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.


Antigens : A substance that when introduced
into the body stimulates the production of an
antibody

Immunoassay: A laboratory technique that
makes use of the binding between an antigen
and its homologous antibody in order to
identify and quantify the specific antigen or
antibody in a sample


Analyte: The sample being analyzed and in
immunoasssays the analyte is either Antibody
or Antigen


It is a biochemical technique used mainly in
immunology to detect the presence of an
antibody or an antigen in a sample.
ELISA is so named because the technique
involves the use of an immunosorbent, an
absorbing material specific for one of the
components of the reaction, the antigen or
antibody.
ELISA is usually done using 96-well microtitre
plate suitable for automation


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.

Substrate
Primary
antibody
Secondary
antibody
Different antigens in sample
Coloured
product
The ELISA plate is coated with Antibody to
detect specific antigen

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.




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


e.g : Assay of thyroid hormone (T4)
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



Then the serum is added, which contains a mixture
of the serum antibodies, of unknown
concentration, some of which may bind
specifically to the test antigen that is coating the
well.

Afterwards, a secondary antibody is added, which
will bind to the antibody bound to the test antigen
in the well. This secondary antibody often has an
enzyme attached to it



A substrate for this enzyme is then added. Often, this
substrate changes colour upon reaction with the
enzyme. The colour change shows that secondary
antibody has bound to primary antibody, which strongly
implies that the donor has had an immune reaction to
the test antigen.

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


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



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


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 with
linked enzyme will bind.

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.


Detection of antibodies in blood sample for
past exposure to disease.
e.g. Lyme Disease, trichinosis, HIV, bird flu

ELISA can also be used in toxicology as a
rapid presumptive screen for certain classes of
drugs.


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)





ELISA tests are generally relatively accurate
tests.
Highly sensitive and specific
Antigens of very low or unknown
concentration can be detected since capture
antibody only grabs specific antigen
Generally safe: do not require radioactive
substances, contains diluted sulfuric acid
Used in wide variety of tests




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



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