ELISA evolved in the late 1960s from RIA (radioimmunoassay) with the observation that either the antibody or the analyte (antigen) could be adsorbed to a solid surface and still participate in specific high affinity binding. The adsorption process facilitated the separation of bound and free analyte, a situation that proved difficult to engineer for many analytes with RIA. Over the intervening years, the term ELISA has been applied to a wide variety of immunoassays, some of which do not employ enzymes and some of which do not require the separation of bound and free analyte. The distinguishing feature of all of these assays remains the use of antibodies to detect an analyte.

The term ELISA was first used by Engvall & Perlma in 1971

direct elisa

Protocol step by step

APPLICATIONDr Dennis E Bidwell and Alister Voller created the ELISA test to detect various kind of diseases, such

as malaria, Chagas disease, and Johne's disease.[11] 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

Because the ELISA can be performed to evaluate either the presence of antigen or the presence of antibody in

a sample, it is a useful tool for determining serum antibody concentrations (such as with the HIV test[9] or West

Nile virus). It has also found applications in the food industry in detecting potential food allergens, such

as milk, peanuts, walnuts, almonds, and eggs.[10] ELISA can also be used in toxicology as a rapid presumptive

screen for certain classes of drugs.

The ELISA was the first screening test widely used for HIV because of its high sensitivity. In an ELISA, a

person's serum is diluted 400 times and applied to a plate to which HIV antigens are attached. If antibodies to

HIV are present in the serum, they may bind to these HIV antigens. The plate is then washed to remove all

other components of the serum. A specially prepared "secondary antibody" — an antibody that binds to other

antibodies — is then applied to the plate, followed by another wash. This secondary antibody is chemically

linked in advance to an enzyme.

Thus, the plate will contain enzyme in proportion to the amount of secondary antibody bound to the plate. A

substrate for the enzyme is applied, and catalysis by the enzyme leads to a change in color or fluorescence.

ELISA results are reported as a number; the most controversial aspect of this test is determining the "cutoff"

point between a positive and a negative result.

A cutoff point may be determined by comparing it with a known standard. If an ELISA test is used for drug

screening at the workplace, a cutoff concentration, 50 ng/ml, for example, is established, and a sample

containing the standard concentration of analyte will be prepared. Unknowns that generate a stronger signal

than the known sample are "positive." Those that generate weaker signal are "negative".

LIMITATAIONSOne limitation of the ELISA technique is that it provides information on the presence of an

analyte but no information on its biochemical properties, such as molecular weight or its spatial

distribution in a tissue. To obtain this information one needs to perform other types of assays.

For example, blotting assays combine separations based on physical properties of the analyte

with detection techniques Immunohistochemical assays performed on tissue and cells provide

information on the specific location of an analyte. (see Immunohistochemical Staining:

Principles and Practices).Both of these techniques can also provide some quantitation of the

analyte, but not as accurately as ELISA.