ELISA AND IMMUNE

REACTIONS USEFUL IN
BIOASSAYS
AANCHAL GHAI
MSc –II (NUCLEAR
MEDICINE)
 INTRODUCTI
ON field of
Immunology is a rapidly developing study
concerned with the discovery of the mechanisms of
the immune system, the development of vaccines and
the regulatory procedures for manipulating the
immune response.

Immune responses are processes in which animals

form specifically reactive proteins(antibodies) and
cells in response to a great variety of foreign organic
macromolecules.
Immune recognition is remarkable for its specificity.
The Immune system is able to recognize a foreign
particle(ANTIGEN) . Furthermore the system is able to
discriminate between foreign molecules and the body’s
own cells and proteins.

Once a foreign organism has been recognised,the

immune system recruits a variety of cells(e.g
macrophages) and molecules(e.g ANTIBODIES) to
eliminate or neutralize the foreign organism.
ANTIGEN
 An antigen is a substance which when
introduced into the vertebrate
host,stimulates the production of
antibodies and reacts with preformed
antibodies if already present.

TWO PROPERTIES OF ANTIGENS

1. Specificity

2. Immunogenicity
 ANTIBODY
 Antibodies are the antigen binding proteins
present on the B-cell membrane and secreted by
plasma cells.
 Secreted antibodies circulate in the blood,
where they serve as the effectors of humoral
immunity by searching out and neutralizing
antigens or marking them for elimination.
It has been known since the time of the century
that antibodies reside in the serum. The first
evidence that antibodies were contained in
particular serum protein fractions came from a
classic experiment by A. Tiselius and E.A. Kabat, in
1939.
 All antibodies share structural features, bind to
antigen and participate in a limited number of
effector functions.
Schematic diagram of structure of
immunoglobulin
 CHAIN COMPOSITION OF FIVE
IMMUNOGLOBULIN CLASSES IN HUMANS
ANTIBODY
AFFINITY
 The combined strength of the non-covalent
interactions between a single antigen binding site
on an antibody and a single epitope is the affinity
of the antibody for that epitope.
 Low affinity antibodies bind antigen weakly and
tend to dissociate readily whereas high affinity
antibodies bind antigen more tightly and remain bound
longer.
 ANTIBODY
AVIDITY
 The strength of multiple interactions between a
multivalent antibody and antigen is called the avidity.
 The avidity of an antibody is a better measure of its
binding capacity within biological systems than the
affinity of its individual binding sites. High avidity can
compensate for low affinity
 PRECIPITATION REACTIONS

 Antibody and soluble antigen interacting in

aqueous solution form a lattice that eventually
develops into a visible precipitate.
 Antibodies that aggregate soluble antigens are
called precipitins.
 Formation of an Ag-Ab lattice depends on the
valency of both the antibody and antigen:
 The antibody must be bivalent as precipitate will
not form with monovalent Fab fragments.
The antigen must be either bivalent or
polyvalent;i.e it must have atleast two copies of the
same epitope or have different epitopes that react
with different antibodies present in polyclonal
antisera.

 Experiments with myoglobin illustrate the

requirement that protein antigens be bivalent or
polyvalent for a precipitin reaction to occur.
Precipitation reactions:
Polyclonal antibodies can form
lattices or large aggregates that
precipitate out of solution
however, if each antigen
molecule contains only a single
epitope recognized by a given
monoclonal antibody the
antibody can link only two
molecules of antigen and no
precipitate is formed
PRECIPITATION REACTIONS IN FLUIDS YIELD A PRECIPITIN CUR

A precipitation curve for a system of one antigen & its antibodies.

FROM THE GRAPH

This plot of the amount of the antibody

precipitated vs. increasing antigen concentrations
reveals 3 zones:

A zone of antibody excess in which precipitation is

inhibited and Ab not bound to Ag can be detected in
the supernatant.

An equivalence zone of maximal precipitation in

which Ab and Ag form large and soluble complexes
and neither Ab nor Ag can be detected in the
supernatant.
 A zone of antigen excess in which precipitation is
inhibited and antigen not bound to antibody can be
detected in the supernatant.
PRECIPITATION REACTIONS IN GELS YIELD
VISIBLE PRECIPITIN LINES
Immune precipitates can be formed in an agar matrix
also.
When Ag and Ab diffuse towards one another in agar
a visible line of precipitation will form.
Two types of immunodiffusion reactions can be used
to determine relative concentration of Ab’s or Ag’s,or
to determine the relative purity of an Ag preparation.
They are:

Radial immunodiffusion

Double immunodiffusion
 In this an antigen sample is placed in well & is allowed to diffuse
into agar containing a suitable dilution of an antiserum.
 The area the precipitin ring is proportional to the concentration of
the antigen.
 In this method,both antigen & antibody diffuse radially from
wells towards each other thereby establishing a concentration
gradient.
 As equivalence is reached, a visible line of precipitation, a
precipitin line forms.
AGGLUTINATION REACTIONS
The interaction between Ab and a particulate Ag results
in visible clumping called agglutination.
Antibodies that produce such reactions are called
agglutinins.
An excess of Ag or Ab can also inhibit agglutination
reactions. This inhibition is called as PROZONE
EFFECT.
Hem agglutination is used in blood typing.
 Agglutination reactions are routinely
performed to type RBC’s. In typing for the ABO
antigens, RBC’s are mixed on a slide with
antisera to the A or B blood group antigens.
If the antigen is present on the cells, they agglutinate,
forming a visible clump on the slide.
Determination of which antigens are present on donor
and recipient RBCs are the basis for matching blood
types for transfusions.
BACTERIAL AGGLUTINATION
TO DIAGNOSE INFECTION
The presence of serum antibodies specific for
surface antigens on the bacterial cells can be detected
by bacterial agglutination reactions.

Serum from a patients thought to be infected with a

given bacteria is serially diluted in an array of tubes to
which the bacteria is added.

The last tube showing visible agglutination will

reflect the serum antibody titer of the patient.
BACTERIAL AGGLUTINATION
TO DIAGNOSE INFECTION
The agglutinin titer of an antisera can be used to
diagnose a bacterial infection.

Example : Patients with typhoid fever show a

significant rise in the agglutination titer to salmonella
typhi.
 Passive Agglutination is Useful With
Soluble Antigens
In this technique, antigen coated RBCs are prepared by
mixing a soluble antigen with RBCs that have been treated
with tannic acid or chromium chloride.
Serum containing antibody is serially diluted into microtiter
plate wells and the antigen coated RBCs are then added to
each well; agglutination is accessed by the size of the
characteristic spread pattern of agglutinated RBCs on the
bottom of the well.
 In agglutination Inhibition, Absence of Agglutination Is
Diagnostic of Antigen:
A modification of the agglutination reaction, called
agglutination inhibition, provides a highly sensitive assay
for small quantities of an antigen.
Example-one of the early types of home pregnancy test kits
included latex particles coated with human HCG and
antibody to HCG. the addition of urine from a pregnant
women which contained HCG,inhibited agglutination of the
latex particles when the anti HCG antibody was added. Thus
absence of agglutination indicated pregnancy.
Fig. shows the pregnancy test employed hapten inhibition to determine the presence
or absence of HCG
 INTRODUCTION TO ELISA
ELISA, or enzyme-linked immunosorbent assay, is
an immunoassay technique involving the reaction
of antigen and antibody in vitro. ELISA is a
sensitive and specific assay for the detection and
quantitation of antigens or antibodies. ELISA tests
are usually performed in microwell plates.

The ELISA test, or the enzyme immunoassay

(EIA), was the first screening test commonly
employed for HIV. It has a high sensitivity.
INTRODUTION contd…

Certain enzymes (such as peroxidase) react with

appropriate substrates (such as 3,3’,5,5’-
Tetramethylbenzidine), they can result in changes
in color, which can be used as a signal.

This signal has to be associated with the presence

of antibody or antigen.

This linking process was independently developed

by Stratis Avrameas and G.B. Pierce
A 96-WELL MICROTITRE PLATE USED FOR
ELISA
 ELISA COMPONENTS
Antibody: IgG fraction of serum purified by affinity
chromatography.

Enzyme: Horse Radish Peroxidase (HRP) MW 44,

000, glycoprotein with 4 lysine residues.

Substrate: TMB (3,3',5,5', tetramethylbenzidine) The

enzyme acts as a catalyst to oxidize substrate in the
presence of Hydrogen peroxide to produce a blue
color. Reaction stopped with dilute acid to cause
complex to turn yellow.
 Principle Of ELISA
An enzyme conjugated with an antibody reacts with a
colorless substrate to generate a coloured reaction
product.
Such a substrate is called a chromogenic substrate.
The principle is based on two observations:
1.Antibodies and some antigens can attach to
polystyrene plastic plates and still maintain their full
immunologic capabilities.
2.Antigens and antibodies can be bonded to enzymes
and the resulting complexes are still fully functional
both immunologically and enzymatically.
It is the enzyme activity which is the measure of
quantity of antigen or antibody present in the test
sample.
Enzymes used in ELISA include galactosidase
,glucose oxidase ,Peroxidase and alkaline
phosphatase.
USES:
1.ELISA is as sensitive as RIA.
2.Enzyme immunoassays are less expensive, safer and
just as reliable and accurate as radioimmunoassays.
A number of variations of ELISA have been
developed, allowing qualitative detection or
quantitative measurement of either antigen or
antibody.
Each type of ELISA can be used qualitatively to
detect the presence of antibody or antigen.


INDIRECT ELISA

Antibody can be detected or quantitatively determined with an indirect ELISA.

Indirect ELISA method is used to detect the

presence of serum antibodies against HIV.
SANDWICH ELISA
Antigen can be detected or measured using Sandwich ELISA.

In this the antibody is immobilized on the microtitre well

COMPETITIVE ELISA
 This method is used to measure amounts of antigen.
Standard curve for elisa
CURVES OF ELISA
 CHEMILUMINESCEN
CE
The measurement of light produced by
chemiluminescence during certain chemical reactions
provide a convenient and highly sensitive alternative to
absorbance measurements in ELISA assays.
In versions of the ELISA using chemiluminescence , a
luxogenic substrate takes the place of the chromogenic
substrate in conventional ELISA reactions.
The advantages of chemiluminescence assays over
chromogenic ones is enhanced sensitivity.
 Immunoflourescence
In 1944, Albert Coons showed that antibodies could be
labeled with molecules that have the property of
fluorescence.

Fluorescent molecule absorb light of one wavelength and

emit light of another wavelength.

If antibody molecules are tagged with a fluorescent dye or

fluorochrome, immune complexes containing these
fluorescently labeled antibodies can be detected by colored
light emission when emission by light of appropriate
wavelength.
In Immunoflourescence technique, fluorescent
substances are also routinely used, such as fluorescein and
rhodamine, are in common use, but other highly
fluorescent substances such as phycoerythrin, an intensely
colored and highly fluorescent pigment obtained from
algae.

These molecules can be conjugated with the Fc region of

the antibody without effecting the specificity of the
antibody.
• Two methods:

1. Direct

2.Indirect

IN DIRECT staining the specific antibody is directly

conjugated with fluorescein.
IN INDIRECT staining, the primary antibody is
unlabeled and is detected with an additional
fluorochrome labeled reagent.
INDIRECT METHOD OVER DIRECT
METHOD.

The primary antibody does not need to be conjugated

with a fluorochrome in indirect method.
Indirect methods increase the sensitivity of staining
because multiple molecules of the fluorochrome
reagent bind to each primary antibody molecule which
increase the amount of light emitted at the location of
each primary antibody molecule.
 Applications of Elisa
RIA (RADIOIMMUNO ASSAY)

IRMA (IMMUNORADIOMETRIC ASSAY)

RIA
Radioimmunoassay is used to measure very low
concentrations of analytes (a substance to be
measured) in biological fluids .It belongs to a class of
assay procedures called saturation analysis.

It is used for the quantitative measurement of

triiodothyronine and thyroxine.
IRMA
In IRMA an excess of radiolabelled antibody is
allowed to react with the analyte either from the
standard or from the sample.
At the end of the reaction, the antigen bound fraction
is counted for radioactivity and the concentration of
the unknown antigen can be read from a standard
curve.
IRMA is used for the quantitative measurement of
TSH in human serum or plasma.
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