Immunoassays

Immunoassays
Laboratory techniques that makes use of
the binding between an antigen and its
homologous antibody in order to identify
and quantify the specific antigen or
antibody in a sample.

ELISA technique
Is a biochemical technique used mainly in
immunology to detect the presence of an
antibody or an antigen in a sample.
The technique is divided into
1- Competitive ELISA
2- Sandwich ELISA (also called direct ELISA)
3- Indirect ELISA

ELISA
An ELISA test uses components of the
immune system and chemicals to detect
Ag-Ab reactions
The ELISA test involves an enzyme (a
protein that catalyzes a biochemical
reaction). It also involves an antibody or
antigen (immunologic molecules).

Types of ELISA
Qualitative ELISA
Positive or Negative results

Quantitative ELISA
optical density or fluorescent units of the sample is
interpolated into a standard curve, which is
typically a serial dilution of the target.

ELISA
Principle:
Bound antigen is detected by an antibody that is covalently
coupled with an enzyme such as peroxidase or alkaline
phosphatase
Quantification of antigen-antibody binding is achieved by
measuring the colour intensity of the coloured product
generated by the enzyme and the added substrate.
The intensity of the colour is proportional to the amount of
the labelled antibody bound to the antigen.
Direct detection of an antigen with the labelled antibody is not
popular because of low sensitivity.
In the indirect method, the antibody with specificity for the
desired antigen is unlabelled, and a second enzyme labelled
antibody with a specificity for the first antibody (an antibody
to an antibody) is then added to the mixtur.e

Basic principles of ELISA

the antibodies fixed to a
solid surface, such as
the inner surface of a
microtitre plate;
a preparation of the
same antibodies
coupled to an enzyme.
that produces a
coloured product after
reaction with a
colourless substrate.

Direct ELISA

Other types of ELISAs

Outline of procedure e.g Sandwish ELISA

Coat an appropriate surface with a known quantity of capture
antibody and allow time for binding
Wash to remove any unbound antibody
Block any non specific binding sites(empty sites ) on the surface with
a an irrelevant protein
Apply the antigen-containing sample(TEST sample) to the plate.
Wash the plate, so that unbound antigen in the test sample is
removed.
Apply enzyme linked primary antibodies as detection antibodies
which also bind specifically to the antigen captured by first antibody.
Wash the plate, so that the unbound antibody-enzyme conjugates are
removed.
Apply a chemical substrate which reacts with enzyme in the
conjugate to form a a coloured product.
Measure the absorbance of the plate wells to determine the presence
and quantity of antigen

Sandwich ELISA
The ELISA plate is coated with Antibody to
detect specific antigen

Results-ELISA
This standard curve is used to determine
the unknown concentration of each
sample by finding the opposite
concentration to the absorbance

Absorptio
n nm

Concentration ng/ml

Advantages of ELISA
ELISA tests are generally relatively accurate
tests.
They are considered highly sensitive and
specific and compare favorably with other
methods used to detect substances in the body,
such as radioimmuno assay (RIA) tests.
They have the added advantages of not needing
radioisotopes (radioactive substances) or a
costly radiation counter (a radiation-counting
apparatus).

APPLICATIONS OF ELISA
Diagnostic Assays
Serum Antibody Concentrations
Disease outbreaks- tracking the spread of disease HIV,
Detections of antigens in body fluids e.g pregnancy

hormones, drug allergens

Detecting potential food allergens
(milk, peanuts, walnuts, almonds and eggs)
Research and industrial applications

ELISPOT-

Set similar to ELISA but used to identify antigen secreting cells

e.g lymphocytes secreting a particular cytokine using anticytokine
labelled monoclonal antibody.

ELISPOT Assays
PBMC are plated on a filter-bottom 96-well plate coated
with anti-cytokine antibody.
The plate is cultured 24-48 hours to allow cytokine
secretion and capture on the plate.
Cells are washed off and detector antibody is added,
followed by enzyme substrate.
Cytokine-secreting cells are identified as spots of
secreted cytokine.

SDS-PAGE (sodium dodecyl sulfate

polyacrylamide gel electrophoresis
SDS-PAGE, is a technique widely used in
biochemistry, forensics, genetics and molecular
biology:
Separate proteins according to their
electrophoretic mobility (a function of length of
polypeptide chain or molecular weight).
Separate proteins according to their size

SDS
SDS (sodium dodecyl sulfate) is a
detergent (soap) that can dissolve
hydrophobic molecules but also has a
negative charge (sulfATE) attached to it
Therefore, if a cell is incubated with SDS,
the membranes will be dissolved, all the
proteins will be solubIlized by the detergent
and all the proteins will be covered with
NET negative charges.

PAGE( polyacrylamide gel

electrophoresis ).
If the proteins are denatured and put into an electric
field, they will all move towards the positive pole at the
same rate, with no separation by size.
However, if the proteins are put into an environment that
will allow different sized proteins to move at different
rates e.g polyacrylamide
Small molecules move through the polyacrylamide
faster than big molecules.
Big molecules are slower and remain close to the
starting point
The entire process is called polyacrylamide gel
electrophoresis (PAGE). ddd

Protein gel (SDS-PAGE) that has

been stained with Coomassie
Blue.

Sample of SDS- PAGE(Silver

stain)

After electrophoresis?
1. Fix the proteins in the gel followed by
staining
2. Electrophorectic transfer to a
membrane and then probe with
antibodies- (Western blotting)

..Western blotting
Western blot analysis can detect specific
protein in a mixture of any number of proteins
while giving you information about the size of the
protein.
This method is, however, dependent on the use
of a high-quality antibody directed against a
desired protein.
This antibody is used as a probe to detect the
protein of interest.

Western Blot followed by SDS

Proteins are separated using SDS-polyacrylamide gel
electrophoresis which separates proteins by size.
Separated components transferred from gel to nitrocellulose
paper by blotting
Nitrocellulose membrane is placed on the gel. The actual
blotting process may be active (electroblotting) or passive
(capillary).
Electroblotter is used for faster and more efficient transfer of
protein from gel to membrane
Sandwich of filter paper, gel, membrane and more filter
paper is prepared in a cassette, which is placed between
platinum electrodes.
An electric current is passed through the gel causing the
proteins to electrophorese out of the gel and onto the
nitrocellulose membrane.

..Western Blot
The blot is incubated with a generic
protein (such as milk proteins) to bind to
any remaining sticky places on the
nitrocellulose.
An antibody is then added to the solution
which is able to bind to its specific protein.
The antibody has an enzyme (e.g.
alkaline phosphatase or horseradish
peroxidase) or invasible dye attached to it

..Western Blot
The location of the antibody is revealed
by incubating it with a colourless substrate
which reacts with the attached enzyme
and is concerted to a colored product that
can be seen and photographed.

Western blotting

Western Blot

Radioimmunoassay (RIA),
-A very sensitive method, but is becoming less attractive, because
it is potentially hazardous and tedious.
-The assay was originally developed to detect trace amounts of
peptides and hormones in plasma or biological fluids.
-In this assay, antibodies are immobilized and the radiolabeled
(usually 125I) and cold antigen are allowed to compete for
binding to the immobilized antibodies

Radioimmunoassay (RIA)
Very sensitive test; used for measuring hormones,
serum proteins, drugs, etc. at low [C]s (
0.001ug/ml)
measures competitive binding of radiolabelled
Ag + unlabelled (test) Ag to high affinity Ab

Other immunological assays:

Immunoprecipitation.
Provides a quick and sensitive test for identifying small amounts of
antigens (low conc) in a complex mixture. Antigen is isolated from a
radiolabelled mixture by specific precipitation with antibody and
analysed by polyacrylamide gel electrophoresis followed by auto
radiography
Chemiluminescence
Set up similar to ELISA technique but uses a different detection
system.
The system uses Luminol, hydrogen peroxide and horseradish
peroxidase which react to emit light.
Complement Fixation Uses the ability of Ab-Ag complexes to fix
complement because an Ag/Ab complex will "consume" complement if it
is present whereas free Ag's or Ab's do not. Test only works with
complement fixing antibodies.