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Antibodies (also known as immunoglobulins

abbreviated Ig) are gamma globulin proteins

that are found in blood and are used by the

immune system to identify and neutralize

foreign objects, such as bacteria and viruses.


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Antigens : A substance that when introducedinto the body stimulates the production of anantibody

Immunoassay: A laboratory technique thatmakes use of the binding between an antigen

and its homologous antibody in order toidentify and quantify the specific antigen orantibody in a sample


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Analyte: The sample being analyzed and in

immunoasssays the analyte is either Antibody

or Antigen


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It is a biochemical technique used mainly inimmunology to detect the presence of anantibody or an antigen in a sample.

ELISA is so named because the techniqueinvolves the use of an immunosorbent, anabsorbing material specific for one of thecomponents of the reaction, the antigen or

antibody. ELISA is usually done using 96-well microtitre

plate suitable for automation


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Technique used to detect (assay) specific

molecules (e.g. proteins & carbohydrates) in

samples.

Immunological technique: uses antibodies.

Quantitative.

Very sensitive.Commonly used in medicine and scientific

research.


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Substrate

Primary

antibody

Secondary

antibody

Different antigens in sample

Coloured

product


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The ELISA plate is coated with Antibody to

detect specific antigen


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Prepare a surface to which a known quantity of

capture antibody is bound.

Block any non specific binding sites on the

surface

Apply the antigen-containing sample to the

plate.

Wash the plate, so that unbound antigen is

removed.


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Apply enzyme linked primary antibodies as

detection antibodies which also bind

specifically to the antigen.

Wash the plate, so that the unbound antibody-

enzyme conjugates are removed.

Apply a chemical which is converted by the

enzyme into a coloured product.

Measure the absorbency of the plate wells to

determine the presence and quantity of antigen


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e.g : Assay of thyroid hormone (T4)


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The protein antigen to be tested for is added to

each well of ELISA plate, where it is given

time to adhere to the plastic through charge

interactions

A solution of non-reacting protein is added to

block any plastic surface in the well that

remains uncoated by the protein antigen


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Then the serum is added, which contains a mixtureof the serum antibodies, of unknownconcentration, some of which may bind

specifically to the test antigen that is coating thewell.

Afterwards, a secondary antibody is added, which

will bind to the antibody bound to the test antigenin the well. This secondary antibody often has anenzyme attached to it


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A substrate for this enzyme is then added. Often, thissubstrate changes colour upon reaction with theenzyme. The colour change shows that secondaryantibody has bound to primary antibody, which strongly

implies that the donor has had an immune reaction tothe test antigen.

The higher the concentration of the primary antibody

that was present in the serum, the stronger the colourchange. Often a spectrometer is used to givequantitative values for colour strength


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This method is largely used to measure

antibodies in almost all human infections

e.g : HIV antibody detection

In patients with AIDS, the human immuno

deficiency virus(HIV) produces specific

antibody.

To detect the HIV antibody, indirect ELISA

method is used


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Serum Antibody Concentrations

Detecting potential food allergens

(milk, peanuts, walnuts, almonds and eggs)

Disease outbreaks- tracking the spread of

disease

e.g. HIV, bird flu, common, colds, cholera, STD etc


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Detections of antigens

e.g. pregnancy hormones, drug allergen, , mad cow disease

Human chorionic gonadotropin (HCG), the commonly

measured protein which indicates pregnancy.

A mixture of purified HCG linked (coupled) to an enzyme and

the test sample (blood, urine, etc) are added to the test system.

If no HCG is present in the test sample, then only HCG withlinked enzyme will bind.


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The more HCG which is present in the test

sample, the less enzyme linked HCG will bind.

The substrate the enzyme acts on is then

added, and the amount of product measured,

such as a change in color of the solution.


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Detection of antibodies in blood sample forpast exposure to disease.

e.g. Lyme Disease, trichinosis, HIV, bird flu

ELISA can also be used in toxicology as arapid presumptive screen for certain classes of

drugs.


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To monitor diabetes, glucose concentrations will be

checked.

Bacterial left-over in milk can be determined with an

ELISA test.

ELISA assays are as well employed in foodstuff

protection through signifying the occurrence of

salmonella

ELISA assays can also be utilised to diagnose several

cancers(eg:bladdercancer)


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ELISA tests are generally relatively accuratetests.

Highly sensitive and specific

Antigens of very low or unknownconcentration can be detected since captureantibody only grabs specific antigen

Generally safe: do not require radioactivesubstances, contains diluted sulfuric acid

Used in wide variety of tests


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Only monoclonal antibodies can be used as matched

pairs

Monoclonal antibodies can cost more than polyclonal

antibodies

Monoclonal antibodies are more difficult to find

Negative controls may indicate positive results if

blocking solution is ineffective [secondary Ab or antigen

can bind to open sites in well]

Enzyme/substrate reaction is short term so microwells

must be read as soon as possible


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http://www.edumedia-sciences.com/en/a543-

direct-enzyme-linked-immunosorbent-assay-

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