Goat anti mouse peroxidase conjugated antibody

Antigen capture ELISA was performed as previously described with some modifications (15 (link), 16 (link)). Rabbit immune sera raised against the homologous virus was used as capture antibody. Immulon II plates (VWR, PA, USA) were coated in carbonate-bicarbonate buffer (50 mM sodium carbonate, 50 mM sodium bicarbonate, pH 9.6; Thermo Fisher, MA, USA) and incubated overnight at 4°C. Nonspecific binding sites were blocked with StartingBlock (200 μL/well; Thermo Fisher, MA, USA) before cell culture supernatant was added to the plates (50 μL/well) and incubated for 2 h at 37°C, after which plates were washed five times with PBS/0.1% Tween wash buffer with an automatic plate washer. Virus-specific MHIAF diluted in PBS was mixed with 5% nonfat dry milk (Thermo Fisher, MA, USA) in PBS (50 μL/well) and was incubated on the plate for 1 h at 37°C. Plates were washed again, and peroxidase-conjugated goat anti-mouse antibody (Jackson ImmunoResearch, PA, USA) diluted 1:8,000 in 5% nonfat milk/PBS (50 μL/well) was incubated at 37°C for 1 h. After a final wash, 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (KPL, MA, USA) was added (100 μL/well) and incubated for 10 min at room temperature. The reaction was stopped with 2 N H₂SO₄ (50 μL/well; VWR, PA, USA), and optical density (OD) was measured as a ratio of 450/630 nm.