1. Dilute the capture antibody to 1 ^g/mL in coating buffer pH 9.0. Add 100 ^L 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 (see Note 6).
3. Wash plate: discard unbound antibody by inverting and flicking the plate over a sink. Fill each well with washing buffer and leave for a couple of seconds before discarding once more. Ensure all liquid has been removed between each wash by repeatedly tapping the plate onto clean paper towels. Repeat the washing process three times to ensure that all unbound antibody is removed from the plate.
4. Add 200 |L of blocking buffer to each well. Seal plate and incubate for 1 h at room temperature.
5. Discard blocking buffer. Wash plate three times (see step 3). (Optimization of the concentration of the enzyme-conjugated antigen is required for initially establishing the competitive ELISA; see Subheading 3.2.).
6. Prepare a 2X solution of the conjugated antigen (having previously optimized this antigen concentration to result in a final OD reading approx 90% of the maximal obtained).
7. Prepare a titration series of known unconjugated standards (for example, in 1.5 mL of polypropylene tubes) diluted in a matrix representing that of the samples (e.g., culture medium or human serum). For this example the antigen is conjugated with biotin (see Note 7) which is then detected using streptavidin-HRP. Include a negative control (i.e., culture medium only). Incubate the samples and standards with the conjugated antigen. Mix well with at a 1:1 ratio with the 2X conjugated antigen. Transfer to the ELISA plate in triplicate at 100 |L per well.
8. Seal plate and incubate at room temperature for 3-4 h or overnight at 4°C for increased sensitivity.
9. Wash plate three times (see step 3).
10. Dilute streptavidin-HRP according to manufacturer's instructions. Add 100 |L per well. Incubate at room temperature for 30 min.
11. Wash plate four times (see step 3).
12. Add 100 |L per well of "one-step" TMB. Allow color to develop between 5 and 60 min (10 min is usually sufficient). 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.
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