Online Inquiry

Protocol for Indirect ELISA

What is Indirect ELISA?

Indirect ELISA (Enzyme-Linked Immunosorbent Assay) is an immunological technique used to detect the presence and quantity of a specific antibody in a sample. The process involves coating a solid surface such as a microplate with the antigen of interest. The sample containing the antibody is then added to the coated surface, where it binds to the antigen.

Next, a secondary antibody labeled with an enzyme and specific to the primary antibody is added to the surface. The secondary antibody binds to the primary antibody forming an antigen-antibody-enzyme complex. After washing away unbound secondary antibody, a substrate solution is added that reacts with the enzyme to produce a detectable signal, typically a color change.

The signal generated is proportional to the amount of the primary antibody in the sample. Therefore, Indirect ELISA is a quantitative assay that can be used to measure the concentration of the antibody in the sample.

Indirect ELISA is a highly sensitive and specific technique that can be used to detect the presence of a wide range of antibodies. It is particularly useful in the detection of autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus, and in monitoring the immune response to infections or vaccinations.


  • Coating buffer (e.g., carbonate-bicarbonate buffer)
  • Blocking buffer (e.g., 5% BSA in PBS or 3% milk in TBST)
  • Primary antibody (diluted in blocking buffer)
  • Secondary antibody conjugated with HRP (diluted in blocking buffer)
  • Substrate solution (e.g., TMB or DAB)
  • Stop solution (e.g., 2M H2SO4 or 2M HCl)
  • Wash buffer (e.g., PBS or TBST)


1. Dilute the primary antibody in the blocking buffer at the desired concentration.

2. Coat the wells of a 96-well ELISA plate with the antigen or capture antibody in the coating buffer at 4°C overnight or for 2 hours at room temperature.

3. Wash the plate 3 times with the wash buffer to remove any unbound antigen or antibody.

4. Block the plate with the blocking buffer for 1-2 hours at room temperature to prevent non-specific binding.

5. Add the diluted primary antibody to each well and incubate for 1-2 hours at room temperature or overnight at 4°C.

6. Wash the plate 3 times with the wash buffer to remove any unbound primary antibody.

7. Add the diluted secondary antibody conjugated with HRP to each well and incubate for 1 hour at room temperature.

8. Wash the plate 3 times with the wash buffer to remove any unbound secondary antibody.

9. Add the substrate solution to each well and incubate for 10-30 minutes at room temperature in the dark.

10. Add the stop solution to each well to terminate the reaction.

11. Measure the absorbance of each well at the desired wavelength using a microplate reader.

Note: The exact protocol may vary depending on the specific antibodies, substrates, and assay conditions used.

* For Research Use Only. Not for use in diagnostic procedures.
Our customer service representatives are available 24 hours a day, 7 days a week. Inquiry

Online Inquiry

Please submit a detailed description of your project. We will provide you with a customized project plan to meet your research requests. You can also send emails directly to for inquiries.

* Email
* Service & Products of Interest
Services Required and Project Description
* Verification Code
Verification Code