Competitive ELISA Protocol

What is a competitive ELISA?

Competitive ELISA assays are used by researchers determine the amount of analyte in their sample and changes in analyte level under different conditions. Competitive ELISA assays allow researchers to measure a range of metabolites, hormone and proteins in a sample efficiently, quantitatively & cheaply. Similar to a Sandwich ELISA, a competitive ELISA utilizes antibodies to determine the amount of analyte in a sample and uses an enzyme-linked secondary antibody to quantitatively measure the amount of analyte in a sample.

Figure 1: Schematic of a competitive ELISA principle, whereby the primary antibody has bound the analyte plated onto the ELISA plate. The enzyme-linked detection secondary antibody has bound the capture antibody.

The advantages and disadvantages of a competitive ELISA

The competitive ELISA assay is a great tool to measure key analytes including universal hormones & signalling molecules such as cortisol, T3, hydroxyproline, serotonin. As many researchers study these types of molecules, the competitive ELISA provides a cheap, effective and fast way to measure these analytes without the high costs of expensive machinery.


  • Measure your analyte in complex samples: A key advantage that a competitive ELISA assay provides is it's ability to measure analytes of interest in complex biological samples. As the main step in a competitive ELISA is to mix the capture antibody with your sample, it allows for the formation of the capture-analyte complex effectively,.


  • Negative controls can give positive results: If wells are not blocked correctly, antigens or secondary antibodies may bind to the well non-specifically.
  • Signal is inversely related to the amount of antigen in a sample: Meaning that for samples with high concentrations of antigen, a weaker signal is generated and affecting the range of the assay. Dilution of samples may be required to measure samples in a significant range.

Competitive ELISA standard curve

Competitive ELISA assays are based on the principle of the capture antibody competing for an antigen in a sample. Firstly, an unlabeled capture antibody is incubated with a sample containing the antigen of interest. This leads to the formation of an antibody-antigen complex. During this step, excessive antibody is added to the mixture, therefore free antibody is leftover after the incubation step.

Next step is the addition of the antibody-antigen complex to a an ELISA plate pre-coated with the antigen of interest (inhibitor antigen). The free antibody remaining from the initial complex forming step is then able to bind the inhibitor antigen on the plate, while the preformed antibody-antibody complexes are washed off during wash steps.

Next, an enzyme-linked detection antibody is added to the plate, which binds the primary antibody bound to the inhibitor antigen. Finally, a substrate is added and a colorimetric change occurs, whereby the intensity of signal is inversely correlated with the amount of antigen in the sample.

Figure 2: Competitive ELISA standard curve. The amount of analyte in a sample is inverse to absorbance values due to the binding of antigen with capture antibody in the complex formation step.

The below protocol is a sample protocol for a competitive ELISA kits. Competitive ELISA kits allow for the detection and quantification of an analyte in a sample. This allows the researcher to calculate the amount of analyte present in their sample. This can be very useful when looking for increases in protein concentration, phosphorylation of proteins or decreases on protein concentrations.

Competitive ELISA protocol step-by-step

Figure 3: Schematic of a Competitive ELISA protocol.

Step Procedure


Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells.


Add Sample and Biotin-detection antibody: Add 50µL of Standard, Blank (Sample/Standard dilution buffer), or Sample per well. The blank well is added with Sample / Standard dilution buffer. Immediately add 50 µL of Biotin-detection antibody working solution to each well. Cover with the Plate sealer we provided. Gently tap the plate to ensure thorough mixing. Incubate for 45 minutes at 37°C. (Solutions are added to the bottom of micro ELISA plate well, avoid touching plate walls and foaming).


Wash: Aspirate each well and wash, repeating the process x3 according to instructions.


HRP-Streptavidin Conjugate (SABC): Add 100µL of SABC working solution to each well. Cover with a new Plate sealer. Incubate for 30 minutes at 37°C.


Wash: Repeat the aspiration/wash process for x5 times according to instructions.


TMB Substrate: Add 90µL of TMB Substrate to each well. Cover with a new plate sealer. Incubate for about 15-20 minutes at 37°C. Protect from light. The reaction time can be shortened or extended according to the actual color change, but not more than 30 minutes. When apparent gradient appears in standard wells, you can terminate the reaction.


Stop: Add 50µL of Stop Solution to each well. Wells will turn to yellow immediately. The adding order of stop solution should be as the same as the substrate solution.


OD Measurement: Determine the optical density (OD Value) of each well at once, using a microplate reader set to 450 nm. You should open the microplate reader ahead, preheat the instrument, and set the testing parameters.