Enzyme Linked Immuno

Sorbent Assay(ELISA)
PRESENTED BY:
DANIA RASOOL
AYESHA
ZAINAB
 Contents

• Immunoassay
• ELISA
• History
• Required Equipments
• ELISA kit principle
• Medical significance
• Sample separation
• Types of ELISA
• Advantages and disadvantages
 Immunoassay

It is a specific technique or procedure used to detect or measures specific protein or

substance through their properties as antigens or as antibodies.
Types of immunoassay:
There are different types of immunoassay:
 Radioimmunoassay.
 ELISA.
 Fluoroimmunoassay.
 Counting immunoassay.
 Chemiluminescence immunoassay.
 ELISA

ELISA is enzyme linked Immuno sorbent assay.

ELISA test uses the components of immune system (IgG or IgM) and certain other
chemicals for the detection of immune response in body. This test involves the
specific enzyme, antigen and antibody. When specific antigen and antibody react,
antigen and antibody complex form which indicates positive result. If antigen and
antibody are not specific, no antigen-antibody complex form, indicates negative
result.
Substance that can be detected by ELISA includes hormones, an allergens, viral
antigen, bacterial antigen and antibodies that body produce in response to infection
and vaccination.
 History

 In 1974, Peter Perlmann and Eva Engvall developed the test as a substitute for
certain radioimmunoassay.

 The ELISA test is versatile and medical professional can perform this test easily
as compare to other complicated test.
 Required Equipments

Centrifuge machine:
It works by using the principle of sedimentation. This
device spins liquid sample at high speed and causes the
denser particles move to the bottom of the tube. By this
way mixture with different sized particles are
separated.
Shaking incubator:
Shaking of micro plate in ELISA is essential to obtain
reliable results. It helps in the homogenization of
samples, increase the rate of biochemical reaction and
also provide stable temperature conditions.
Microplate washer:
It is a laboratory instrument used to control the procedure
of washing experimental samples. Washers dispense,
soak and aspirate liquids from plate in seconds.
ELISA reader:
It measures the color difference in wells of plate. It emits
light at one wavelength and then measures the light
absorbed and reflected by object such as protein. This
instrument can use multiple samples at a time and smaller
volume of sample can be used.
Biosafety cabinet:
It is a ventilated enclosure, provides protection to the product, person and environment
from the aerosols arising from the handling of potentially hazardous microorganisms. Air is
filtered and then discarded to the environment.
96-well plate:
96-micro titer plates are rectangular multi-well plates used in variety of assay such as in
ELISA. Rows on plate are labelled with numbers. Volume of each well is 0.30mL.
Micropipette
Micro dispenser
 It is a laboratory instrument used for transferring  Instrument used to transfer the accurate
micro/accurate volume of liquid solution. volume liquid sample.
 It works by displacing air from pipette shaft ,  In dispenser we take the liquid in large
allowing the liquid to be drawn into the resultant
quantity at once.
vacuum.
 It is used for those liquids for which we do
 In it we need to change the tips for the transferring
of sample(avoid cross contamination). not need to change the tips.
 Easy to use. Micropipette of different volumes are
available such as 100ղL,25ղL.
 ELISA kit components

• Antibody/antigen coated micro titration plate

• Standard
• Enzyme conjugated antibodies
• Diluent buffer(used to dilute the sample)
• Wash buffer(used to rinse micro plate during the coating
process and between reagent addition steps of ELISA)
• Substrate (react with enzyme and produce color)
• Stop solution(used to terminate enzyme substrate reaction)
• Plate cover
 Principle of ELISA kit

ELISA is typically performed in 96-well [Link] well antibodies or protein

bind passively. It is the binding and immobilization of reagents that makes
ELISA easy to perform and design. As ELISA has immobilized reactants , it
makes easy to separate bound material from unbound material during assay.
The washing process makes WLISA a powerful tool for the measuring of
specific agent.
 Medical significance

An enzyme linked immunosorbent assay is a test that is used to detect and to

measure the antibodies and antigens present in the blood.
Antibodies are the proteins that body produce in response to harmful substance
(enter in body) called antigens.
An ELISA test used to diagnose:
• HCV Abs (IgG and IgM)
• HBsAg
• HIV (causing AIDS)
• HEV, HAV
•
 Sample

 Blood is collected as sample in ELISA.

 Sample Separation

 After the collection of blood allow it

to clot by leaving it undisturbed at
room temperature. This usually
takes 15-20min.
 Serum is separated by centrifugation
at 3500RPM for 15min after
centrifugation the resultant
supernatant is designated as serum.
Why serum is used as sample?

 Serum provide the liquid

portion of the blood
without cells and clotting
factors and, it contains
proteins and other
molecules that present the
whole body system.
 Types of ELISA

There are different types of ELISA:

• Direct ELISA
• Indirect ELISA
• Sandwich ELISA
• Competitive ELISA.
 Direct ELISA

Used for the Antigens in the sample

detection of antigen. absorbed and immobilized in
Sample is added in micro titer wells. Wash to
remove unbound antigens.
the wells.

Enzyme linked Abs

Specific Abs will
specific to target Ags
Substrate react with enzyme Color can be measured by
and produce color. spectrophotometer.
Advantages
• Quick as only one antibody is required.
• Cross reactivity of secondary antibody is eliminated.
Disadvantages
• Low flexibility, as primary antibodies must be labelled.
• Labelling of primary antibodies is expensive and time consuming.
• Low signal amplification(only primary antibody is used and
secondary antibody is not needed).
 Indirect ELISA

Antigens coated wells are taken. Wash the plate to remove unbound
Add primary Abs. incubate the Abs. add enzyme linked secondary
plate. Abs.

Incubate the plate. Then wash the Add substrate. Incubate the plate.
Advantages
• A wide variety of labelled secondary antibodies are commercially
available.
• Maximum immunoreactivity of primary antibody is retained
because it is not labelled. Increased sensitivity as each primary Ab
has several epitopes that bind with secondary labelled Abs,
allowing for signal amplification.
Disadvantages
• An extra incubation step is required.
• Cross reactivity may occur with secondary antibodies lead to non
 Sandwich ELISA

Incubate the plate.

Antibodies coated plate is taken.
Wash the plate to remove unbound
Add Ags containing sample.
antigens.

Add specific primary Abs that Wash the plate. Add enzyme linked
sandwiches the antigen. incubate secondary antibodies. Incubate the
Incubate the plate. Substrate
react with enzyme and Add stop solution.
produce color.

Color can be measured by

spectrophotometer.
Advantages
• Highly specific (the antigen is specifically captured and detected).
Disadvantages
• The antigen of interest must be large enough so that two antibodies
can bind to it at different epitopes.
• It is sometimes difficult ti find two different antibodies that
recognize different epitopes on the antigen.
 Competitive ELISA

 In competitive ELISA, the reaction takes place between the sample antigen and
the antigen bounded to the wells of micro titration plate with the primary
antibody.
 First, the primary antibody is incubated with the sample antigen.
 Result in formation of Ag-Ab complexes, which are added into wells coated with
antigens. Incubate the plate. After incubation wash the plate.
 The more antigens in the sample, the more primary antibodies will be bound to
antigens in sample. So, there will less amount of antibodies available to bind to
Ags coated in the wells.
 Add enzyme conjugated secondary antibodies, followed by addition of substrate.
 Incubate the plate. Substrate react with enzyme and produce color. Add stop
solution, color can be measured.
 Absence of color indicates the presence of antigens in the sample.
Advantages
• High sensitivity.
• Best for the detection of small antigens, even when they are
present in low concentration.
• High flexibility.
Disadvantages
• Relatively complex protocol.