ENZYME-LINKED IMMUNOSORBENT ASSAY

HISTORY• Radioimmunoassay was first described in a scientific

paper by Rosalyn Sussman Yalow and Solomon Berson published in 1960.

• Before the development of the ELISA, the only option for an immunoassay was radioimmunoassay, a technique using radioactively labeled antigens or antibodies.

• Because radioactivity poses a potential health threat, a safer alternative was sought.

• A suitable alternative to radioimmunoassay would substitute a nonradioactive signal in place of the radioactive signal.

INTRODUCTION

• The ELISA is a test that uses antibodies and color change to identify a substance.

• ELISA is a popular format of "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.

• 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 (polystyrene microtiter plate) either non-specifically or specifically .

• 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 enzym through bioconjugation.

• Between each step, the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are non-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.

PRINCIPLE• ELISA involves detection of an "analyte" in a liquid sample

by a method that continues to use liquid reagents during the "analysis" 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, the final detection step in "dry" analysis involves reading of a dried strip by methods such as reflectometry.

• 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 spectrophotometry.

• 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".

TYPES OF ELISA

• Direct ELISA• Indirect ELISA• Sandwich ELISA• Competitive ELISA

DIRECT ELISAThe steps of direct ELISA:1. A buffered solution of the antigen to be tested for is

added to each well of a microtiter plate, where it is given time to adhere to the plastic through charge interactions.

2. A solution of nonreacting protein, such as bovine serum albumin or casein, is added to well (usually 96-well plates) in order to cover any plastic surface in the well which remains uncoated by the antigen.

3. The primary antibody with an attached (conjugated) enzyme is added, which binds specifically to the test antigen coating the well.

4. A substrate for this enzyme is then added. Often, this substrate changes color upon reaction with the enzyme.

5. The higher the concentration of the primary antibody present in the serum, the stronger the color change. Often, a spectrometer is used to give quantitative values for color strength

https://en.wikipedia.org/wiki/File:ELISA_diagram.png

• The enzyme acts as an amplifier; even if only few enzyme-linked antibodies remain bound, the enzyme molecules will produce many signal molecules.

• More antibody is bound, the faster the color will develop.

DISADVANTAGE• A major disadvantage of the direct ELISA is the

method of antigen immobilization is not specific.• when serum is used as the source of test

antigen, all proteins in the sample may stick to the microtiter plate well, so small concentrations of analyte in serum must compete with other serum proteins when binding to the well surface.

• The sandwich or indirect ELISA provides a solution to this problem, by using a "capture" antibody specific for the test antigen to pull it out of the serum's molecular mixture.

INDIRECT ELISA• When the presence of an antigen is analyzed, the name

"direct ELISA" refers to an ELISA in which only a labelled primary antibody is used.

• The term "indirect ELISA" refers to an ELISA in which the antigen is bound by the primary antibody which then is detected by a labeled secondary antibody.

• In the latter case a sandwich ELISA is clearly distinct from an indirect ELISA. When the 'primary' antibody is of interest, e.g. in the case of immunization analyses, this antibody is directly detected by the secondary antibody and the term "direct ELISA" applies to a setting with two antibodies.

SANDWICH ELISA The steps are:1. A surface is prepared to which a known quantity of capture

antibody is bound.2. Any nonspecific binding sites on the surface are blocked.3. The antigen-containing sample is applied to the plate, and

captured by antibody.4. The plate is washed to remove unbound antigen.5. A specific antibody is added, and binds to antigen (hence the

'sandwich': the Ag is stuck between two antibodies). This primary antibody could also be in the serum of a donor to be tested for reactivity towards the antigen.

6. Enzyme-linked secondary antibodies are applied as detection antibodies that also bind specifically to the antibody's Fc region (nonspecific).

7. The plate is washed to remove the unbound antibody-enzyme conjugates.

8. A chemical is added to be converted by the enzyme into a color or fluorescent or electrochemical signal.

9. The absorbance or fluorescence or electrochemical signal (e.g., current) of the plate wells is measured to determine the presence and quantity of antigen.

https://en.wikipedia.org/wiki/File:ELISA-sandwich.svg

COMPETITIVE ELISA

The steps for this ELISA:1. Unlabeled antibody is incubated in the presence of

its antigen (sample).2. These bound antibody/antigen complexes are then

added to an antigen-coated well.3. The plate is washed, so unbound antibodies are

removed. (The more antigen in the sample, the more Ag-Ab complexes are formed and so there are less unbound antibodies available to bind to the antigen in the well, hence "competition".)

4. The secondary antibody, specific to the primary antibody, is added. This second antibody is coupled to the enzyme.

5. A substrate is added, and remaining enzymes elicit a chromogenic or fluorescent signal.

6. The reaction is stopped to prevent eventual saturation of the signal.

For the detection of HIV antibodies1.  The wells of microtiter plate are coated with the HIV

antigen. 2. Two specific antibodies are used, one conjugated with

enzyme and the other present in serum (if serum is positive for the antibody).

3. Cumulative competition occurs between the two antibodies for the same antigen, causing a stronger signal to be seen.

4. Sera to be tested are added to these wells and incubated at 37 °C, and then washed.

5. If antibodies are present, the antigen-antibody reaction occurs.

6. No antigen is left for the enzyme-labelled specific HIV antibodies.

7. These antibodies remain free upon addition and are washed off during washing.

8. Substrate is added, but there is no enzyme to act on it, so positive result shows no color change.

Applications It is a useful tool for determining serum antibody

concentrations such as with the HIV test 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 and as serological blood test forcoeliac disease.

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.

• ELISA test to detect various kind of diseases, such as malaria, Chagas disease, andJohne'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 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