Hrp labeled rabbit anti dog igg

ELISA conditions were optimized using a checkerboard titration method with the vaccine antigens and sera. The EgTIM, EgANXB3, EgADK1, EgEPC1, and EgFABP-EgA31 purified proteins were diluted in PBS to 5 μg/mL. The ELISA plates were coated with diluted antigen solutions and incubated overnight at 4°C. The plates were subsequently washed three times with PBST and blocked with 5% skim milk for 2 h at 37°C. After washing the wells three times, 100 μL of the serum samples (diluted 1:100 in PBST) were added and incubated at 37°C for 1 h. The HRP-labeled rabbit anti-dog IgG (diluted 1:3000, Solarbio, Beijing, China) was added and incubated for 1.5 h at 37°C. The wells were subsequently washed again and incubated with TMB (Tiangen, Beijing, China) substrate at 37°C for 20 min. Finally, the reaction was stopped with 100 μL of 2 M H₂SO₄, and the optical density was measured at 450 nm.

The enzyme-linked immunosorbent assay (ELISA) conditions were optimized by checkerboard titration of the rEgHCDH antigen and sera. The purified rEgHCDH protein (5 μg/ml) was diluted in 0.1 M carbonate buffer (pH 9.6). The ELISA plates were coated with the diluted antigen solution overnight at 4 °C, then washed with PBST and incubated with 5% skim milk for 2 h at 37 °C. The wells were then washed thoroughly and incubated with 100 μl of serum samples (1:80) in PBST at 37 °C for 1.5 h. After washing, the HRP-labeled rabbit anti-dog IgG (1:3000; Solarbio, Beijing, China) was added to the plates and incubated at 37 °C for 1.5 h, following which the wells were washed again and incubated with the substrate 3, 3′, 5, 5′-tetramethylbenzidine (Tiangen, Beijing, China) at 37 °C for 15 min. Finally, the reaction was stopped with 100 μl of 1 M H₂SO₄ and the optical density at 450 nm was determined.

The standard checkerboard titration procedure was used to assess the optimal concentrations of the rEg-TSP11 antigen and serum. Carbonate buffer (0.1 M, pH 9.6) was used to dilute the purified rEg-TSP11 protein to 5 μg/mL, which was used as the antigen in the ELISA test. The diluted antigen solution was used to coat ELISA plates at 4°C overnight. The next day, the plate was washed with PBS-Tween-20 (PBST) and then incubated with 5% skim milk at 37°C for 2 h. After thorough washing, 100 μL of serum samples (2-fold dilutions, 1:80) in PBST was added to each well and incubated for 1.5 h at 37°C. After washing, HRP-labeled rabbit anti-dog IgG (1:3,000 dilution; Solarbio, Beijing, China) was added to the plates and incubated for 1.5 h at 37°C. After washing, the substrate, 3, 3′, 5, 5′-tetramethylbenzidine (TMB) (Tiangen, Beijing, China) was added to the wells and incubated for 1.5 h at 37°C. Finally, 1 M H₂SO₄ (100 μL) was added to stop the development of color, and the optical density was measured at 450 nm (OD 450).