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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-
elisa
http://www.sumanasinc.com/webcontent/anim
ations/content/ELISA.html
http://www.biology.ualberta.ca/facilities/multi
media/uploads/procedures/elisa-sound.swf
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