ELISA

From A Z

Introduction-1

ELISA

ELISA (Enzyme-linked immunosorbent assay) is one of

immunoassay method used to
detection of
Antibodies-1
Proteins-2
Peptides-3
Biomolecules-4

?What is immunoassay
The term immunoassay is a
combined term of immuno(=
immunological, practically
immunochemical antigen-antibodyreaction) and assay (=
determination of the purity of a
substance or the amount of any
.constituent of a mixture

Antigen/antibody of interest is absorbed on. 1

.to plastic surface (sorbent)
Antigen is recognised by specific antibody .2
.(immuno)
This antibody is recognised by second .3
antibody (immuno) which has enzyme
.attached (enzyme-linked)
Substrate reacts with enzyme to produce .4
. product, usually coloured

History of Elisa
Radioimmunoassay was first described in a
scientific paper by Rosalyn Sussman Yalow and
. Solomon Berson published in 1960
In 1971, Peter Perlmann and Eva Engvall at
Stockholm University in Sweden, and Anton
Schuurs and Bauke van Weemen in the
Netherlands independently published papers that
synthesized this knowledge into methods to
.perform EIA/ELISA

History of Elisa

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Important components
in immunoassay
Antibody (antiserum)-1

Antigen-2

Labeling materials-3

Antibody-1
(antiserum)
Antibody:
proteins
produced by the immune
SYMBOL
FOR

system ANTIBODY
which help defend against antigens

The variable regions are

though to be the place for
recognition and binding with
.the antigen

Antigen-2

Any molecule that induces production of antibodies

.when introduced in the body is called antigen

OR

Any thing, foreign to the immune system. e.g.

bacteria, viruses, (or their parts), pollen, etc.

SYMBOL FOR ANTIGEN

Labeling materials-3
In immunoassay, it is necessary to
use any marker to know the
antigen-antibody binding. For such
purpose, we label either antigen or
antibody with some materials that
.do not interefere with the binding
e.g:horseradishperoxidase enzyme
substrate: trimethylbenzidine

ELISA
READER

ELISA KIT

Components of Kit
Pre-Coated, Stabilized 96-well Microtiter

Plate.
Sample Diluent
Standards and controls
Conjugated Detection Antibody
10X Wash Solution
Substrate
Stop Solution

Basic principal-3

of

ELISA

ELISA.wmv.MP4

Advantages of ELISA

Reagents are relatively cheap & have a long

shelf life
ELISA is highly specific and sensitive
No radiation hazards occur during labelling or
disposal of waste.
Easy to perform and quick procedures
Equipment can be inexpensive and widely
available.
ELISA can be used to a variety of infections.

Disadvantages of ELISA

Measurement of enzyme activity can be more complex

than measurement of activity of some type of

radioisotopes.
Enzyme activity may be affected by plasma
constituents.
Kits are commercially available, but not cheap
Very specific to a particular antigen. Wont recognize
any other antigen
False positives/negatives possible, especially with
mutated/altered antigen

Types-4
Of
Elisa

Types Of Elisa
Direct ELISA-1
Indirect ELISA-2
Sandwich ELISA-3
Competitive ELISA-4
Ogives ELISA-5

Direct
-1
Direct ELISA
ELISA- 1
The direct detection method uses a labeled
primary antibody that reacts directly with the
antigen. Direct detection can be performed
with antigen that is directly immobilized on
the assay plate . Direct detection is not
widely used in ELISA but is quite common for
immunohistochemical staining of tissues and
.cells

Indirect ELISA- 2
The indirect ELISA utilizes an unlabeled
primary antibody in conjunction with a
labeled secondary antibody.The
secondary antibody has specificity for
the primary antibody

Direct and Indirect ELISA

Sandwich ELISA- 3
The sandwich measures the amount of antigen between two
.layers of antibodies
Sandwich are especially useful if the concentration of
antigens is low or they are contained in a mix of high
concentrations of contaminating protein
To utilize this assay, one antibody (capture) is bound to a
microtiter plate well. Antigen is then added and bound to the
antibody. Unbound products are then removed, and 2ry
antibody is added (detection), then add the 3rd labeled
antibody to complete the sandwich
Major advantages of this technique are that the antigen does
. not need to be purified prior to use, due to its high specificity

competitive ELISA- 4
In this Unlabeled antibody is incubated in the
presence of its antigen. These bound
antibody/antigen complexes are then added to
an antigen coated well. The plate is washed
unbound antibody is removed. The secondary
antibody, specific to the primary antibody is
added. This second antibody is coupled to the
enzyme. A substrate is added, and remaining
enzymes elicit a chromogenic or fluorescent
signal. For competitive ELISA, the higher the
original antigen concentration, the weaker the
.eventual signal

m u l t i p l e a n d p o r t a b l e- 5
ELISA
A newer technique uses an solid phase made
up of an immuno-sorbent 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
with reagents

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Ogiv ack
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The
method

APPLICATIONS

HIV-1 and HIV-2 (presence of anti-HIV-1

antibodies). hepatitis C (presence of
. antibodies)
hepatitis B (testing for both antibodies-2
. and a viral antigen)
Measuring hormone levels HCG (as a test-3
. for pregnancy)
LH (determining the time of ovulation).-4
. TSH, T3 and T4 (for thyroid function)

ELISA
step by step

Reading the kit insert

before working

Coating of Wells with Antibody. 1

. L of antibody diluted in buffer is added to each well 100

.Cover the plate and incubate at 4 C overnight

Washing. 2

wash manually 3 times as follows:Empty the plate by inversion over a

sink. Tap the inverted plate against some layers of soft paper tissue to
remove residual liquid. Wash the plate by filling the wells by
immersion in buffer B. Leave on the table for 3 minutes. Empty the
.plate as described above and repeat washing two more times

A concentrated solution of non-interacting protein, such -2

as bovine serum albumin (BSA) or casein, is added to all
plate wells.This step is known as blocking,because the
serum proteins block nonspecific adsorption of other
.proteins to the plate

.Incubation with Test Samples. 3

L of test sample or standard diluted in buffer is added 100

.per well
Cover the plate and incubate at room temperature for 2
.hours

.Wash as described in step 2. 4

.Incubation with enzyme- Conjugated Antibody. 5

L of enzyme-conjugated antibody diluted in buffer is 100

.added to each well
.Cover the plate and incubate at room temperature for 1 hour
The enzyme-conjugated antibody should be directed against
.the antigen to be determined

The conjugatec antibody must be specific for the

antigen of interest

.Wash as described in step 2. 6

Colour Development. 7

.L of chromogenic substrate is added to each well 100

Cover the plate and incubate for 15 minutes, or until a suitable
colour has developed. The plate should preferably be
.protected against light during this incubation

Stopping the Colour Development. 8

.Stop the reaction by adding 100 L 0.5 M H2SO4 to each well
Reading of Results. 9

Read results directly through the bottom of the microwell

plate using an automated or semiautomated
photometer (ELISA-reader). The subtraction of the
absorbance at a reference wavelength (between 620 and
.650 nm) is recommended

The result

Fundamental techniques
for performing ELISA
?How to use a tip-exchange type pipette. 1

Fundamental techniques for

performing ELISA

Fundamental techniques for

performing ELISA
How to wash a microplate. 6

?HOW TO TREAT WITH THE REAGENTS-7

Use reservoir for each
reagent

Label the reservoi

Dont use the same

reservoir for multiple
regents

Dont return the

reagents to the stock

Shaking of the well-plate for. 8

mixing
Place the plate on the flat and smooth
surface of a laboratory table, hold the
plate and move the plate roundly to draw
circles rapidly for approx. 10 seconds
while lightly pressing the plate on the
.surface. Repeat 3 times

Important points in
performing ELISA and
improvement of assay
performance
.Sample treatment-1
.Stability of assay samples-3
Infleunce of humidity and air-2
.stream

Important points in performing ELISA and

improvement of assay performance

Sampling and treatments of samples. 1

Serum or plasma

sampling
In general, we recommend using

serum
When getting plasma
heparin is most often used as an anticoagulant
Use of fluoride must be avoided because
. fluoride ion is a potent inhibitor of peroxidase

TAKE CARE
An important phenomenon with frozen plasma
is that an insoluble substance (fibrin) will be
formed when thawed. In this case, the sample
must be mixed and centrifuged, then the
insoluble cluster flowing in the plasma should
be taken out by a thin wire needle sharply bent
at an end. If such fibrin remains in the sample,
it may clog the tip of a pipette and influences
assay variability

Hemolysis and Lipemia

pH Of the
sample

Serum or plasma, when fresh, shows pH

near neutral, however, it very quickly
goes to alkaline more than pH 8 by
.losing CO2

In alkaline pH, the antigenantibody reaction is

interfered. resulting in
cancellation of the assay or
giving inaccurate assay

Storage temperature and

.freezing-thawing
Sample storage temperature is better to be
lower than -35 C. Ultra-low temperature such
as -80 C is recommended for a long-term
.storage
Repeated freezing and thawing is also harmful
.to the protein, and may cause inactivation

When samples
are taken out
from the freezer
and thawed,
never forget to
mix these
samples
because the
solution after
thawing is not
homogeneous,
and the bottom
area contains
more solute

.Stability of assay samples-2

In assay, the problem of sample stability, i.e. how long the
substance to be measured can keep its immunoreactivity,
in serum or plasma, is very important. Blood samples also
contain enzymes to destroy peptides or proteins, and
stability against those enzymes differs from substance to
.substance
Freezer of 20C is not trustable for the constancy of
temperature but use of a freezer of 35 C or lower
. temperature is recommended

Avoid air fans

Avoid sunlight

infeluence of humidity and air-3

stream
During all the incubation process, the wellplate should be covered using the attached
plate cover. Plate cover is effective only
under the most suitable condition, i.e. room
temperature, humidity more than 50%, and
.air stream of less than 0.2m/sec

N.B

It is recommend to get
a small semi-transparent plastic box, and
.put moistened paper towel on the bottom

Trouble shooting in
ELISA

Poor or no coloration after-1

last step
.the
The standard
or samples might not be added( 1
Reagents necessary for coloration might not( 2
.be added
Wrong reagents related to coloration might( 3
.have been added
Influence of the temperature under which( 4
.the kits had been stored
Excessive hard washing of the well( 5
.plate

The standard curve)2

.obtained was not smooth
There might be some mistake in the serial
.dilution of the original standard solution

Flat(3
.Standard
solutions are not added
standard
. curve

Big variation between-4

two wells in duplicated
.Scratching
assay was
observed
the bottom
of the well by( 1

aspirator tip during aspiration of washing

.buffer
Scratching the bottom of the well by( 2
pipette tip during addition of standards,
.samples, or reagents
Assay might be started while the well-plate(3
.was still cooler than room temperature

Air stream, warmer or cooler than room( 4

temperature
Air stream from air conditioner or other( 5
instruments
.might dry wells
Insufficient removal of washing buffer from( 6
the wells
might dilute reagent solution added in the
.following step of the procedure
Big variation would be obtained if the sample(7
is not
.homogeneous

Standard curve Of ELISA

Shapes of standard curves depending on scales
.in X-and Y-axes
Standard curve of ELISA prepared by plotting
standard concentration on X-axis and
absorbance on Y-axis, both in normal scale,
looks like a linear line except for lower
.concentration area

Standard curve
Of ELISA