Overview of ELISA

ELISA (enzyme-linked immunosorbent assay) is a plate-based assay technique designed for detecting and quantifying 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 complexed with an antibody that is linked to a reporter enzyme. Detection is accomplished by measuring the activity of the reporter enzyme via incubation with the appropriate substrate to produce a measurable product. The most crucial element of an ELISA is a highly specific antibody-antigen interaction.

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The enzyme linked immunosorbent assay (ELISA) is a powerful method for detecting and quantifying a specific protein in a complex mixture. Originally described by Engvall and Perlmann (1971), the method enables analysis of protein samples immobilized in microplate wells using specific antibodies. ELISAs are typically performed in 96-well or 384-well polystyrene plates, which passively bind antibodies and proteins. It is this binding and immobilization of reagents that makes ELISAs easy to design and perform. Having the reactants of the ELISA immobilized to the microplate surface makes it easy to separate bound from non-bound material during the assay. This ability to use high-affinity antibodies and wash away non-specific bound materials makes 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 depend on the same basic elements:

1. Coating/capture—direct or indirect immobilization of antigens to the surface of polystyrene microplate wells.
2. Plate blocking—addition of irrelevant protein or other molecule to cover all unsaturated surface-binding sites of the microplate wells.
3. Probing/detection—incubation with antigen-specific antibodies that affinity-bind to the antigens.
4. Signal measurement—detection of the signal generated via the direct or secondary tag on the specific antibody.

The most commonly used enzyme labels are horseradish peroxidase (HRP) and alkaline phosphatase (AP). Other enzymes have been used as well; these include β-galactosidase, acetylcholinesterase, and catalase. A large selection of substrates is available commercially for performing ELISA with an HRP or AP conjugate. The choice of substrate depends upon the required assay sensitivity and the instrumentation available for signal-detection (spectrophotometer, fluorometer, or luminometer).

There are several formats used for ELISAs. These fall into either direct, indirect, or sandwich capture and detection methods. The key step is immobilization of the antigen of interest, accomplished by either direct adsorption to the assay plate or indirectly via a capture antibody that has been attached to the plate. The antigen is then detected either directly (labeled primary antibody) or indirectly (such as labeled secondary antibody). The most widely used ELISA assay format is the sandwich ELISA assay, which indirectly immobilizes 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 detecting a different epitope of the antigen–the capture antibody and the detection antibody. The sandwich ELISA format is highly used because of its sensitivity and specificity.

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

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

The indirect detection method uses a labeled 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 critical that the secondary antibody is specific for the detection of the primary antibody only (and not the capture antibody) or the assay will not be specific for the antigen. Generally, this is 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 been cross-adsorbed to remove any secondary antibodies that might have affinity for the capture antibody.

Comparison of direct, indirect, and sandwich ELISA detection methods

Besides the standard direct and sandwich formats described above, several other styles of ELISA exist:

Competitive ELISA is a strategy that is commonly used when the antigen is small and has only one epitope or antibody binding site. One variation of this method consists of labeling purified antigen instead of the antibody. Unlabeled antigen from samples and the labeled antigen compete for binding to the capture antibody. A decrease in signal from the purified antigen indicates the presence of the antigen in samples when compared to assay wells with labeled antigen alone.

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

In-cell ELISA is performed with cells that are plated and cultured overnight in standard microplates. After the cultured cells are fixed, permeabilized, and blocked, target proteins are detected with antibodies. This is an indirect assay, not a sandwich assay. The secondary antibodies are either fluorescent (for direct measurement by a fluorescent plate reader or microscope) or enzyme-conjugated (for detection with a soluble substrate using a plate reader).

ELISA is nearly always performed using 96-well 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.

When developing a new ELISA for a specific antigen, the first step is to optimize the plate-coating conditions for the antigen or capture antibody. Begin by choosing an assay microplate (not tissue culture treated plates) with a minimum protein-binding capacity of 400 ng/cm². It is also important that the CV value (coefficient of variation) of the protein binding be low (<5% is preferred) so that there is limited deviation in values that should be identical in the assay results between wells and plates. The choice of plate color depends upon the signal being detected. Clear polystyrene flat bottom plates are used for colorimetric signals while black or white opaque plates are used for fluorescent and chemiluminescent signals. Visually inspect plates before use as imperfections or scratches in the plastic will cause aberrations when acquiring data from the developed assay. Thermo Scientific ELISA Plates are available with a variety of surfaces to optimize coating with the macromolecule of your choice. These plates are designed to deliver optimal results, lot-to-lot reliability, and well-to-well reproducibility.

Plate coating is achieved through passive adsorption of the protein to the plastic of the assay microplate. This process occurs though hydrophobic interactions between the plastic and non-polar protein residues. Although individual proteins may require specific conditions or pretreatment for optimal binding, the most common method for coating plates involves adding a 2–10 μg/ml solution of protein dissolved in an alkaline buffer such as phosphate-buffered saline (pH 7.4) or carbonate-bicarbonate buffer (pH 9.4). The plate is left to incubate for several hours to overnight at 4–37° C. Typically, after removing the coating solution, blocking buffer is added to ensure that all remaining available binding surfaces of the plastic well are covered (see subsequent discussion). Coated plates can be used immediately or dried and stored at 4° C for later use, depending on the stability of the coated protein.

It is important to note that optimal coating conditions and plate binding capacity can vary with each protein/antibody and must be determined experimentally. With the exception of competition ELISAs, the plates are coated with more capture protein than can actually be bound during the assay in order to facilitate the largest working range of detection possible. Some proteins, especially antibodies, are best coated on plates at a concentration lower than the maximum binding capacity in order to prevent nonspecific binding in later steps by a phenomenon called "hooking". Hooking results from proteins getting trapped between the coating proteins, which prevents effective washing and removal of unbound proteins. When hooking nonspecifically traps detection of primary and secondary antibodies, high background signal results, thus lowering the signal to noise ratio and sensitivity of an assay.

For most antibodies and proteins, coating plates by passive adsorption usually works well. However, problems can arise from passive adsorption, including improper orientation, denaturation, poor immobilization efficiency, and binding of contaminants along with the target molecule. Several types of pre-coated plates can help alleviate these issues. Plates pre-coated with Protein A, G, or A/G can help orient capture antibodies properly and preserve their antigen binding capability. Fusion proteins can be attached to a microplate in the proper orientation using glutathione, metal-chelate, or capture-antibody coated plates. Peptides and other small molecules, which typically do not bind effectively by passive adsorption, can be biotinylated and attached with high efficiency to a streptavidin or NeutrAvidin protein coated plate. Biotinylated antibodies also can be immobilized on plates pre-coated with biotin-binding proteins. Using pre-coated plates in this manner physically separates the antigen or capture antibody from the surface of the plate as a protection from its denaturing effects. Polymer coated and modified surfaces can be used to help increase passive adsorption. There is a wide selection of high-performance surface coated plates (pre-coated and pre-blocked) in 96-well and 384-well formats (black, clear or white). These coated microplates can be used for ELISA development and other plate-based assays with colorimetric, fluorescence, or chemiluminescence plate readers.

Different types of microplates for ELISA

The following example illustrates how variations in polymer coatings may impact protein binding capacities.

Either monoclonal or polyclonal antibodies can be used as the capture and detection antibodies in sandwich ELISA and other ELISA systems. Monoclonal antibodies have inherent monospecificity toward a single epitope that allows fine detection and quantitation of small differences in antigen. Polyclonal antibodies are often used as the capture antibody to pull down as much of the antigen as possible. Then a monoclonal is used as the detecting antibody in the sandwich assay to provide improved specificity. In addition to the use of traditional monoclonal antibodies, recombinant monoclonal antibodies may also be utilized for ELISA. Recombinant antibodies are derived from antibody-producing cell lines engineered to express specific antibody heavy and light chain DNA sequences. Compared to traditional monoclonal antibodies derived from hybridomas, recombinant antibodies are not susceptible to cell-line drift or lot-to-lot variation, thus allowing for peak antigen specificity.

An important consideration in designing a sandwich ELISA is that the capture and detection antibodies must recognize two different non-overlapping epitopes. When the antigen binds to the capture antibody, the epitope recognized by the detection antibody must not be obscured or altered. Capture and detection antibodies that do not interfere with one another and can bind simultaneously are called "matched pairs" and are suitable for developing a sandwich ELISA. Many primary antibody suppliers provide information about epitopes and indicate pairs of antibodies that have been validated in ELISA as matched pairs. Using the same antibody for the capture and detection can limit the dynamic range and sensitivity of the final ELISA.

The binding capacity of microplate wells is typically higher than the amount of protein coated in each well. The remaining surface area 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, mixture of proteins, 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 background signal and improving the signal-to-noise ratio. The ideal blocking buffer will bind to all potential sites of nonspecific interaction, eliminating background altogether, without altering or obscuring the epitope for antibody binding.

When developing any new ELISA, it is important to test several different blockers for the highest signal to noise ratio in the assay. Many factors can influence nonspecific binding, including various protein-protein interactions unique to the samples and 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 as compared to that obtained with a sample without the target analyte. Using inadequate amounts of blocker will result in excessive background and a reduced signal to noise ratio. Using excessive concentrations of blocker may mask antibody-antigen interactions or inhibit the enzyme, again causing a reduction of the signal to noise ratio. No single blocking agent is ideal for every occasion, and empirical testing is essential for true optimization of the blocking step.

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

The final stage in all ELISA systems is a detection step. Unless a radioactive or fluorescent tag was used, this involves the introduction of an enzyme substrate. The enzyme converts the substrate to a detectable product. If an ELISA has been constructed and developed properly, then the intensity of signal produced when the substrate is added will be directly proportional to the amount of antigen captured in the plate and bound by the detection reagents. Enzyme-conjugated antibodies (especially those involving horseradish peroxidase, HRP) offer the most flexibility in detection and documentation methods for ELISA because of the variety of substrates available for chromogenic, chemifluorescent, and chemiluminescent imaging.

Colorimetric substrates form a soluble, colored product that accumulates over time relative to the amount of enzyme present in each well. When the desired color intensity is reached, the product absorbance is either measured directly or in some cases a stop solution is added to provide a fixed end point for the assay. Colorimetric substrates are available for both horseradish peroxidase (TMB, OPD, ABTS) and alkaline phosphatase (PNPP). Though not as sensitive as fluorescent or chemiluminescent substrates, chromogenic ELISA substrates allow direct visualization and enable kinetic studies to be performed. Furthermore, chromogenic ELISA substrates are detected with standard absorbance plate readers common to many laboratories.

Chemiluminescence is a chemical reaction that generates energy released in the form of light. Most chemiluminescent substrates are HRP-dependent, although some AP equivalents are available. The most common approach is to use luminol in the presence of HRP and a peroxide buffer. The luminol is oxidized and forms an excited state product that emits light as it decays to the ground state. Light emission occurs only during the enzyme-substrate reaction, therefore when the substrate becomes exhausted, the signal ceases. Chemiluminescent detection is generally considered to be more sensitive than colorimetric detection. One drawback of using chemiluminescent substrates for ELISA is that the signal intensity can vary more than with other substrates. For assays requiring many plates to be read, this can present a problem if the signal begins to decay before plates are read. For this reason, it is important to make sure the assay has been optimized with the substrate in order to avoid misinterpreting signal-fade in a sample as low antigen abundance. Chemiluminescent substrates for HRP include Thermo Scientific SuperSignal ELISA Pico and ELISA Femto substrates.

Fluorescent ELISA substrates are not as common and require a fluorometer that produces the correct excitation beam to cause signal emission to be generated from the fluorescent tag. Chemifluorescent detection is also enzyme-based, but the generated product is fluorescent rather than colorimetric. The signal is measured using a fluorometer with the appropriate excitation and emission filters. Chemifluorescence reactions are either measured over time in kinetic assays or halted using a stop solution for direct measurement. Examples of chemifluorescent substrates for HRP are Thermo Scientific QuantaRed and QuantaBlu substrates.

In addition to the individual components and general principles of ELISA discussed in this article, ready-to-use ELISA kits are commercially available for detection of hundreds of specific cytokines, chemokines, growth factors, neurobiology analytes, and phosphorylated proteins that are common targets of research interest.

For many targets, two kit types are available:

• Coated ELISA kits—containing pre-coated antibody-plates, detection antibodies, buffers, diluents, standards, and substrates. In addition to traditional ELISA kits, instant ELISA kit plates are also available that contain all the necessary components including capture antibody and lyophilized detection antibody, streptavidin-HRP, and sample diluent. In addition, strip wells containing the standard for the standard curve are provided separately to allow full use of the 96 wells for assay samples.

• Uncoated ELISA kits—These kits come with all the reagents needed to coat your own plate and to run the assay with the exception of the stop solution and wash buffer.
• Antibody pair kits—These kits contain optimized matched antibodies for sandwich ELISA development and standards (no plates or detection reagents).

This ELISA format selection guide compares characteristics of Invitrogen antibody pair kits and ELISA kits.

1. John R. Crowther, Methods in Molecular Biology, The ELISA Guidebook. Second Edition. Humana Press, a part of Springer Science + Business Media, LLC 2009.
2. Butler J.E. The Behavior of Antigens and Antibodies Immobilized on a Solid Phase. In: M.H.V. Van Regenmortel, ed. Structure of Antigens. Boca Raton, FL: CRC Press, 1992: 209-259. Vol.1, 209; CRC Press, Inc.
3. Lequin, Rudolf M. Enzyme immunoassay (EIA)/enzyme-linked immunosorbent assay (ELISA). Clinical chemistry 51.12 (2005): 2415-2418.
4. Engvall, Eva, and Peter Perlmann. Enzyme-linked immunosorbent assay (ELISA) quantitative assay of immunoglobulin G. Immunochemistry 8.9 (1971): 871-874.
   
   

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