Development of cELISA

Optimized dilution of PCV2 VLP antigen and horseradish peroxidase-conjugated PCV2-specific MAb were established by systematic checkerboard titrations. The polystyrene microliter ELISA plates were coated with 1.6 μg PCV2 VLP in phosphate buffer (0.02 mol/L, pH7.4) at 4 °C for 16 ~ 24 h. After three times washing, the plates were blocked with 200 μl of 20 % calf bovine serum in phosphate buffer (0.02 mol/L, pH7.4) for 2 h at 37 °C. After three times washing, 50 μl of the serum samples were added to each well, then 50 μl of 1:2000 dilution MAb 3H11 conjugated with HRP (Sigma, USA) were added to the wells except the blank well. The plates were incubated at 37 °C for 30 min. After five times washing, 100 μl of the substrate solution (0.2 mg/ml of TMB and 0.2 % H₂O₂ in 0.05 mol/L citrate buffer, pH4.6) was added and the colorimetric reaction was developed at 37 °C for 15 min. The reaction was stopped by adding 50 μl of 2 mol/L sulphuric acid. The optical density (OD) was measured at 450 nm. The controls included positive control (in triplicate), negative control (in triple) and one blank control. The OD₄₅₀ of the samples were converted to a percent inhibition (PI) value using the following formulation: PI (%) = (OD₄₅₀ value of negative value − OD₄₅₀ value of sample)/OD₄₅₀ value of negative value × 100 %.