ELISA ( Enzyme-linked immunosorbent assay)

23.03.2018

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Enzyme-Linked Immunosorbent Assay (ELISA)
A sought after marker in a biological sample can be quantified using the Enzyme-linked immunosorbent assay, or ELISA, method. The technique works for peptide, hormone, protein or antibody markers. The ELISA method can analyse a number of kinds of samples of across a breadth of origins. The use of ELISA methods in specific marker quantification has many advantages over qualitative methods such as Western Blotting. ELISA is quicker, more flexible, easier to replicate, as well as being highly sensitive with high throughput.

A popular format of “wet-lab” analytic biochemistry assay, ELISA is used in quality-control for various industries, and as diagnostic tool in plant pathology and medicine. To discover the presence of matter, usually an antigen, the method uses a solid-phase enzyme immunoassay or EIA, in a wet sample.

The method requires antigens to be taken from the sample and attached to a surface. A particular antibody is then applied to the surface so that it is bound to the antigen. After linking the enzyme to the antibody, a substance is added that contains the enzyme’s substrate. A colour change in the substrate is then produced as a result of the reaction to this final step.

A minimum of one antibody relating uniquely to a specific antigen is involved in ELISA. A polystyrene microtiter plate is used as a support to immobilise a sample with an unknown quantity of the antigen. The immobilisation occurs either specifically, in a “sandwich”, through capture by another antibody particular to that antigen, or, non-specifically, through surface adsorption. 

The detection antibody is introduced after the immobilisation process has been applied to the antigen, forming a complex. A supplementary antibody linked to an enzyme through bioconjugation can be used to detect the detection antibody itself, or this can be linked to an enzyme covalently. 

The plate is usually washed with mild detergent between each step to remove non-specifically bound antibodies or proteins. An enzymatic substrate is added to develop the plate after washing for the last time. This produces a visible signal indicating the amount of antigen that exists in the sample.

The word “immuno” is in included in ELISA because of the development history of the technique. However, ELISA is not restricted to “immuno” assays and has the ability to execute other forms of ligand binding assays. In essence, the method requires the immobilisation of a ligating reagent on the solid phase applied to a detections reagent to specifically bind and use an enzyme to return a properly quantifiable signal.

The non-specific or unbound components are washed away in between washes, with only the ligand and its specific binding equivalents remaining specifically bound by antigen-antibody interactions to the solid phase. ELISA plates are not easily reusable because they have the reaction products immunosorbed on the solid phase. In this way, ELISA differs from other spectrophotometric wet lab assay formats that use a cuvette, and the same reaction well can be reused after washing.

ELISA is an analytic biochemistry assay that involves the detection of the particular physical entity whose existence is being analysed either qualitatively or quantitatively in a liquid state. This physical entity is also known as the “analyte.” 

The technique uses a controlled sequence of biochemical reactions to generate an easily quantifiable signal that can be interpreted as a measurement of the quantity of analyte present in the sample. This is achieved through the use of liquid reagents that remain within the reaction chamber to contain the reactants. This process differs from “dry lab” techniques that use dry strips even when analysing liquid samples. These dry strips are read using reflectometry and a reaction containment chamber is not required to prevent samples becoming mixed or spilling over.

ELISA is a heterogeneous assay and absorbs some components onto a solid, physically immobilised phase. Thus, some components of the analytical reaction mixture are separated. A stationary solid phase with particular binding properties is combined with a liquid sample and several liquid reagents, which are added in sequential order, washed and incubated. This produces a visible change in the final liquid such as an enzymatic reaction producing colour development. The quantity of the analyte is measured from this result. This provides a quantifiable reading based on the intensity of light transmitted by spectrophotometric processes. Such processes involve the precise measurement of particular wavelength transmission of light through the liquid. This is also measure through the bottom of the well, which is transparent, in the multiple-well plate format.

Detection sensitivity is dependent on amplification of the signal during analytic reactions. The signal is created by enzyme reactions, which are well known amplification processes, linked to detection reagents in set proportions, facilitating accurate measurement. This gives the name “enzyme linked.” 

Because the analyte will bind or ligate to a detection reagent, it is known as the ligand. In this way, ELISA is categorised among the bigger ligand binding assays. The ligand-specific binding reagent is usually coated and dried onto the diaphanous bottom and occasionally to a side wall of a well to immobilise it. This is known as the “ELISA” plate and is usually constructed as a multiple-well plate. The ELSA plate is the stationary solid phase or substrate, not to be confused with the solid microparticles or beads that can be washed away. 

As with other forms of immunoassays, the particular antigen-antibody type reaction is used because it is easy to raise an antibody targetted at bulk antigen reagent. A target antigen can be used as the binding reagent if the analyte itself is an antibody.

Article last time updated on 09.04.2018.

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