Hrp conjugated sheep anti mouse igg secondary antibodies

Serum antibody titer was measured by enzyme-linked immunosorbent assay (ELISA) as in our previous report^{25 (link)}. In brief, 96-well ELISA plates were coated with 2 µg/ml rHA at 4 °C overnight. After blocking with 5% non-fat milk, two-fold serial dilutions of immune sera were added and incubated at room temperature for 90 min. After washing in PBS supplemented with 0.05% Tween 20 (PBST), horseradish peroxidase (HRP)-conjugated sheep anti-mouse IgG secondary antibodies (1:2500, NA931, GE Healthcare Life Sciences) were added and incubated at room temperature for 1 h. After washing in PBST, TMB substrates were added and reactions were then stopped by addition of 3 N H₂SO₄. Optical absorbance (OD_450nm) was determined using a microplate reader (Molecular Device). Serum antibody titer was defined as the reciprocal dilution factor that resulted in OD_450nm ~ 3 times higher than the background values.

Serum antibody titer was measured by enzyme-linked immunosorbent assay (ELISA). In brief, vaccine antigens (1–10 µg/ml) were coated into 96-well ELISA plates at 4˚C overnight. After blocking with 5% non-fat milk, 2-serial dilutions of immune sera were added and incubated at room temperature for 90 min. After washing in phosphate-buffered saline (PBS) supplemented with 0.05% Tween 20 (PBST), horse-radish peroxidase (HRP)-conjugated sheep anti-mouse IgG secondary antibodies (1:5000, NA931, GE Healthcare Life Sciences) were added and incubated at room temperature for 1 h. After washing in PBST, TMB substrates were added and reactions were then stopped by addition of 3 M H₂SO₄. Optical absorbance (OD450/490 nm) was read in a microplate reader (Molecular Device). Serum antibody titer was defined as the reciprocal dilution factor that resulted in OD450/490 nm that was ~ 3 times higher than the background values. For detection of subtype antibody titer, HRP-conjugated anti-mouse IgG1 and IgG2a or IgG2c secondary antibodies were used.

Serum antibody titer was measured by enzyme-linked immunosorbent assay (ELISA) as in our previous report [19 (link)]. In detail, ELISA plates were coated with NP or M1 (0.5 μg/mL) at 4 °C overnight. After blocking with 5% non-fat milk, 2-serial dilutions of immune sera were added and incubated at room temperature for 90 min. After washing in PBS supplemented with 0.05% Tween 20 (PBST), horseradish peroxidase (HRP)-conjugated sheep anti-mouse IgG secondary antibodies (1:2500, NA931, GE Healthcare Life Sciences) were added and incubated at room temperature for 1 h. After washing in PBST, 1-step ultra TMB substrates (34028, Thermo Scientific) were added and reactions were then stopped by addition of 1M H₂SO₄. Optical absorbance (OD_450nm) was read in a microplate reader (Molecular Devices). Serum antibody titer was defined as the reciprocal dilution factor that resulted in OD_450nm that was ~3 times higher than the background values. For detection of subtype antibody titer, HRP-conjugated anti-mouse IgG1 (046120, Invitrogen, Thermo Fisher Scientific) and IgG2c (A90136P, Bethyl Laboratories) secondary antibodies were used.