at 37 °C. After incubation, 100 μl of the antibody-virus mixtures were inoculated in duplicate onto A549 monolayers in 48-well plates and incubated for 1 h at 37 °C. The inoculum was removed prior to adding the infection medium containing 0.8% methylcellulose. Plates were incubated for 5 to 7 days at which time the overlay medium was removed and cell monolayers fixed with 100% methanol; plaques were detected by immunostaining with rabbit RSV polyclonal anti-F sera, followed by addition of alkaline phosphatase goat anti-rabbit IgG (H + L) (Jackson, 111-055-144) antibody. The reactions were developed by using Vector Black Alkaline Phosphatase (AP) substrate kit (Vector Labs, Burlingame, CA). Numbers of plaques were counted per well and the neutralization titers were calculated by adding a trend line to the neutralization curves and using the following formula to calculate 50% endpoints: antilog of [(50+y-intercept)/slope].