ELISA
ELISA (ENZYME-LINKED
IMMUNOSORBENT ASSAY)
DEFINITION
The enzyme-linked immunosorbent assay is a common laboratory
technique which is used to measure the concentration of an
analyte(usually antibodies or antigens) in solution.
INTRODUCTION
Enzyme-linked Immunosorbent Assay is biochemical assay technique
used mainly in immunology.
It is a plate-based assay designed for detecting and quantifying
substances such as peptides, proteins, antibodies and hormones.
First and most basic test to determine if an individual is positive for
selected pathogen.
PRINCIPLE
In ELISA various antigen-antibody combinations are used, always
including an enzyme-labelled antigen or antibody and enzyme activity
is measured colorimetrically.
The enzyme activity is measured using a substrate that changes color
when modified by the enzyme.
The most commonly used enzyme labels are horseradish peroxidase
(HRP) and alkaline phosphatase (AP). Other enzymes have been used
as well; these include β-galactosidase, acetylcholinesterase, and
catalase.
Substrates for AP:
• BCIP (5-bromo-4-chloro-3-indolyl-phosphate) is used in conjunction
with NBT (nitro blue tetrazolium) for the colorimetric detection of
alkaline phosphatase activity.
• PNPP (p-Nitrophenyl Phosphate, Disodium Salt)
Substrates for HRP:
• ABTS (2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]
• 3,3′,5,5′-Tetramethylbenzidine or TMB
bovine serum albumin (BSA) as a blocking agent to prevent
non-specific binding of antigens and antibodies to the microtiter well.
TYPES OF ELISA
Depending on antigen-antibody combination, the assay is divided into
following;
1.Direct ELISA
2.Indirect ELISA
3.Sandwich ELISA
4.Competitive ELISA
1.DIRECT ELISA
Antigen is immobilized onto the wells of a 96-well plate.
An enzyme-labelled primary antibody specific for the target
antigen added to the wells and directly binds to the antigen.
A respective enzyme substrate is added, which upon reaction with
the enzyme, produces a visible colorimetric output that can be
measured by a spectrophotometer or absorbance microplate
reader
1.DIRECT ELISA
2.INDIRECT ELISA
Micro-well plates are incubated with antigens, washed up and
blocked with BSA.
Samples with antibodies are added and washed.
Enzyme linked secondary antibody are added and washed.
A substrate is added, and enzymes on the antibody elicit a
chromogenic or fluorescent signal.
2.INDIRECT ELISA
3.SANDWICH ELISA
It measures the amount of antigen between two layers of antibodies.
1. Prepare a surface to which a known quality of antibody is bound.
2. Apply the antigen containing sample to the plate
3. Apply the enzyme linked antibodies which are also specific to the
antigen.
4. Apply the substrate which is converted by enzyme into fluorescent
signal.
5. Results can b viewed by spectrophotometry
3.SANDWICH ELISA
4.COMPETITIVE ELISA
The central event of competitive ELISA is a competitive binding process
executed by original antigen (sample antigen) and add-in antigen.
Primary antibody (unlabelled) is incubated with sample antigen
Antibody-antigen complexes are then added to 96-well plates which
are pre-coated with the same antigen. Unbound antibody is removed
by washing the plate. (The more antigen in the sample, the less
antibody will be able to bind to the antigen in the well, hence
"competition.")
The secondary antibody that is specific to the primary antibody and
conjugated with an enzyme is added.
A substrate is added, and remaining enzyme elicit the chromogenic
or fluorescent signal.
4.COMPETITIVE ELISA
APPLICATION OF ELISA
1. Presence of antigen or the presence of antibody in a sample can be evaluated.
2. Determination of serum antibody concentrations in a virus test.
3. Used in food industry when detecting potential food allergens.
4. Applied in disease outbreaks- tracking the spread of disease
ADVANTAGES AND DISADVANTAGE
DISEASES
INFETIOUS BRONCHITIS
ILT
INFLUENZA
INFECTIOUS BURSAL DISEASE
FOWL CHOLERA
ADENOVIRUS INFECTIONS
THANK YOU .
