Enzyme Linked Immunosorbent Assay ELISA

Enzyme-linked immuno sorbent assay (ELISA is a test that uses antibodies and color change to identify a substance. ELISA is a popular format of a "wet-lab" type analytic biochemistry assay that uses a 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. Antigens from the sample are attached to a surface. Then, a further specific antibody is applied over the surface so 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

ELISAs are typically performed in 96-well (or 384-well) polystyrene plates, which will passively bind antibodies and proteins. It is this binding and immobilization of reagents that makes ELISAs so easy to design and perform. Having the reactants of the ELISA immobilized to the microplate surface makes it easy to separate bound from nonbound material during the assay. This ability to wash away nonspecifically bound materials makes the ELISA a powerful tool for measuring specific analytes within a crude preparation.

Principle 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 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 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.

Types of ELISA Direct ELISA An antigen coated to a multiwell plate is detected by an antibody that has been directly conjugated to an enzyme. This can also be reversed, with an antibody coated to the plate and a labeled antigen used for detection, but the second option is less common. This type of ELISA has two main advantages: It is faster, since fewer steps are required It is less prone to error, since there are fewer steps and reagents

IN direct ELISA Antigen coated to a polystyrene multiwell plate is detected in two stages or layers. First an unlabeled primary antibody, which is specific for the antigen, is applied. Next, an enzyme-labeled secondary antibody is bound to the first antibody. The secondary antibody is usually an anti-species antibody and is often polyclonal. This method has several advantages: Increased sensitivity, since more than one labeled antibody is bound per primary antibody Flexibility, since different primary detection antibodies can be used with a single labeled secondary antibody Cost savings, since fewer labeled antibodies are required

Sandwich ELISA Sandwich ELISAs typically require the use of matched antibody pairs, where each antibody is specific for a different, non-overlapping part (epitope) of the antigen molecule. The first antibody, termed the capture antibody, is coated to the polystyrene plate. Next, the analyte or sample solution is added to the well. A second antibody layer, the detection antibody, follows this step in order to measure the concentration of the analyte. Polyclonals can also be used for capture and/or detection in a sandwich ELISA provided that variability is present in the polyclonal to alow for both capture and detection of the analyte through different epitopes. If the detection antibody is conjugated to an enzyme, then the assay is called a direct sandwich ELISA. If the detection antibody is unlabeled, then a second detection antibody will be needed resulting in an indirect sandwich ELISA. This type of assay has several advantages: High specificity, since two antibodies are used the antigen/analyte is specifically captured and detected Suitable for complex samples, since the antigen does not require purification prior to measurement Flexibility and sensitivity, since both direct and indirect detection methods can be used

advantages of this technique is the ability to obtain quick and accurate results 1. 2.They are considered highly sensitive, specific and compare favorably with other methods used to detect substances in the body, such as radioimmune assay (RIA) tests. 3.The sample volume can be increased to improve the test sensitivity in clinical (blood, saliva, urine), food (bulk milk, pooled eggs) and environmental (water) samples. 4 .not needing radioisotopes (radioactive substances) or a costly radiation counter (a radiation-counting apparatus).

DISADVANTAGES: Only monoclonal antibodies can be used as matched pairs (recognize one specific bindng site also called epitope) Monoclonal antibodies can cost more than polyclonal antibodies Monoclonal antibodies more difficult to find Negative controls may indicate positive results if blocking solution is ineffective [secondary antibody or antigen (unknown sample) can bind to open sites in well] Enzyme/substrate reaction is short term so microwells must be read as soon as possible

Applications ELISA test to detect various kind of diseases, such as malaria, Chagas disease, and Johne's disease .ELISA tests also are used as in in vitro diagnostics in medical laboratories. The other uses of ELISA include: detection of Mycobacterium antibodies in tuberculosis detection of rotavirus in feces detection of hepatitis B markers in serum detection of enterotoxin of E. coli in feces detection of HIV antibodies in blood samples

The ELISA tests have also found applications in the food industry in detecting food allergens, such as milk, peanuts, walnuts, almonds, and eggs ELISA tests can also be used in toxicology as a screen for certain classes of drugs.