Determination of specific anti-Mycobacterium tuberculosis complex IgY by indirect ELISA

In brief, wells of Microtiter plates were coated with 100 μl of antigen solution appropriately diluted with 0.05 M carbonate buffer (pH 9.6). After overnight incubation at 4°C, the plates were washed, and 200 μ1 of PBS (pH 7.4) containing bovine serum albumin (1% in PBS) was added to the wells to block the uncoated surface. After being blocked, each well was washed three times with 200 μL of PBS-Tween (PBS-T; 0.85% NaCl - 0.01 M phosphate buffer, pH 7.2) (containing 0.05% Tween 20), and IgY from immunized hens at different time intervals was applied to the well in triplicate for reaction with the antigen for 2 h at 37°C. After each well was washed again with 200 μL of PBS-T, 100 μL of horseradish peroxidase-conjugated rabbit anti-chicken IgG (Sigma Chemical Co.) diluted (1:1000) with PBS-T was added to each well, and the plate was incubated at 37°C for 2 h. Each well was washed again with 200 μL of PBS-T, and then 100 μL of TMB solution with H₂O₂. The reaction was stopped after 20 min with 4N H2SO4 (50 μl per well), and the intensity of color developed was measured at 490 nm with a microplate reader. IgY anti-TBC titer was defined as the maximum dilution multiple of the sample with an OD. Crude IgY from nonimmunized hens was used as the control.