1. For most applications, a polyvinylchloride (PVC) microtiter plate is best; however, consult manufacturer guidelines to determine the most appropriate type of plate for protein binding.
2. Add 50 μL of diluted primary antibody (capture) to each well. The appropriate dilution should be determined using an 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 hrs. 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:
a. 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).
b. Incubate the plate overnight at 4°C to allow complete binding.
c. Add primary capture antibody (as above).
3. 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. 4. 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
5. Wash wells twice with PBS.
6. 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 or horseradish peroxidase. Do not include sodium azide in buffers or wash solutions, if an HRP-labeled conjugate will be used for detection. 7. 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 hrs. at room temperature in a humid atmosphere.
8. Wash the plate four times with PBS.
9. 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.