Alkaline phosphatase conjugated goat anti mouse igg secondary antibody

Serum was collected at D11 and D31 post vaccination. HA-specific antibodies in the sera from individual mice were quantified by ELISA assays. Plates (Corning Costar, Tewksbury, MA, USA) were coated with 200 ng of purified HA protein from influenza B/Brisbane/60/08. Wells were rinsed with PBS, incubated with blocking buffer (3% BSA in PBS), and then diluted serum samples (in 0.5% BSA–PBS) were added to the plates and incubated for 2 h at room temperature. The wells were washed and incubated sequentially with 100 μL/well alkaline phosphatase conjugated goat anti-mouse IgG secondary antibody (SouthernBiotech, Birmingham, AL, USA) and one p-nitrophenyl phosphate substrate (Sigma, Burlington, MA, USA). After washing, absorbance at 405 nm was read.

C3H female mice, 6–8 weeks old, were purchased from Jackson Laboratories. All mice were maintained in the pathogen-free facility at the University of Rochester according to institutional guidelines.
Mice were immunized subcutaneously in the hind footpads with 50 μl of Fluzone 2014–15 formula or Flucelvax 2015–16 emulsified in alum (Invivogen). Influenza vaccine was dialyzed and concentrated, and each mouse was immunized with 3 μg HA. At 13 days postimmunization, serum was collected. NA-, M1-, and NP-specific antibodies in the sera from individual mice were quantified by ELISA assays. Plates (Costar) were coated with 200 ng of purified protein. Wells were rinsed with PBS, incubated with blocking buffer (3% BSA in PBS), and then diluted serum samples (in 0.5% BSA–PBS) were added to the plates and incubated for 2–3 h at RT. The wells were washed and incubated sequentially with 100 μl/well alkaline phosphatase-conjugated goat anti-mouse IgG secondary antibody (SouthernBiotech) and p-nitrophenyl phosphate substrate. After washing, absorbance at 405 nm was read.