ELISA

reader &
microplate Farheen Saba
Ph.D Ist year
washer Deptt. of
Zoology
MANUU
Elisa reader Microplate washer
Below is a brief outline of some equipment
available for performing ELISA testing.

• Pipettes :
• Single-channel, fixed-volume and adjustable-volume (1–20 µL, 10–100 µL,
20–200 µL, etc.)
• Multichannel, 8- and 12-channel
• Semiautomated dispensing units
• Fully automated systems that can process multiple plates .
• Dilutors :
• Single-channel
• Multichannel
• Automated dispensing units
• Washer Systems:
• Manual systems that wash one row or column at a time
• Semiautomated systems that handle one strip or plate at a time
• Fully automated systems that can process multiple plates

• ELISA Plate Readers:

• Manual readers that read one row or well at a time
• Semiautomated readers that read one plate at a time
• Fully automated systems that can process multiple plates simultaneously
• Other :
• humidity chamber (not required for all ELISAs)
• Plate sealers for assays that have long incubation times (to avoid
evaporation)
• Incubator or plate shaker incubator (not required for all ELISAs)
ELISA Reader?

• An ELISA reader, is a highly sophisticated instrument whose job it is

to detect the presence of a protein in a liquid sample.
• ELISA stands for enzyme-linked immunosorbent assay.
• The ELISA reader is one of the most popular versions of a device
called a plate reader which is designed to determine the presence
of any sort of biological, chemical or physical presences in a small
microtiter sample.
Microplate washer
• Microplate washers are laboratory instruments designed to control
the procedure of washing experimental samples arranged in plate-
based formats.
• Users load a plate and select a program; microplate washers then
dispense, soak and aspirate liquids from the plate in seconds.
• Compared to manual alternatives, microplate washers
tremendously improve the speed and accuracy of many different
washing procedures, and are particularly useful for Enzyme-Linked
Immunosorbent Assays (ELISAs).
• Microplate washers are also employed to wash cell cultures,
protein arrays, Western blots and beads as well as applied in
DNA purification protocols.
• Microplate format compatibility (48, 96 or 384 wells), the
number of channel heads (6, 8, 16, 96 or 384), the number of
liquids dispensed (1 to 5) and the range of volumes dispensed
and aspirated (10 microliters to 3 milliliters) all vary by
instrument model.
• All microplate washers enable users to program tank selection,
volumes, step times, and cycles for customized procedures.
History :

• In 1971,two scientists Eva Engvall and Peter Perlman invented a test

that revolutionized medicine.
• Before the development of the ELISA, the only option for conducting
an immunoassay was radioimmunoassay, a technique using
radioactively labeled antigens or antibodies.
• As radioactivity poses a potential health threat, a safer alternative
was sought.
• A suitable alternative to radioimmunoassay would substitute a
nonradioactive signal in place of the radioactive signal. When
enzymes (such as horseradish peroxidase) react with appropriate
substrates (such as ABTS* or TMB*), a change in color occurs, which is
used as a signal. However, the signal has to be associated with the
presence of antibody or antigen, which is why the enzyme has to be
linked to an appropriate antibody.

(ABTS : 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)

(TMB : Trimethyl benzidine)
Commonly used enzymatic markers

• OPD (o-phenylenediamine dihydrochloride)

• TMB (3,3',5,5'-tetramethylbenzidine)
• ABTS (2,2'-Azino bis [3-ethylbenzothiazoline-6-sulfonic acid])
• PNPP (p-Nitrophenyl Phosphate, Disodium Salt).
ELISA KIT COMPONENTS:
• Coated Plates: The 96-well plates are made of polystyrene and coated
with either inactivated antigen or antibody. This coating is the binding
site for the antibodies or antigens in the sample. unbound antibodies
or antigens in the sample are washed away after incubation.
• Sample Diluent: Most assays require a specific dilution of the
sample. Samples are added to the sample diluent and mixed prior to
putting them onto the coated plates.
• Controls: The positive control is a solution that contains antibody or
antigen. The negative control is a solution without antibody or
antigen. The controls help to normalize or standardize each plate.
controls are also used to validate the assay and to calculate sample
results. In most tests, the controls are prediluted and ready to use. Be
sure to follow the instructions in the package insert.
• Conjugate: ELISA conjugates are enzyme-labeled antibodies or
antigens that react specifically to plate-bound sample analytes.
unbound conjugate is washed away after incubation and before the
addition of substrate. The optical density of the colorimetric substrate
is directly proportional to the quantity of bound enzyme present.

• Substrate: For peroxidase conjugates, the substrate is a mixture of

hydrogen peroxide and a chromogen that reacts with the enzyme
portion of the conjugate to produce color.
• Wash Concentrate: The wash concentrate is a buffered solution
containing detergent used to wash away unbound materials from the
plates.

• Stop Solution: The stop solution stops the enzyme-substrate reaction

and, thereby, the color development.
Introduction to ELISA
(enzyme linked
immunosorbent assay)
• A test that uses antibodies and color
change to identify a substance.
• Elisa is a popular format of a “wet lab” type
analytical biochemistry assay.
• ELISA involves at least one antibody with
specificity for a particular antigen.
• ELISA can perform other forms of ligand
binding assays instead of strictly “immune”
assays.
components Of ELISA

• Antibody: IgG fraction of serum.

• Enzyme : Horse radish peroxidase (HRPO) MW 44000 daltons

glycoprotein with 4 lysine residues.

• Substrate: TMB (Tetramethyl benzidine). The enzyme acts as a

catalyst to oxidize substrate in the presence of hydrogen peroxidase
to produce a blue color. Reaction stopped with dilute acid to cause
complex to turn yellow.
Basic principle of ELISA

• Use an enzyme to detect the binding of antigen (Ag) antibody (Ab).

• The enzyme converts a colorless substrate to a colored product ,
indicating the presence of Ag: Ab binding.
• An ELISA can be used to detect either the presence of antigens or
antibodies in a sample depending how the test is designed.
.
.
How does ELISA work

• There are variations of the ELISA test but the most basic type consist
of an antibody attached to the solid surface. This antibody has affinity
for the substance of interest.
• For example … human chorionic gonadotropin (HCG), the commonly
measured protein which indicates pregnancy.
• A mixture of purified HCG linked to an enzyme and sample (blood,
urine) under test are added to the test system. If no HCG is present in
the test sample the only HCG with linked enzyme will bind. The more
HCG which is present in the test sample the less enzyme linked HCG
will bind.
 NON COMPETITIVE ELISA
- DIRECT ELISA
Types of
ELISA
- INDIRECT ELISA
- SANDWICH ELISA
COMPETETIVE ELISA
 • Used to detect the antigen
in given biological sample.
• Microtiter wells are
initially coated with
antigen to be detected
which is followed by an
antibody linked to an
enzyme conjugate. This
follows the addition of
substrate which produces
DIRECT ELISA color detection using ELISA
detector.
 • Used for detection of an
antibody in the given
sample.
• Microtiter wells are
initially coated with
antigen specific for
antibody to be detected ,
followed by the addition
of sample. Enzyme
conjugated secondary
antibody is added
followed by the substrate
Indirect ELISA which forms a colored
reaction product.
 • Used for detection of antigen in
the given sample.
• Microtiter wells are initially
coated with monoclonal
antibodies (called capture
antibodies) raised against
antigen to be detected ,
followed by addition of sample.
Any trace of antigen is detected
by adding primary antibody ,
followed by enzyme conjugated
secondary antibody and a
chromogenic substrate ; or by
directly adding an enzyme
SANDWICH ELISA conjugated primary antibody.
 • The variation of ELISA is used to
quantitatively estimate the amount
of antigen in the given sample.
• Ag and Ab are initially incubated so
that they form Ag-Ab complex. This
mixture is then added to micro titer
wells coated with synthetic analogue
of antigen to be detected, any free
antibody binds to these antigens.
This complex is estimated by enzyme
conjugated secondary antibody by
chromogenic detection. More the
amount of antigen in the sample ,
lesser is he antibody available to bind
to microtiter wells.
COMPETETIVE ELISA
.
APPLICATIONS
• Since ELISA can detect both antigen and antibody it is a useful tool for
determining serum antibody concentrations.
• It has also found applications in the food industry in detecting
potential food allergens such as milk, peanuts, almonds etc.
• The other uses of ELISA include:
a) detection of mycobacterium antibodies in tuberculosis.
b) detection of hepatitis B markers in serum.
c) detection of enterotoxin of E. Coli in feces.
d) detection of HIV antibodies in blood samples.
Advantages
• Sensitive assay equipments are widely available.
• No radiation hazards.
• Reagents are cheap with long shelf life.
• ELISA can be used on most types of biological samples such as
plasma, serum, urine, and cell extracts.
DISADVaNTAGES

• Only monoclonal antibodies can be used as matched pairs.

• Monoclonal antibodies can cost more than polyclonal antibodies.
• Negative controls may indicate positive results if blocking solution is
ineffective [secondary antibody or antigen (known sample) can bind
to open sites in well].
• Enzyme/ substrate reaction is short term hence color must be read as
soon as possible.
Thank
you
.