Description:
General Competitive ELISA Principle: The microtiter well-plate is pre-coated with an antibody against the target Antigen. Sample or standards are added to the wells along with a fixed quantity of biotinylated Antigen and incubated. The Antigen found in the sample or standards competes with the biotinylated Antigen for limited binding sites on the immobilized anti-Antigen antibody. Excess unbound biotinylated Antigen and sample or standard is washed from the plate. Avidin-HRP conjugate is added, incubated and washed. An enzymatic reaction is then produced through the addition of TMB substrate which is catalyzed by the immobilized HRP to generate a blue color product that changes to yellow after adding acidic stop solution. The density of yellow coloration is measured by reading the absorbance at 450 nm which is quantitatively proportional to the amount of biotinylated Antigen captured in the well and inversely proportional to the amount of Serotonin which was contained in the sample or standard.
Why Competitive Assays?
ELISA Kits that have a competitive principle have an output that is inversely proportionate to the amount of analyte in test samples. This format is preferred when developing kits for small molecules. Below are common advantages and disadvantages for competitive principles:
Advantages | Disadvantages |
| Typically shortest and easiest assay protocol, simple method provides a very robust assay. | Development can be difficult to optimize competitive assay components |
| Very low reagent cost | Shortest dynamic range and provides primarily qualitative measurements |
| Good reproducibility from small number of assay processing steps | Lowest level of sensitivity, although novel amplification methods are available with extra cost and development |
| Common for, but not limited to, small molecule and biochemical measurements |
0 ng/mL vs. Absolute Blank
A common inquiry for competitive assays is the difference between a given 0 ng/mL standard and the absolute blank.
For a competitive assay, the well for 0 ng/mL detects how much signal the Antigen-Biotin-Complex is reacting with the kit without the competition of your samples/standard. Since this well will only contain the Antigen-Biotin-Complex, it will have the strongest reaction with TMB, thus having the deepest blue shade. The purpose of a 0 ng/mL well is to have all the reagents and buffers in the assay in an environment without the standard so you can confirm that your standard and Antigen-Biotin-Complex is working competitively.
On the other hand, the blank for a competitive assay will be a well with no sample and no Antigen-Biotin-Complex (Generally Step 10.4 in the manual), which will detect nonspecific binding of the kit for you to subtract from. It should only contain sample diluent.
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