Blood samples were taken by puncture of the saphenous vein. Serum was obtained by centrifugation at 17,000 × g for 10 min, and then the supernatant was transferred to a new microcentrifuge tube and centrifuged again at 17,000 × g for 5 min. Sera were stored at −20°C.
For ELISA, 96-well microtiter plates (Costar 3590, Corning, NY, USA) were coated with one of the following recombinant influenza HA proteins: A/Puerto Rico/8/1934(H1N1) (cat. no. 11684-V08H, Sino Biological), A/Viet Nam/1194/2004(H5N1) (cat. no. 11062-V08H1, Sino Biological), or A/Hong Kong/1073/99(H9N2) (cat. no. 11229-V08H, Sino Biological) (0.5 μg/mL). Plates were blocked by 1% BSA in PBS at room temperature for 1 h. Serially diluted sera from individual mice were then incubated at 4°C for 16 to 18 h. Murine IgG was detected by incubation for 1 h with either horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Fc specific) (1:5,000; cat. no. A2554, Merck, New Jersey, USA), biotinylated anti-mouse IgG1[a] (1:500; cat. no. 553500, BD Biosciences, New Jersey, USA), or biotinylated anti-mouse IgG2a[a] (1:500; cat. no. 553504, BD Biosciences). For biotinylated antibodies, a secondary incubation with HRP-conjugated streptavidin (1:5,000; cat.no. 7105-05, SouthernBiotech, Alabama, USA) at room temperature for 30 min was performed. The plates were developed by addition of 3,3′,5,5′-tetramethylbenzidine (TMB) solution (cat. no. CL07-1000ML, Merck), and the reaction was stopped after 10 min by addition of 1 M H2SO4. Absorbance at 450 nm was read using a Wallac EnVision 2104 Multilabel Reader (PerkinElmer, Massachusetts, USA).
For ELISA, 96-well microtiter plates (Costar 3590, Corning, NY, USA) were coated with one of the following recombinant influenza HA proteins: A/Puerto Rico/8/1934(H1N1) (cat. no. 11684-V08H, Sino Biological), A/Viet Nam/1194/2004(H5N1) (cat. no. 11062-V08H1, Sino Biological), or A/Hong Kong/1073/99(H9N2) (cat. no. 11229-V08H, Sino Biological) (0.5 μg/mL). Plates were blocked by 1% BSA in PBS at room temperature for 1 h. Serially diluted sera from individual mice were then incubated at 4°C for 16 to 18 h. Murine IgG was detected by incubation for 1 h with either horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Fc specific) (1:5,000; cat. no. A2554, Merck, New Jersey, USA), biotinylated anti-mouse IgG1[a] (1:500; cat. no. 553500, BD Biosciences, New Jersey, USA), or biotinylated anti-mouse IgG2a[a] (1:500; cat. no. 553504, BD Biosciences). For biotinylated antibodies, a secondary incubation with HRP-conjugated streptavidin (1:5,000; cat.no. 7105-05, SouthernBiotech, Alabama, USA) at room temperature for 30 min was performed. The plates were developed by addition of 3,3′,5,5′-tetramethylbenzidine (TMB) solution (cat. no. CL07-1000ML, Merck), and the reaction was stopped after 10 min by addition of 1 M H2SO4. Absorbance at 450 nm was read using a Wallac EnVision 2104 Multilabel Reader (PerkinElmer, Massachusetts, USA).
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