Detection of specific antibodies by the indirect enzyme-linked immunosorbent assay (iELISA)

The titers or capacities of antisera against rOmpF and bacteria (E. coli CVCC 1515, E. coli CICC 21530, S. dysenteriae CMCC 51252, S. flexneri CMCC 51571, S. enteritidis CVCC 3377, S. pullorum CVCC 503, P. aeruginosa CICC 21630, and P. aeruginosa CICC 10419) were measured by iELISA (Guan et al. 2015; Hu et al. 2010). rOmpF was dissolved in coating buffer (pH 9.6, 0.015 M sodium carbonate, 0.035 M sodium bicarbonate). The 96-well plates were coated with 2 μg/mL of the rOmpF solution or 10⁶ CFU/mL bacteria solution (each well 100 μL), incubated overnight at 4 °C, and washed four times with 0.01 M PBS (containing 0.05% Tween 20). The plates were blocked for 2 h at 37 °C by adding 0.01 M PBS (containing 5% BSA), washed three times, and then incubated with serial dilutions of mice serum at 37 °C for 1.5 h. After washing as above, 100 μL of horseradish peroxidase (HRP) conjugated goat anti-mouse IgG (1:5000) was added into each well and incubated for 30 min at 37 °C. The plates were washed three times again. 100 μL of 3, 3′, 5, 5′-tetramethylbenzidine (TMB) was added to each well and incubated for 20 min in the dark at room temperature. Finally, the color reaction was stopped by adding 2 M H₂SO₄ (50 μL/well). The absorbance of each well at 450 nm was determined by an automatic ELISA plate reader (Perlong Medical, Beijing). The result was considered as positive when the ratio of the test group and negative control group was greater than 2.1 (Lunin et al. 2009; Xu et al. 2011).