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Protocol for Direct ELISA

What is Direct ELISA?

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

Next, a detection antibody labeled with an enzyme is added to the surface, which binds to the captured antigen forming an antigen-antibody complex. After washing away unbound detection 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 antigen in the sample. Therefore, Direct ELISA is a quantitative assay that can be used to measure the concentration of the antigen in the sample.

Direct ELISA is a simple and straightforward technique that can be used to detect the presence of a wide range of antigens. However, it may have limitations in terms of sensitivity and specificity, particularly when dealing with complex samples containing multiple antigens or interfering substances. In such cases, other types of ELISA, such as Sandwich or Indirect ELISA, may be more appropriate.


  • 96-well ELISA plate
  • Coating buffer (e.g. carbonate-bicarbonate buffer)
  • Antigen or capture antibody (e.g. polyclonal or monoclonal)
  • Blocking buffer (e.g. 5% BSA or 1% casein in PBS)
  • Primary detection antibody (e.g. labeled polyclonal or monoclonal)
  • Detection buffer (e.g. HRP-conjugated secondary antibody in blocking buffer)
  • Substrate solution (e.g. TMB or ABTS)


1. Coat the ELISA plate with the antigen or capture antibody in coating buffer at the appropriate concentration and incubate overnight at 4°C.

2. Discard the coating solution and wash the plate 3 times with washing buffer (e.g. PBS with 0.05% Tween-20).

3. Block the plate with blocking buffer for 1 hour at room temperature to prevent nonspecific binding.

4. Discard the blocking buffer and wash the plate 3 times with washing buffer.

5. Add the primary detection antibody to the wells at the appropriate concentration and incubate for 1 hour at room temperature.

6. Discard the primary detection antibody and wash the plate 3 times with washing buffer.

7. Add the detection buffer to the wells at the appropriate dilution and incubate for 30 minutes at room temperature.

8. Discard the detection buffer and wash the plate 3 times with washing buffer.

9. Add the substrate solution to the wells and incubate for 10-30 minutes at room temperature until the desired signal intensity is reached.

10. Stop the reaction by adding stop solution (e.g. 2N sulfuric acid or 1N hydrochloric acid).

11. Read the absorbance at the appropriate wavelength using a microplate reader.


  • It is important to optimize the antigen/capture antibody and detection antibody concentrations to achieve a strong signal and low background.
  • It is recommended to include positive and negative controls on each plate to ensure the accuracy and consistency of the assay.
  • The choice of substrate and detection buffer will depend on the labeling system used for the detection antibody.
* For Research Use Only. Not for use in diagnostic procedures.
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