1. Dilute the capture antibody to 1 pg/mL in coating buffer pH 9.5. Add 100 pL to each well of a high capacity protein binding 96-well microtiter plate.
2. Seal the plate to avoid evaporation and incubate overnight (12-18 h) at 2-8°C.
3. Wash plate: discard unbound antibody by inverting and flicking the plate over a sink. Fill each well with 300 pL of washing buffer, then discard, ensuring all liquid has been removed by tapping the plate onto clean paper towels. Repeat 3 times.
4. Add 200 pL of blocking buffer to each well. Seal plate and incubate for at least 2 h at room temperature.
5. Discard blocking buffer. Wash plate three times (see step 3).
6. Prepare a titration series of known standards (for example, in 1.5-mL Eppendorf tubes) diluted in a matrix representing that of the samples (e.g., culture medium). Include a negative control (i.e., culture medium only). Transfer samples and antigen standards to the ELISA plate in duplicate at 100 |L per well. Seal plate and incubate at room temperature for at least 2 h (see Note 5), or overnight at 4°C for increased sensitivity.
7. Wash plate three times (see step 3).
8. Dilute biotinylated detection antibody to 1 |g/mL in PBS, pH 7.4. Add 50 |L per well. Incubate at room temperature for 2 h. If problems with non-specific binding of the biotinylated antibody to the plate occur, dilute the antibody in PBS/Tween/1%BSA rather than just PBS.
9. Wash plate three times (see step 3).
10. Dilute streptavidin-HRP according to manufacturers instructions. Add 100 |L per well. Incubate at room temperature for 30 min.
11. Wash plate three times (see step 3).
12. Add 100 |L per well of "one-step" TMB. Allow color to develop between 5-20 min. OD may be monitored at this stage at 650 nm as the color develops.
13. Add 100 |L of 0.5 M H2SO4 to each well to stop the reaction. Read OD at 450 nm.
14. Estimate amount of antigen within samples by comparing ODs to those of known standards (see Note 6).
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