Competitive ELISA Assay

For small molecule analytes, typically less than 10 kDa, the competitive ELISA is used. An advantage of this assay over the Immunometric ELISA is that non-purified primary antibodies may be used. Although there are several different configurations for competitive ELISAs, below is an example for one such configuration. In order to utilize a competitive ELISA, one reagent must be conjugated to a detection enzyme, such as horseradish peroxidase. The enzyme may be linked to either the immunogen or the primary antibody.

The protocol below uses a labeled antigen as the competitor. In this procedure, the plate is coated with a generic antibody that will capture your antigen-specific antibody. That is, if you have a mouse monoclonal against your target antigen, you would coat the plate with an anti-mouse antibody. The antigen conjugate will compete with the antigen in your standards and samples for binding to the specific antibody. In order to optimize the assay, both the antibody and conjugate will have to be titrated using a checkerboard titration.

 

Before the assay, an enzyme conjugate of the target antigen must be made. There are various conjugation chemistries available, consult the literature to find a chemistry that is appropriate for your molecule. For most applications, a polystyrene microtiter plate is best. However, consult manufacturer guidelines to determine the most appropriate type of plate for protein binding.

1. Add 50 µl of diluted generic antibody (capture) to each well. Allow to incubate for 4 hours at room temperature or 4°C overnight.
2. Wash the wells twice with PBS. A 500 ml squirt bottle is convenient for this purpose. The antibody solution washes can be removed by flicking the plate over a suitable container.
3. 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 4 hrs. to overnight in a humid atmosphere at room temperature.
   Note:Sodium azide is an inhibitor or horseradish peroxidase. Do not include sodium azide in buffers or wash solutions, if an HRP-labeled conjugate will be used for detection. Always use ELISA grade BSA.
4. Wash wells twice with PBS.
5. 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).
6. Add 50 µl antibody solution to the wells. All dilutions should be done in the Blocking Buffer (3% BSA/PBS with 0.05% Tween-20).
7. Add 50 µl enzyme conjugate to the wells.
8. Incubate the plate for 2 hours at room temperature in a humid atmosphere.
9. Wash the plate 4 times with PBS.
10. 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 degree of color change.

Volumes and incubation times may need to be changed to ensure optimal binding for each assay.