Exploring the Fundamentals of ELISA Assay: Understanding Its Purpose, Principles, and Applications in Biomedical Research and Diagnostics

The enzyme-linked immunosorbent assay (ELISA) is a laboratory test that identifies and measures proteins, antigens, antibodies, peptides, and hormones in biological samples. Widely regarded as the gold standard in immunoassays, ELISA testing is used in pregnancy tests and in diagnosing human immunodeficiency virus (HIV) infection.

ELISA Flowchart. Credit: Dhamad and Abdal Rhida

ELISA technology is continually evolving. This has led to the development of portable sensing devices with rapid turnaround time and lower contamination, which are critical in pandemic situations.

In this article, you’ll learn more about what is an ELISA assay, the main types, how it works, its various applications in research and clinical diagnostics, and its limitations.

What Is ELISA?

An antigen-antibody reaction is the basis of the biochemical assay known as ELISA. A particular antigen must be immobilized on a surface, like a microtiter plate, and then incubated with a sample containing the appropriate antibody. A secondary antibody linked to an enzyme, such as alkaline phosphatase or horseradish peroxidase, is then used to detect the antibody.

A spectrophotometer can quantify the colored product the enzyme creates from a colorless substrate. The amount of antigen or antibody in the sample directly correlates to the color produced.

Main Types Of ELISA

ELISA assays differ in their design and the specific application for which they are intended. Here are the four major types of ELISA assays:

Direct ELISA: A labeled primary antibody specific to the antigen detects the antigen in a direct ELISA, immobilizing the antigen onto the microtiter plate’s surface. Although this procedure is rapid and easy, it might not be as sensitive as other ELISA techniques.
Indirect ELISA: The antigen is fixed on the surface of the microtiter plate in an indirect ELISA, and an unlabeled primary antibody specific to the antigen is used to detect the antigen. The signal is then amplified by a secondary antibody tagged with an enzyme and bound to the original antibody. Compared to a direct ELISA, this approach is more sensitive.
Competitive ELISA: A tagged antigen and the antigen in the sample compete for binding to a small quantity of immobilized antibody in a competitive ELISA. This technique helps determine the antigen present in a sample and detect tiny compounds like medicines or hormones.
Sandwich ELISA: In a sandwich ELISA, a primary antibody bound on the surface of the microtiter plate captures the antigen. Afterward, a tagged secondary antibody that attaches to the second primary antibody is added, followed by a second primary antibody specific to a distinct epitope on the antigen. This technique is exact and sensitive.

The best ELISA assay will depend on the application and the antigen or antibody being evaluated. Each type of ELISA assay has its corresponding benefits and drawbacks.

How Does ELISA Work?

ELISA detects and quantifies proteins, peptides, antibodies, and hormones in biological samples using an antigen-antibody reaction. The ELISA process is described in detail below:

Coating: In microtiter plates, antigens are immobilized on the surface of plastic, glass, or other materials.
Blocking: An antigen-blocking solution is applied to the microtiter plate surface to prevent non-specific binding.
Detection: The corresponding antibody is added to the microtiter plate and allowed to bind to the immobilized antigen. The secondary antibody is then added to the plate and recognizes and attaches to the primary antibody with the help of an enzyme, such as horseradish peroxidase or alkaline phosphatase.
Quantification: The enzyme converts colorless substrates into colored products, which spectrophotometers can measure. Antigens and antibodies in the sample produce color in proportion to their amount.

The ELISA assay is a sensitive and specific tool to diagnose diseases, develop new drugs, and conduct basic research.

What Are The Applications Of ELISA?

ELISA is a versatile laboratory technique in research, clinical diagnostics, and industrial testing. The following are some of the most common applications of ELISA:

Disease Diagnosis: An ELISA test is used to detect and quantify specific antibodies or antigens in patient serum or other body fluids, enabling the diagnosis of infectious diseases like HIV, hepatitis B and C, Lyme disease, and COVID-19.
Food Safety Testing: Food samples can be tested for allergens, pathogens, and other contaminants using ELISAs, ensuring quality and safety.
Drug Discovery: A variety of diseases can be treated with the help of ELISA by screening large numbers of compounds for binding to specific proteins or inhibiting enzymatic activity.
Immunology Research: An ELISA can measure cytokines and growth factors in serum or tissue samples, providing insight into the immune response to infections.
Veterinary Diagnostics: Infectious diseases in livestock and pets can be detected and quantified with ELISA tests by measuring specific antibodies or antigens in serum or other body fluids.
Environmental Monitoring: To support environmental monitoring and remediation activities, ELISA can be used to identify and measure ecological pollutants in samples of soil, water, and other media, including pesticides, heavy metals, and toxins.

ELISA is a valuable tool for research, clinical diagnostics, and industrial applications since it can identify and quantify particular proteins, peptides, antibodies, and hormones in a range of samples.

What Are The Limitations Of ELISA?

Although ELISA is a powerful laboratory technique, it has some limitations. Its limitations are as follows:

False Positives: False positive results can occur if the sample contains interfering substances, such as heterophile antibodies, which bind to the detection antibody.
False Negatives: ELISA results can be falsely negative if the target antigen or antibody is not present at detectable levels in the sample or if the assay conditions are not optimized.
Limited Dynamic Range: It’s not possible for ELISA to accurately quantify samples with very high or deficient concentrations of the target antigen or antibody due to its limited dynamic range.
Limited Multiplexing: A single ELISA can only detect one target antigen or antibody per well, which limits its ability to see multiple targets simultaneously.
Cross-Reactivity: Cross-reactivity between ELISA and other proteins with similar epitopes can lead to inaccurate results.
Labor-Intensive: ELISA can be a time-consuming and labor-intensive assay when multiple samples and dilutions are necessary.
High Cost: ELISA kits and reagents can be expensive compared to other assays, such as Western blotting or polymerase chain reaction (PCR).

Despite these limitations, ELISA remains a widely used and essential laboratory technique, particularly in the fields of clinical diagnostics and biomedical research, where it provides a sensitive and specific tool for detecting and quantifying specific antigens and antibodies in a variety of sample types.

Conclusion

ELISA is a popular and adaptable tool in both clinical diagnostics and biomedical research. It’s vital for illness diagnosis, drug development, and fundamental research due to its capacity to identify and quantify proteins, peptides, antibodies, and hormones in biological samples.

Researchers and physicians can obtain accurate and trustworthy results by understanding the principles and applications of ELISA technology. With the technology’s continued evolution, more efficient and sensitive ELISA assays will be developed, significantly reducing detection time and sample volume.