Hrp conjugated goat anti mouse iga

iELISA was performed to measure PCV-2-specific IgG levels in serum and IgA concentrations in saliva as well as lung and intestinal lavage (mucosal) samples [29 ]. In brief, 96-well ELISA plates (Corning, USA) were coated with 0.2 µg PCV-2 antigen in 0.05 M carbonate buffer (pH 9.6) per well and incubated overnight at 4℃. After blocking with 5% skimmed milk (Bio-Rad, USA) in PBS plus 0.5% Tween 20 (PBST) at 37℃ for 1 h, 100 µL of serum sample diluted 200-fold or undiluted mucosal samples were added to each well and the plates were incubated at 37℃ for 1 h. The plates were then washed three times with PBST to remove unbound antibodies. Horseradish peroxidase (HRP)-conjugated sheep anti-mouse IgG (1 : 2,000; Baosen Biotechnology, China) or HRP-conjugated goat anti-mouse IgA (1 : 10,000; Bethyl Laboratories, USA) was subsequently added to each well. After incubating at 37℃ for 1 h, the plates were washed three times and developed with 3,3',5,5'-tetramethylbenzidine (TMB; Sigma, USA) for 15 min. The reaction was stopped by adding 1 M H2SO4, and the optical density (OD) was measured at a wavelength of 450 nm using a Thermo plate reader (Thermo Scientific, USA).