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
ELISA is usually done using 96-well microtitre
plate suitable for automation
What is ELISA?
• Technique used to detect (assay) specific
molecules (e.g. proteins & carbohydrates) in
• Immunological technique: uses antibodies.
• Very sensitive.
• Commonly used in medicine and scientific
Different antigens in sample
The technique is divided into :
1- Competitive ELISA
2- Sandwich ELISA (also called direct ELISA)
3- Indirect ELISA
The labelled antigen competes for primary antibody
binding sites with the sample antigen (unlabeled).
The more antigen in the sample, the less labelled
antigen is retained in the well and the weaker the
The ability to use crude or impure samples and still
selectively bind any antigen that may be present.
Used for the detection of human T-cell leukemia-
lymphoma virus type III (HTLV-III).
COLOR OF SOLUTION &
COLOR OF SOLUTION ARE
COMPLEMENTARYCOLOR OF FILTER WAVELENGTH COLOR OF
VIOLET 420 BROWN
BLUE 470 YELLOWISH BROWN
GREEN 520 PINK
YELLOW 580 PURPLE
RED 680 GREEN/BLUE
This method is largely used to measure
antibodies in almost all human infections
Eg : HIV antibody detection
In patients with AIDS, the human immuno
deficiency virus(HIV) produces specific
To detect the HIV antibody, indirect ELISA
method is used
MULTIPLE & PORTABLE ELISA
This new technique uses a solid phase made
up of an immunosorbent polystyrene rod with
8-12 protruding ogives.
The entire device is immersed in a test tube
containing the collected sample and the
following steps (washing, incubation in
conjugate and incubation in chromogenous)
are carried out by dipping the ogives in
microwells of standard microplates pre-filled
The advantages of this technique are as follows:
The ogives can each be sensitized to a different
reagent, allowing the simultaneous detection of
different antibodies and/or different antigens for multi-
The sample volume can be increased to improve the
test sensitivity in clinical (saliva, urine), food (bulk milk,
pooled eggs) and environmental (water) samples
One ogive is left unsensitized to measure the non-
specific reactions of the sample
The use of laboratory supplies for dispensing sample
aliquots, washing solution and reagents in microwells
is not required
APPLICATIONS OF ELISA
Serum Antibody Concentrations
Detecting potential food allergens
(milk, peanuts, walnuts, almonds and eggs)
Disease outbreaks- tracking the spread
e.g. HIV, bird flu, common, colds, cholera,
Detections of antigens
e.g. pregnancy hormones, drug
, mad cow disease
•human chorionic gonadotropin (HCG), the
commonly measured protein which indicates
•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
ELISA can also be used in toxicology as
a rapid presumptive screen for certain
classes of drugs.
To monitor diabetes, glucose concentrations will be
Bacterial left-over in milk can be determined with an
ELISA assays are as well employed in foodstuff
protection through signifying the occurrence of
ELISA assays can also be utilised to diagnose
several cancers (eg: bladder cancer)
ADVANTAGES OF ELISA
ELISA tests are generally relatively accurate
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
Monoclonal antibodies can cost more than
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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