Enzyme-Linked Immunosorbent Assay (ELISA) is a powerful and widely used laboratory test that detects and measures specific biomolecules—like proteins, peptides, hormones, or antibodies—in a sample. In fact, due to its high specificity and versatility, ELISA is considered the "gold standard" of immunoassays.
The test works by using antibodies linked to enzymes to produce a measurable signal. When the target molecule is present, an enzyme-driven reaction causes a color change, with the intensity of the color being proportional to the amount of the target molecule.
Its reliability and cost-effectiveness make it a cornerstone in many fields:
Detecting infections (HIV, Lyme disease), confirming pregnancy, and monitoring hormone levels.
Quantifying biomarkers like cytokines or growth factors in drug development.
Ensuring the safety and efficacy of vaccines and biologics.
While there are different formats, nearly every ELISA follows this fundamental four-stage process to isolate and measure a target analyte. We'll use the common Sandwich ELISA as an example:
A "capture" antibody is immobilized onto the surface of a polystyrene microplate well, specifically chosen to bind to the target antigen.
The remaining surface is coated with an irrelevant protein to prevent non-specific binding that can create false positives.
The sample is added, the target antigen is captured, and an enzyme-linked detection antibody creates the "sandwich" structure.
A substrate is added, causing an enzyme reaction that creates a colored product proportional to the antigen amount.
Choosing the Right Format for Your Assay
How it Works: The target antigen is coated on the plate, and an enzyme-linked primary antibody directly binds to it.
Best For: Quickly detecting the presence of a high-abundance antigen.
How it Works: An unlabeled primary antibody binds the antigen, followed by an enzyme-linked secondary antibody.
Best For: Detecting specific antibodies in a sample (serology) and maximizing signal sensitivity.
How it Works: The antigen is captured between two antibodies (a capture and a detection antibody).
Best For: Measuring low-concentration antigens in complex samples. Very high specificity and sensitivity.
Most common format for quantitative research
Find Your Sandwich ELISA KitHow it Works: Sample antigen competes with reference antigen for binding. Lower signal means more antigen present.
Best For: Detecting small molecules like hormones or toxins where sandwiching isn't possible.
The Right Tool for the Job: Protein vs. Genetic Material. Researchers often need to decide between ELISA and PCR. While both are powerful diagnostic tools, they answer fundamentally different questions.
| Aspect | ELISA (Immunoassay) | PCR (Molecular Assay) |
|---|---|---|
| Target | Proteins (antigens, antibodies, hormones) | Nucleic Acids (DNA, RNA) |
| Principle | Antigen–antibody binding; enzyme generates color signal | Exponential amplification of a target gene sequence |
| Sensitivity | High for proteins, but can miss very early infections | Ultra-high; can detect minute amounts of genetic material |
| Application | Measuring protein levels, detecting antibody responses (HIV, COVID-19 antibodies), pregnancy tests | Detecting active infections (SARS-CoV-2), genetic disease testing, forensics |
From Color Change to Concrete Conclusion
An ELISA result is more than just a color. Interpretation depends on the assay type:
Many diagnostic kits give a yes/no answer. A sample's signal is compared to a pre-determined cutoff value. This is common for infectious disease screening.
Research and hormone ELISAs provide precise concentration (e.g., pg/mL) calculated from standard curves and evaluated against normal ranges.
The cutoff minimizes overlap between populations, but false positives/negatives are possible.
You understand the theory. Now get the tools to put it into practice. MyBioSource supplies thousands of ready-to-use, validated ELISA kits for a vast range of targets across dozens of species.
Explore Our ELISA KitsIt perfectly describes how the test works:
In a Direct ELISA, a single enzyme-linked primary antibody binds to the target antigen. In an Indirect ELISA, an unlabeled primary antibody binds the antigen first, followed by an enzyme-linked secondary antibody that binds to the primary antibody. The indirect method adds an amplification step, generally making it more sensitive.
For detecting the genetic material of a pathogen, yes. PCR's ability to amplify tiny amounts of DNA or RNA makes it ultra-sensitive, especially early in an infection when antigen or antibody levels might be too low for ELISA to detect. However, for detecting proteins, ELISA is extremely sensitive.
The Sandwich ELISA is popular because it is highly specific and sensitive. Since it requires two different antibodies to bind to the antigen (capture and detection), the chance of a non-specific signal is very low. This makes it ideal for accurately measuring low concentrations of an antigen in complex biological samples like serum or plasma.
Yes. Although highly specific, false positives can occur due to technical errors or antibody cross-reactivity (where the antibody binds to a molecule similar to the intended target). This is why a positive result from a screening ELISA (like for HIV) is always confirmed using a different, highly specific method.