ELISA Kits / ELISA Assays

Enzyme-linked immunosorbent assay (ELISA) kits make it easier to use ELISAs for detecting molecules or performing ligand-binding assays successfully. Many ELISA kits use the detection enzymes horseradish peroxidase (HRP) or alkaline phosphatase (AP), along with chromogenic, fluorescent, or chemiluminescent substrates. Alternative detection strategies, such as fluorescent tags or other detectable probes, are also accepted under the umbrella term of ELISAs if they are plate-based methods, regardless of whether they use reporter enzymes or antibodies for detection. There are several ELISA formats, including the “sandwich,” competitive, direct and indirect detection assays. In the robust sandwich assay, the target is adsorbed to the assay plate, then bound by two primary antibodies. The competitive assay is often used for smaller targets that may have only one binding site. In direct detection, a labeled primary antibody binds to the target. In indirect detection, the most common protocol for ELISAs, the target is bound by the primary antibody, and detected when a labeled secondary antibody binds to the primary antibody. Related articles:elisa kits(http://www.creative-diagnostics.com/ELISA-Assays.html)

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Principle of ELISA

As a "wet lab" analytic biochemistry assay, ELISA involves detection of an "analyte" (i.e. the specific substance whose presence is being quantitatively or qualitatively analyzed) in a liquid sample by a method that continues to use liquid reagents during the "analysis" (i.e. controlled sequence of biochemical reactions that will generate a signal which can be easily measured quantified and interpreted as a measure of the amount of analyte in the sample) that stays liquid and remains inside a reaction chamber or well that is needed to keep the reactants contained; It is opposed to "dry lab" that can use dry strips - and even if the sample is liquid (e.g. a measured small drop), the final detection step in "dry" analysis involves reading of a dried strip by methods such as reflectometry and does not need a reaction containment chamber to prevent spillover or mixing between samples. As a heterogenous assay, ELISA separates some component of the analytical reaction mixture by adsorbing certain components onto a solid phase which is physically immobilized. In ELISA a liquid sample is added onto a stationary solid phase with special binding properties and is followed by multiple liquid reagents that are sequentially added, incubated and washed followed by some optical change (e.g. color development by the product of an enzymatic reaction) in the final liquid in the well from which the quantity of the analyte is measured. The qualitative "reading" usually based on detection of intensity of transmitted light by spectrophotometric which involves quantitation of transmission of some specific type of light through the liquid (as well as the transparent bottom of the well in the multi-well plate format). The sensitivity of detection depends on amplification of the signal during the analytic reactions. Since enzyme reactions are very well known amplification processes, the signal is generated by enzymes which are linked to the detection reagents in fixed proportions to allow accurate quantification - thus the name "Enzyme linked". The analyte is also called the ligand because it will specifically bind or ligate to a detection reagent and thus ELISA falls under the bigger category of Ligand Binding Assays. The ligand-specific binding reagent is "immobilized" i.e. usually coated and dried onto the transparent bottom and sometimes also side wall of a well (the stationary "solid phase'/"solid substrate" here as opposed to solid microparticle/beads that can be washed away), which is usually constructed as a multi-well plate known as the "Elisa Plate". Conventionally, like other forms of immunoassays the specificity of Antigen-Antibody type reaction is used because it is easy to raise an antibody specifically against an antigen in bulk as a reagent. Alternatively if the analyte itself is an antibody its target antigen can be used as the binding reagent.

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Enzyme-linked immunosorbent assay (ELISA), is a popular format of a "wet-lab" type analytic biochemistry assay that uses one sub-type of heterogeneous, solid-phase enzyme immunoassay (EIA) to detect the presence of a substance, usually an antigen, in a liquid sample or wet sample. The ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as a quality-control check in various industries. Attempting to detect (and quantify) the presence of the antigen in the sample proceeds as follows: Antigens from the sample are attached to a surface. Then a further specific antibody is applied over the surface so that it can bind to the antigen. This antibody is linked to an enzyme, and, in the final step, a substance containing the enzyme's substrate is added. The subsequent reaction produces a detectable signal, most commonly a color change in the substrate. Performing an ELISA involves at least one antibody with specificity for a particular antigen. The sample with an unknown amount of antigen is immobilized on a solid support (usually a polystyrene microtiter plate) either non-specifically (via adsorption to the surface) or specifically (via capture by another antibody specific to the same antigen, in a "sandwich" ELISA). After the antigen is immobilized, the detection antibody is added, forming a complex with the antigen. The detection antibody can be covalently linked to an enzyme, or can itself be detected by a secondary antibody that is linked to an enzyme through bioconjugation. Between each step, the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are not specifically bound. After the final wash step, the plate is developed by adding an enzymatic substrate to produce a visible signal, which indicates the quantity of antigen in the sample. It is worth noting that ELISA can perform other forms of ligand binding assays instead of strictly "immuno" assays, though the name carried the original "immuno" because of the common use and history of development of this method. The technique essentially requires any ligating reagent that can be immobilized on the solid phase along with a detection reagent that will bind specifically and use an enzyme to generate a signal that can be properly quantified. In between the washes only the ligand and its specific binding counterparts remain specifically bound or "immunosorbed" by antigen-antibody interactions to the solid phase, while the nonspecific or unbound components are washed away. Unlike other spectrophotometric wet lab assay formats where the same reaction well (e.g. a cuvette) can be reused after washing, the ELISA plates have the reaction products immunosorbed on the solid phase which is part of the plate and thus are not easily reusable.

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