The Enzyme-Linked Immunosorbent Assay, widely known as ELISA, is a powerful laboratory technique used to detect and measure various substances present in a liquid sample. These include proteins, antibodies, hormones, and small molecules. While several ELISA variations exist, this article explains the indirect competitive ELISA method.
The Core Mechanism of Competition
Indirect competitive ELISA operates on a principle of biological competition. A known quantity of antigen is pre-coated onto the testing plate. The target antigen from a sample is then introduced. This sample antigen competes with the pre-coated antigen for binding to a limited amount of primary antibody.
The more target antigen present in the sample, the more primary antibodies will bind to it in solution, leaving fewer free primary antibodies available. These remaining free primary antibodies are then the only ones able to bind to the antigen pre-coated on the plate. Therefore, the amount of primary antibody that ultimately binds to the plate is inversely proportional to the initial concentration of the target antigen in the sample.
The Step-by-Step Laboratory Process
Indirect competitive ELISA involves a series of laboratory steps.
The procedure begins with Antigen Coating, where a purified antigen is affixed to the bottom surface of each well within a microplate, often incubated for several hours or overnight at 4°C. Next, a Blocking step adds a blocking buffer, such as bovine serum albumin (BSA) or ovalbumin, to fill unoccupied binding sites. This prevents non-specific binding of subsequent components, which could lead to false positive signals.
The Competition phase follows. The test sample, containing the unknown target antigen, is first mixed with a limited quantity of primary antibody. This mixture is then added to the antigen-coated wells, allowing sample antigens to compete with plate-bound antigen for available primary antibodies. After incubation, a Washing step removes any unbound antigens, primary antibodies, or antigen-antibody complexes.
An Enzyme-Linked Secondary Antibody is then introduced. This antibody specifically recognizes and binds to primary antibodies attached to the plate-coated antigen. This is the “indirect” aspect of the assay, as the detection antibody does not directly bind to the target antigen.
A Final Wash step follows, removing excess or unbound secondary antibodies. The final step is Substrate Addition & Signal Generation, where a chromogenic substrate reacts with the enzyme attached to the secondary antibody. This reaction produces a measurable signal, typically a color change, whose intensity is quantified by measuring light absorption, often at 450 nm.
Analyzing the Results
Interpreting indirect competitive ELISA results involves understanding the inverse relationship between the measured signal and target antigen concentration. A strong signal, often an intense color, indicates very little target antigen was present. Conversely, a weak signal suggests a high concentration of target antigen.
This inverse correlation arises from the competition mechanism. When a sample contains high target antigen, most limited primary antibodies bind to these antigens in solution before encountering the plate. Consequently, fewer primary antibodies bind to the plate-coated antigen, leading to fewer secondary antibodies binding and a weaker signal. For accurate quantification, the measured signal is compared against a standard curve. This curve is generated by running the assay with known concentrations of the target antigen, allowing precise determination of the unknown sample’s antigen level.
Key Applications
Indirect competitive ELISA is valuable for detecting and quantifying small molecules, which are challenging to measure using other ELISA formats due to limited binding sites.
In endocrinology, this method measures small hormones, such as cortisol or testosterone, in biological fluids like blood serum or saliva. The assay’s sensitivity allows for accurate monitoring of these compounds.
The technique also finds use in food safety, detecting small molecule toxins like mycotoxins, such as aflatoxin, in grain products. This ensures the safety of agricultural commodities by identifying contaminants.
In pharmacology and toxicology, indirect competitive ELISA screens for drugs or their metabolites in biological samples, useful in drug discovery, therapeutic drug monitoring, and forensic analysis. Environmental monitoring also benefits from this assay’s capability to detect small-molecule pollutants, such as pesticides, in water samples.