The Enzyme-Linked Immunosorbent Assay (ELISA) assay is a commonly used laboratory technique in immunology to detect the presence of an antibody or antigen in a sample. ELISA allows for parallel analysis of many samples, making it a useful diagnostic tool in medicine and a quality control check in various industries. This article will explain the science behind how the ELISA technique works and why it’s an important assay in healthcare and beyond.
What Is ELISA?
ELISA stands for enzyme-linked immunosorbent assay. It is a plate-based assay technique that uses antibodies and color change to detect the presence of a substance in a liquid sample. An ELISA can test for either antibodies or antigens in the sample.
ELISA relies on an enzyme attached to an antibody or antigen to catalyze a color change reaction. The color change can then be measured and correlated to the amount of antigen or antibody present in the initial sample. This dual capability of being both quantitative and qualitative is what sets ELISA apart as a reliable and preferred method for various analytical purposes, particularly when it’s critical to rely on the ELISA assay for accurate results.
ELISA Components
Four main components make up an ELISA assay:
- Coated Plate – The plate is coated with an antigen to test for the presence of antibodies or coated with an antibody to test for a specific antigen. The antigen or antibody will bind to the plate where it is immobilized.
- Sample and Conjugate – The liquid sample is added to the wells, followed by an enzyme-conjugated antibody or antigen. This conjugate will bind to any antigen or antibody from the sample captured on the plate.
- Substrate – After washing off any unbound samples, a substrate is added. The enzyme converts the substrate into a detectable color-change product.
- Stop Solution – To end the reaction, a stop solution is added before reading the final amount of color change.
ELISA Plate Preparation
ELISA plates can be purchased pre-coated with antigens or antibodies. Alternatively, the researchers can coat the plates themselves. This involves:
- Incubating the plate with a capture antigen or antibody overnight at 2-8°C allows the protein to adsorb to the plastic well surfaces.
- Blocking any remaining surface area in the wells to prevent non-specific binding of the antibodies or antigens. Common blocking buffers are milk, serum albumin, or solutions of detergents.
- Washing the plate multiple times to remove any unbound capture proteins before adding the samples.
How Does ELISA Work?
Once the ELISA plate is prepared, here are the steps involved:
- Samples and enzyme-conjugates are added to the antigen or antibody-coated wells and incubated for one hour at 37°C. Any antigen or antibody in the sample binds to the immobilized capture protein on the plate. The conjugated enzyme also binds to the captured antigen/antibody complex.
- Unbound materials are washed away using a wash buffer. This separates any sample components that did not specifically bind to the plate.
- The substrate is added and incubated for 15-30 minutes at 37°C. The substrate will change color if the enzyme-antibody/antigen conjugate is present.
- A stop solution ends the enzyme-substrate reaction.
- The optical density or fluorescence is measured using a spectrophotometer or fluorometer. The more antigen or antibody present in the sample, the stronger the color or fluorescent signal.
Why ELISA Is Important
The ELISA technique offers several advantages that make it a versatile immunoassay used in many applications:
- High sensitivity – ELISA can detect very low concentrations of antigens or antibodies down to the picogram per milliliter range.
- High specificity – Using antibodies or antigens for detection ensures high specificity for the target being analyzed.
- High-throughput – The 96-well plate format means many samples can be rapidly analyzed in parallel. Automated equipment allows further high-throughput testing.
- Quantitative data – By measuring the color intensity or fluorescence, ELISA provides quantitative antigen/antibody concentration results.
- Cost-effective – ELISA is relatively inexpensive compared to other immunoassay options.
- Quick and easy – Once ELISA plates have been coated, the assay takes only two to three hours to complete. The procedure is straightforward without multiple complicated steps.
Applications Of ELISA
ELISA’s sensitivity, specificity, and ease have made it a workhorse of clinical immunodiagnostics. It is used to test for:
- Infectious diseases like HIV, malaria, and Lyme disease are used to detect antibodies against pathogens.
- Allergies through detecting IgE antibodies against specific allergens.
- Hormones like insulin or human chorionic gonadotropin (pregnancy test).
- Cancer biomarker proteins to aid in diagnosis or disease
Outside of human healthcare, ELISA is also used for food safety testing, environmental monitoring, and quality control in manufacturing processes like biopharmaceutical production.
The ELISA technique provides researchers with an accurate, efficient, cost-effective immunoassay option. While the basic principles have remained unchanged, continuous improvements in ELISA sensitivity, dynamic range, and flexibility will ensure this assay remains a cornerstone of clinical diagnosis and life science research for years to come. The accuracy and utility of the ELISA assay cannot be understated.
Conclusion
The ELISA assay uses antibodies and enzyme conjugates to detect antigens or antibodies in a sample. Its high sensitivity and specificity, ease of use, quantitative capability, and cost-effectiveness have cemented its role as a critical tool in the clinic and laboratory. From infectious disease diagnosis to allergy testing and monitoring cancer biomarkers, ELISA provides clinicians and researchers with accurate immunodetection and quantification. Thanks to its versatility, ELISA will continue advancing immunoassay-based diagnosis and research across healthcare and the life sciences.