General Protocol for the Competitive ELISA Method
- For most applications, a polystyrene microtiter plate is best; however, consult manufacturer guidelines to determine the most appropriate type of plate for protein binding.
- Add 50 µL of diluted primary antibody (capture) to each well. The appropriate dilution should be determined using a checkerboard titration prior to testing samples. PVC will bind approximately 100 ng/well (300 ng/cm2). The amount of antibody used will depend on the individual assay, but if maximal binding is required, use at least 1 µg/well. This is well above the capacity of the well, but the binding will occur more rapidly, and the binding solution can be saved and used again. Allow to incubate for 4 hours. at room temperature or 4°C overnight. Note: If a purified capture antibody is not available, the plate should first be coated with a purified secondary antibody directed against the host of the capture antibody according to the following procedure: (1) Bind the unlabeled secondary antibody to the bottom of each well by adding approximately 50 µL of antibody solution to each well (20 µg/mL in PBS). (2) Incubate the plate overnight at 4°C to allow complete binding. (3) Add primary capture antibody (as above).
- Wash the wells twice with PBS. A 500 mL squirt bottle is convenient. The antibody solution washes can be removed by flicking the plate over a suitable container.
- The remaining sites for protein binding on the microtiter plate must be saturated by incubating with blocking buffer. Fill the wells to the top with 3% BSA/PBS with 0.02% sodium azide. Incubate for 2 hrs to overnight in a humid atmosphere at room temperature.
- Wash wells twice with PBS.
- Add 50 µL of the standards or sample solution to the wells. All dilutions should be done in the blocking buffer (3% BSA/PBS with 0.05% Tween-20).
Note: Sodium azide is an inhibitor of horseradish peroxidase. Do not include sodium azide in buffers or wash solutions, if an HRP-labeled conjugate will be used for detection.
- Add 50 µL of the antigen-conjugate solution to the wells (the antigen solution should be titrated). All dilutions should be done in the blocking buffer (3% BSA/PBS with 0.05% Tween-20). Incubate for at least 2 hours. at room temperature in a humid atmosphere.
- Wash the plate four times with PBS
- Add substrate as indicated by manufacturer. After suggested incubation time has elapsed, optical densities at target wavelengths can be measured on an ELISA reader.
Note: Competitive ELISAs yield an inverse curve, where higher values of antigen in the samples or standards yield a lower amount of color change.
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