Enzyme-Linked Immunosorbent Assay: Overview

Enzyme-linked immunosorbent assay, or ELISA, is a plate-based biochemical assay used in labs to detect and quantify substances in a complex mixture, such as serum or blood samples.

It involves enzyme-linked antibodies that help in the detection of soluble molecules, such as hormones, proteins, peptides, and other antibodies. The assay is also known as enzyme immunoassay (EIA).

The ELISA assay is performed in 96-well or 384-well plates. The molecules, such as antibodies or antigens are immobilized on the surface of the plates, which makes this assay easy to design and perform in labs. It’s extensively used in labs to detect and measure analytes in crude preparations. Before ELISA, radioimmunoassay was a popular choice among scientists to conduct immunoassays.

Though today different types of ELISA techniques have been developed, they all involve four common steps:

1. Coating/capture: Immobilize specific antigens to the surface of the ELISA plate.
2. Plate blocking: Cover the remaining blocks and surface of the plates using some proteins.
3. Probing/detection: Incubate the ELISA plates with antigen-specific antibodies. The specific antibodies bind to the target antigen.
4. Signal measurement: Detect signals obtained after the reaction of the enzyme label antibodies and antigens and measure their amount using a plate reader. The sensitivity of detection is determined by how the signal is amplified during analytic reactions.

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The enzyme conjugates used to label secondary antibodies during the assay reaction are horseradish peroxidase (HRP), β-galactosidase, alkaline phosphatase (AP), catalase, and acetylcholinesterase.

The enzyme-conjugated antibodies need to be flexible in detection and documentation because of the availability of a spectrum of substrates for chemifluorescent, chromogenic, and chemiluminescent imaging. The substrates are easily visualized by using standard absorbance plate readers.

How Does Enzyme-Linked Immunosorbent Assay Work?

The ELISA test is performed in polystyrene 96-well plates, in which capture antibodies or target antigens are attached or immobilized. Then, incubation with the appropriate substrate produces a measurable product or color change based on the activity of the reporter enzyme.

The change in color can be analyzed by a plate reader machine, which measures the reporter enzyme activity. Accurate and reliable ELISA results depend on highly specific antibody-antigen interactions.

Incubation with the appropriate substrate produces a measurable product based on the activity of the reporter enzyme.

Types of Enzyme-Linked Immunosorbent Assay

The four major types of ELISA are as follows:

• Direct ELISA (antigen-coated plate; screening antibody): In this assay, antigens are immobilized in the solid phase. Then, a reporter enzyme-conjugated, antigen-specific primary antibody is used to detect a specific analyte. The technique is widely used for immunohistochemical staining workflows. The assay has fewer steps, thus performed rapidly, and eliminates cross-reactivity during assay due to the absence of secondary antibodies
• Indirect ELISA (antigen-coated plate; screening antigen/antibody): The assay involves immobilizing antigen on the surface of the well plate. Then, a primary antibody bind to the antigen, and for detection, a labeled secondary antibody against the host species of the primary antibody is used. It’s extensively used to detect specific antibodies in serum.
• Sandwich ELISA (antibody-coated plate; screening antigen): Here, the plate is coated with a capture antibody, which binds to a specific antigen when incubated with a crude sample. Then, a detecting antibody is added, which binds to the antigen followed by the addition of an enzyme-linked secondary antibody, which binds to the detecting antibody. Finally, the incubation of the complex with a suitable substrate facilitates detection. For research applications, sandwich ELISAs require validation to reduce the chance of false positives.
• Competitive ELISA (screening antibody): Also known as competitive immunoassay. Antigens with only one epitope or antibody binding site are commonly used in this method. Here, two types of antigens, unlabeled antigens from samples and labeled antigens, compete to bind to the capture antibody.

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Essential Components of an ELISA Assay

ELISA is a high-throughput experiment that requires labs to work with high-quality reagents and high-tech equipment. Here’re some essentials of ELISA assay that we have listed for you:

• ELISA or Immuno plates: Precoated ELISA plates with Protein A/G, A, or G, assists in orienting capture antibodies properly and preserving their antigen-binding ability.
• Primary antibodies: In sandwich ELISAs and other ELISA systems, monoclonal or polyclonal antibodies can be used as capture and detection antibodies. The monospecificity of monoclonal antibodies towards a single epitope allows the detection and quantification of even the smallest differences in antigens. The capture antibody is often a polyclonal antibody that ensures that as much antigen as possible is removed.
• Blocking buffers and washing buffers: Blocking buffers are solutions of irrelevant proteins or mixtures of proteins that passively adsorb to all remaining binding surfaces. Wash buffers are used to remove unbound compounds and reduce background noise during the assay.

Ready-to-use ELISA kits are also available in the market to make your lab life easier. You can detect many molecules using the kit such as neurobiology analytes, chemokines, and cytokines.

In quantitative ELISA, the positive and negative results are determined by comparing the optical density (OD) of the sample to a standard curve—usually, a solution of the target molecule diluted to a known concentration.

What Industries Use Enzyme-Linked Immunosorbent Assay and How?

ELISA methods have a myriad of applications as diagnostic tools in plant pathology, medicine, and biotechnology. Additionally, it’s also used as a quality check in some life science industries.

Biotechnology

• ELISA is extensively used to study and identify cancer biomarkers. They have also been used clinically to test early-stage ovarian or breast cancers.
• ELISA can determine the concentrations of illicit drugs such as amphetamines, opiates, cannabinoids, and cocaine in urine samples.
• Food companies commonly use ELISA to detect allergens for legally required ingredient labels.

Medical

• ELISA is commonly used as a diagnostic tool in clinical labs to diagnose a range of diseases such as pregnancy tests, HIV infection, and blood typing.
• ELISAs are used in a variety of fields, including the detection of autoimmune diseases, forensic toxicology, the detection of other fungi, viruses, and bacteria, and laboratory and clinical research.  It is necessary to perform Western blot testing to confirm the results of disease diagnosis due to the possibility of false positives.

Plant Pathology

• ELISA is used to detect viral diseases in plants. A sandwich of antibody-antigen-antibody layers is created using protein A. The protein A layer is applied first to prepare the plate for the coating antibody layer. Then, an enzyme is conjugated to the second protein A layer, which detects the second antibody layer. In the coating layer of antibodies, the orientation of IgG prevents unwanted reactions with conjugated protein A at later stages.

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ELISA or Enzyme-linked immunosorbent assay is a technique used in labs for quantitative or qualitative assessment of certain substances in complex biological mixtures. The assay is commonly used in life sciences labs and industries to detect many viruses and diagnose a spectrum of diseases.

Three things that should be highly considered while performing an ELISA test are the quality of reagents used in the process, the use of high-tech equipment, and the accurate implementation of the procedure. These three factors ensure better results with reduced false positives.

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