Section11:Development of a Competitive Immunoassay

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Initial Development Experiment

Goal: Determine the optimal coating concentration of the antibody used for capture and the labeled ligand.

Reagents:

1. Antibody- mono or polyclonal, specific to the analyte.
2. Buffers- same as for a competitive assay.
3. Labeled ligand-the enzyme or biotin is labeled directly to the analyte or ligand.

Experiment: Coat the ELISA plate with various antibody concentrations to determine the optimal concentration of antibody and labeled ligand.

Protocol:

1. Determine the desired analyte working range.
2. Titrate capture antibody using high, low and zero analyte concentration levels.
3. Dilute the capture antibody in coating buffer at 0.1, 0.5, 1 and 2 mg/ml and add 100 µl to each well of the 96-well microtiter plate. The capture antibody may need to be titrated down further. The amount of antibody coated on the plate will be proportional to the sensitivity of the assay.
4. Incubate the plate containing the primary capture antibody overnight at 4°C and use the next day.
5. Stability of the primary capture antibody bound to the plate can be determined in later experiments.
6. Remove the primary coating antibody solution from the microtiter plates by aspirating or dumping the plate.
7. Add 200 µl of blocking buffer to each well of the 96-well microtiter plate.
8. Incubate the plate for one hour at RT.
9. Remove the blocking buffer from the plate by aspirating or dumping the plate.
10. Dilute the labeled standard in antibody dilution buffer over a wide range. The desired result is the condition, which gives a readable signal with the least amount of antibody coated, in combination with the least amount of labeled standard.
11. Zero concentration will give you the NSB.
12. Add 100 µl of the labeled standard to each well in the microtiter plate and incubate for 2.5 hours at RT. (The standard can either be directly labeled with the enzyme or biotinylated).
13. Wash the plates 3 times with wash buffer.
14. If a biotinylated standard is used, Dilute streptavidin-HRP according to manufacturer’s instructions in antibody diluent and add 100 µl to each well in the microtiter plate and incubate for 1 hr at RT.
15. For HRP readout add either OPD or TMB as substrate to allow color development and incubate for 10-20 minutes at RT.
16. Add acid stop reagent to stop the enzyme reaction.
17. Read at 405 nm for TMB/HRP.
18. Determine the linearity of the instrument being used for the readout the same way as described for a sandwich ELISA.

Second Development Experiment

Goal: Determine the potential dynamic range and sensitivity. Take the conditions established in the initial experiment for the concentration of the antibody and labeled ligand and incubate with a wide range of unlabeled analyte. The resulting standard curve and precision profile calculation will give an estimate of the sensitivity and dynamic range of the assay.

Reagents: Reagents are the same as in initial experiment.

Protocol:

1. Dilute the capture antibody in coating buffer at the concentration determined in the initial experiment. Add 100 µl to each well of the 96-well microtiter plate.
2. Incubate the plate containing the primary capture antibody overnight at 4°C and use the next day.
3. Remove the primary capture antibody solution from the microtiter plates by aspirating or dumping the plate.
4. Add 200 µl of blocking buffer to each well of the 96-well microtiter plate.
5. Incubate the plate for one hour at RT.
6. Remove the blocking buffer from the plate by aspirating or dumping the plate.
7. Dilute the labeled standard in antibody dilution buffer at the concentration determined in the initial experiment.
8. Dilute the unlabeled ligand in antibody dilution buffer over a wide range of concentrations.
9. Add 100 µl of the labeled standard to each well in the microtiter plate and 100 µl of the various dilution of the unlabeled ligand. Incubate for 2.5 hours at RT. This is the competitive part of the assay and will allow for the competition between the labeled and unlabeled ligand to compete for the sites on the antibody.
10. Wash the plates 3 times with wash buffer.
11. If a biotinylated standard is used, Dilute streptavidin-HRP according to manufacturer’s instructions in antibody diluent and add 100 µl to each well in the microtiter plate and incubate for 1 hr at RT.
12. For HRP readout add either OPD or TMB as substrate to allow color development and incubate for 10-20 minutes at RT.
13. Add acid stop reagent to stop the enzyme reaction.
14. Read at 405 nm for TMB/HRP.

Third Development Experiment

Goal: Determine the optimal buffers, incubation periods, temperatures, matrix effects, and other variables that may affect the assay.

Reagents: Reagents are the same as in initial experiment.

Protocol: Same as in previous experiment except for the following changes at steps 8 and 9:

• Dilute the unlabeled ligand in antibody dilution buffer, and the matrix appropriate for the experiment, over a wide range of concentrations. Again the dilution buffer can be varied here according to the experimental design.
• Add 100 µl of the labeled standard to each well in the microtiter plate and 100 µl of the various dilution of the unlabeled ligand. Incubate for 2.5 hours at RT. This incubation time can be varied for longer and shorter periods of time to potentially increase the sensitivity and dynamic range of the assay.

Results: Analysis of the results is by use of JMP or other appropriate statistical software to determine the optimal conditions for incubation timing, buffers for dilution, and matrix effects.