1. Before the assay, both antibody preparations should be purified and one must be labeled.

2. 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.

3. Bind the unlabeled antibody to the bottom of each well by adding approximately 50 μL of antibody solution to each well (20 μg/ml in PBS). 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.

4. Incubate the plate overnight at 4°C to allow complete binding.

5. 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.

6. 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 h 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 antibody will be used for detection.

7. Wash wells twice with PBS.

8. Add 50 μL of the antigen solution to the wells (the antigen solution should be titrated). All dilutions should be done in the blocking buffer (3% BSA/PBS). Incubate for at least 2 h at room temperature in a humid atmosphere.

9. Wash the plate four times with PBS.

10.Add the labeled second antibody. The amount to be added can be determined in preliminary experiments. For accurate quantitation, the second antibody should be used in excess. All dilutions should be done in the blocking buffer.

11.Incubate for 2 h or more at room temperature in a humid atmosphere.

12.Wash with several changes of PBS.

13.Add substrate as indicated by manufacturer. After suggested incubation time has elapsed, optical densities at target wavelengths can be measured on an ELISA plate reader.

Note: Some enzyme substrates are considered hazardous, due to potential carcinogenicity. Handle with care and refer to Material Safety Data Sheets for proper handling precautions.

More ELISA Protocols:  Indirect ELISA Protocol 丨 Direct ELISA Protocol 丨 Competitive ELISA Protocol 丨 ELISPOT Protocol