Guide ELISA (Enzyme-linked Immunosorbent Assay) 23rd November 2021 – Tags:

¿What is a Enzyme-linked Immunosorbent Assay (ELISA)?

ELISA (Enzyme-linked Immunosorbent Assay) is a plate-based assay technique designed to detect and quantify soluble substances such as peptides, proteins, antibodies and hormones. Other names, such as enzyme immunoassay (EIA), are also used to describe the same technology.

In an ELISA, the antigen (target macromolecule) is immobilized on a solid surface (microplate) and then forms a complex with an antibody that is bound to a reporter enzyme. Detection is carried out by measuring the activity of the reporter enzyme by incubation with the appropriate substrate to produce a measurable product.

The most crucial element of an ELISA is a highly specific antibody-antigen interaction.


ELISA (Enzyme-linked Immunosorbent Assay) is a powerful method for detecting and quantifying a specific protein in a complex mixture. Originally described by Engvall and Perlmann (1971), the method allows the analysis of protein samples immobilized in microplate wells using specific antibodies.

ELISAs are usually performed in 96- or 384-well polystyrene plates, which passively bind antibodies and proteins. This binding and immobilization of reagents is what makes ELISAs easy to design and perform.

The immobilization of ELISA reagents on the surface of the microplate facilitates the separation of bound from unbound material during the assay. This ability to use high-affinity antibodies and to wash out non-specifically bound materials makes the ELISA a powerful tool for measuring specific analytes within a crude preparation.

Although many variants of ELISA have been developed and used in different situations, they all rely on the same basic elements:

  1. Coating/capture: direct or indirect immobilization of antigens on the surface of the wells of polystyrene microplates.
  2. Plate locking: addition of an irrelevant protein or other molecule to cover all unsaturated surface binding sites in the wells of the microplate.
  3. Probe/detection: incubation with antigen-specific antibodies that bind by affinity to antigens.
  4. Signal-detection measurement of the signal generated through direct or secondary labeling of the specific antibody.

The most commonly used enzyme labels are horseradish peroxidase (HRP) and alkaline phosphatase (AP). Other enzymes, such as β-galactosidase, acetylcholinesterase and catalase, have also been used.

There is a large selection of commercially available substrates to perform ELISA with an HRP or AP conjugate. The choice of substrate depends on the sensitivity required for the assay and the instrumentation available for signal detection (spectrophotometer, fluorometer or luminometer).

Types of ELISA - Direct ELISA, Indirect ELISA and Sandwich ELISA

There are several formats used for ELISAs. These are divided into direct, indirect or sandwich capture and detection methods. The key step is the immobilisation of the antigen of interest, which is done by direct adsorption to the test plate or indirectly via a capture antibody that has been bound to the plate. The antigen is then detected either directly (as labelled primary antibody) or indirectly (as labelled secondary antibody).

The most commonly used ELISA assay format is the sandwich ELISA, which immobilises and indirectly detects the presence of the target antigen. This type of capture assay is called a sandwich assay because the analyte to be measured is bound between two primary antibodies, each of which detects a different epitope of the antigen: the capture antibody and the detection antibody. The sandwich ELISA format is widely used because of its sensitivity and specificity.

ELISA Enzyme linked Immunosorbent Assay

Direct versus indirect ELISA detection strategies

Among the standard assay formats discussed and illustrated above, where differences in both capture and detection were the concern, it is important to differentiate between the particular strategies that exist specifically for the detection step.

Regardless of the method by which an antigen is captured on the plate (by direct adsorption to the surface or via a pre-coated ‘capture’ antibody, as in a sandwich ELISA), it is the detection step (as direct or indirect detection) that largely determines the sensitivity of an ELISA.

The direct detection method uses a primary antibody labelled with a reporter enzyme or a label that reacts directly with the antigen. Direct detection can be performed with an antigen directly immobilised on the test plate or with the capture assay format. Direct detection, although not widely used in ELISA, is quite common for immunohistochemical staining of tissues and cells.

The indirect detection method uses a labelled secondary antibody or a biotin-streptavidin complex for amplification and is the most popular format for ELISA. The secondary antibody has specificity for the primary antibody.

In a sandwich ELISA, it is essential that the secondary antibody is specific for detection of the primary antibody only (and not the capture antibody) or the assay will not be specific for the antigen. This is usually achieved by using capture and primary antibodies from different host species (e.g. mouse IgG and rabbit IgG, respectively).

For sandwich assays, it is beneficial to use secondary antibodies that have cross-adsorbed to eliminate any secondary antibodies that may have affinity for the capture antibody.

Direct detection by ELISA


  • Fast because only one antibody and fewer steps are used.
  • Cross-reactivity of the secondary antibody is eliminated.


  • The immunoreactivity of the primary antibody may be adversely affected by labelling with reporter enzymes or labels.
  • The labelling of primary antibodies for each specific ELISA system is time-consuming and expensive
  • Limited number of commercially available primary antibody conjugates.
  • There is no flexibility in the choice of primary antibody label from experiment to experiment.
  • Minimum signal amplification.

Indirect ELISA detection


  • There is a wide range of commercially available labelled secondary antibodies.
  • Versatile because many primary antibodies can be made in one species and the same labelled secondary antibody can be used for detection.
  • The maximum immunoreactivity of the primary antibody is preserved because it is not labelled.
  • Sensitivity is increased because each primary antibody contains several epitopes that can be bound by the labelled secondary antibody, allowing amplification of the signal.
  • Different detection methods can be used with the same primary antibody (colorimetric, chemiluminescent, etc.).


  • Cross-reactivity with the secondary antibody could occur, resulting in a non-specific signal.
  • An additional incubation step is required in the procedure.

Sandwich ELISA


  • High sensitivity and high specificity for the target antigen, as two antibodies are used for capture and detection.
  • Different detection methods can be used with the same capture antibody.


  • It requires further optimisation to identify antibody pairs and to ensure that there is limited cross-reactivity between capture and detection antibodies.

Competitive ELISA and other formats

In addition to the standard direct and sandwich formats described above, other ELISA styles are available:

The competitive ELISA is a strategy often used when the antigen is small and has a single epitope or antibody binding site. A variation of this method is to label the purified antigen instead of the antibody.

The unlabelled antigen in the samples and the labelled antigen compete for binding to the capture antibody. A decrease in the signal of the purified antigen indicates the presence of the antigen in the samples when compared to the test wells with labelled antigen alone.

Summary of the competitive ELISA method

In competitive ELISA, also called inhibition ELISA, the concentration of the target antigen is determined by detection of signal interference. The target antigen in the sample competes with a labelled reference or standard to bind a limited amount of immobilised antibody on the plate.

ELISPOT (enzyme-linked immunospot assay) refers to the ELISA-type capture and measurement of proteins secreted by cells that are plated onto PVDF membrane-backed microplate wells. It is a sandwich assay in which proteins are captured locally as they are secreted by the seeded cells, and detection is performed with a precipitating substrate. ELISPOT is like a western blot where the result is spots on a membrane surface.

“In-cell ELISA” is performed with cells grown overnight in standard microplates. After fixing, permeabilising and blocking the cultured cells, the target proteins are detected with antibodies. This is an indirect assay, not a sandwich assay. Secondary antibodies are either fluorescent (for direct measurement using a fluorescent plate reader or microscope) or enzyme-conjugated (for detection with a soluble substrate using a plate reader).

ELISA is almost always performed using 96- or 384-well polystyrene plates and samples in solution (i.e. biological fluids, culture media or cell lysates). This is the platform discussed in the remainder of this article.

ELISA primary antibodies

Monoclonal antibodies or polyclonal antibodies can be used as capture and detection antibodies in sandwich and other ELISA systems. Monoclonal antibodies have an inherent monospecificity towards a single epitope that allows fine detection and quantification of small differences in antigen.

Polyclonal antibodies are often used as capture antibodies to extract as much antigen as possible. A monoclonal is then used as a detection antibody in the sandwich assay to improve specificity. In addition to the use of traditional monoclonal antibodies, recombinant monoclonal antibodies can also be used for ELISA.

Recombinant antibodies are derived from antibody-producing cell lines engineered to express antibody-specific heavy and light chain DNA sequences. Compared to traditional hybridoma-derived monoclonal antibodies, recombinant antibodies are not susceptible to cell line drift or batch-to-batch variation, allowing for maximum antigen specificity.

An important consideration in the design of a sandwich ELISA is that the capture and detection antibodies must recognise two different epitopes that do not overlap. When the antigen binds to the capture antibody, the epitope recognised by the detection antibody must not be obscured or altered.

Capture and detection antibodies that do not interfere with each other and can bind simultaneously are called ‘matched pairs’ and are suitable for developing a sandwich ELISA. Many primary antibody suppliers provide epitope information and indicate pairs of antibodies that have been validated in ELISA as matched pairs. The use of the same antibody for capture and detection may limit the dynamic range and sensitivity of the final ELISA.

Blocking buffers and wash buffers

The binding capacity of the microplate wells is usually greater than the amount of protein coated in each well. The remaining surface must be blocked to prevent antibodies or other proteins from adsorbing to the plate during subsequent steps. A blocking buffer is a solution of irrelevant protein, protein mixture or other compound that passively adsorbs to all remaining binding surfaces of the plate. The blocking buffer is effective if it improves the sensitivity of an assay by reducing the background signal and improving the signal-to-noise ratio. The ideal blocking buffer will bind to all potential non-specific interaction sites, completely eliminating the background, without altering or obscuring the epitope for antibody binding.

When developing any new ELISA, it is important to test several different blockers to obtain the highest signal-to-noise ratio in the assay. Many factors can influence non-specific binding, including the various protein-protein interactions specific to the samples and the antibodies involved.

The most important parameter when selecting a blocker is the signal-to-noise ratio, which is measured as the signal obtained with a sample containing the target analyte compared to that obtained with a sample without the target analyte. The use of inadequate amounts of blocker will result in excessive background and a reduced signal-to-noise ratio. The use of excessive concentrations of blocking agent may mask antibody-antigen interactions or inhibit the enzyme, again causing a reduced signal-to-noise ratio. No blocking agent is ideal for all occasions, and empirical testing is essential for true optimisation of the blocking step.

In addition to blocking, it is essential to perform thorough washes between each ELISA step. Wash steps are necessary to remove unbound reagents and decrease the background, thus increasing the signal-to-noise ratio. Insufficient washing will result in high background, while excessive washing may result in decreased sensitivity caused by elution of antibody and/or antigen from the well. Washing is performed in a physiological buffer such as Tris-buffered saline (TBS) or phosphate buffered saline (PBS) without any additives. Typically, a detergent such as 0.05% Tween-20 is added to the buffer to help remove non-specifically bound material. Another common technique is to use a dilute solution of the blocking buffer together with some added detergent. The inclusion of the blocking agent and the addition of a detergent in the wash buffers helps to minimise background in the assay. For best results, use high purity detergents to avoid the introduction of impurities that interfere with the assay, such as enzyme inhibitors or peroxides.


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