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Protocol for Competitive ELISA

Competitive ELISA is a technique used to measure the concentration of a specific antigen in a sample by assessing its ability to compete with a labeled antigen for binding to a limited amount of immobilized capture antibody. The degree of competition between the labeled and unlabeled antigen is directly proportional to the concentration of the unlabeled antigen present in the sample. Here, we provide a comprehensive protocol for conducting a competitive ELISA.


  • Coating buffer (e.g. carbonate-bicarbonate buffer, pH 9.6)
  • Blocking buffer (e.g. 3% BSA in PBS)
  • Wash buffer (e.g. 0.05% Tween-20 in PBS)
  • Standard/sample dilution buffer (e.g. 1% BSA in PBS)
  • Primary antibody (e.g. monoclonal or polyclonal antibody against the target analyte)
  • Detection antibody (e.g. HRP-conjugated secondary antibody against the primary antibody)
  • Substrate solution (e.g. TMB or ABTS substrate)
  • Stop solution (e.g. 2N H2SO4 for TMB substrate or 1% SDS for ABTS substrate)
  • Competitor antigen (e.g. synthetic peptide or recombinant protein)


1. Prepare the coating buffer and use it to coat the wells of a microtiter plate with the primary antibody against the target analyte. Incubate overnight at 4°C or for at least 2 hours at room temperature.

2. Discard the coating buffer and wash the wells three times with the wash buffer.

3. Block the wells with the blocking buffer for at least 1 hour at room temperature.

4. Prepare a series of standard solutions of the target analyte in the standard/sample dilution buffer, ranging from high to low concentrations.

5. Prepare the samples to be tested in the same standard/sample dilution buffer.

6. Mix the standard solutions or samples with the competitor antigen in appropriate proportions, and incubate for at least 1 hour at room temperature.

7. Add the standard/sample/competitor antigen mixture to the coated and blocked wells of the microtiter plate. Incubate for 2 hours at room temperature or overnight at 4°C.

8. Discard the sample solution and wash the wells three times with the wash buffer.

9. Add the detection antibody to the wells and incubate for 1 hour at room temperature.

10. Discard the detection antibody solution and wash the wells three times with the wash buffer.

11. Add the substrate solution to the wells and incubate for appropriate time (usually 10-30 minutes) at room temperature in the dark.

12. Stop the reaction by adding the stop solution to the wells.

13. Read the absorbance of each well at the appropriate wavelength (usually 450 nm for TMB substrate or 405 nm for ABTS substrate) using a microplate reader.

14. Calculate the concentrations of the target analyte in the samples using the standard curve generated from the absorbance values of the standard solutions.

Data analysis:

  • Calculate the average absorbance value for each set of standards and samples in duplicate.
  • Create a standard curve by plotting the average absorbance value for each standard on the y-axis versus the concentration of each standard on the x-axis.
  • Determine the concentration of the unknown samples by comparing their average absorbance value to the standard curve. This can be done by drawing a horizontal line from the average absorbance value on the y-axis to the standard curve and then dropping a vertical line down to the x-axis to read off the corresponding concentration.
  • Calculate the percentage of inhibition for each sample by using the formula:

% inhibition = (1 - (average absorbance value of sample / average absorbance value of control)) x 100

where the control is a sample without any inhibitor.

  • Calculate the IC50 value, which is the concentration of inhibitor that causes 50% inhibition of the binding of the antigen to the antibody. This can be done by plotting the percentage of inhibition versus the log concentration of the inhibitor and then using a non-linear regression curve-fitting program to determine the IC50 value.
  • Analyze the data statistically to determine the significance of any differences observed between the test samples and controls. This can be done using a variety of statistical tests, such as the t-test or analysis of variance (ANOVA).

Note: The competitor antigen competes with the target analyte for binding to the primary antibody, resulting in a decreased signal in the presence of higher concentrations of the competitor. The degree of inhibition is proportional to the concentration of the competitor, which can be used to quantify the amount of target analyte in the samples.

* For Research Use Only. Not for use in diagnostic procedures.
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