Usefulness of enzyme

Linked Immune Sorbent

Assay :
various methods for disease
diagnosis and monitoring
Shivaraj. D.B.
PhD scholar
Dept of Veterinary
Microbiology

INTRODUCTION
Enzyme-linked immunosorbent assay (ELISA),
also known as an enzyme immunoassay (EIA),
is a biochemical technique used mainly in
immunology to detect the presence of an antibody
or an antigen in a sample
Enzyme immunoassays provide highly sensitive
and precise methods for the estimation of
biological parameters with advantage of handling
and analysis of large number of samples with
automation

The enzyme immunoassays, began with

work of AVRAMEAS and URIEL who
conceived the idea of labelling antigens
and antibodies with enzymes for use in
serological techniques.

The basic ELISA test depends on two

assumptions:
1. The antigen or antibody can be attached to a
solid support, yet retain immunological activity.
2. That, either antigen or antibody can be linked
to an enzyme and the complex retain both
immunological and enzymatic activity.

Principle:
In simple terms, in ELISA, an unknown amount of
antigen is affixed to a surface, and then a specific
antibody is applied over the surface so that it can
bind to the antigen. This antibody is linked to an
enzyme, and in the final step a substrate is added
that the enzyme can convert to some detectable
signal, most commonly a colour change in a
chemical substrate

ELISA-Principle

ELISA -Introduction
Primary
immunological test

Antigen
Epitope
Antibody Antigen binding
site
Fc region
Anti antibody

ELISA- Objectives
To identify the causative agent
of infectious/parasitic origin
To identify the presence of
specific antibodies
To quantify the antibodies or
antigen, hormones, enzymes etc.

ELISA- General
requirements

Solid phase preferably 96 well micro tire

plates.
Buffers
Coating, blocking, washing, substrate buffer

Stopping reagents
Single and multi channel pipettes
Disposable tips, wash bottles
Ag, Ab
Enzyme labeled antibody
Substrate
Incubator
ELISA reader

ELISA plates
Flat, Solid support
supports the Ag and
Ab binding
Flat surface helps
equal distribution of
reagents
Eg: cellulose,
polysterene plastic,
glass
Polystyrene most
commonly used

Coating

buffers

Coating buffer:
For initial coating of Ag or Ab on
to the
solid surface
Eg: 0.05 M carbonate buffer of pH 9.6
Coating buffer: {carbonate bi carbonate
buffer, 0.05M, pH9.6}
Sodium carbonate (Na2CO3 )
Sodium bicarbonate (NaHCO3)
Distilled water
1000ml

1.59g
2.93g

Used to coat the antigen or

Washing Buffer:
used to wash the plates in between the
steps to remove the unbound reagent
Eg: PBS plus 0.05% Tween -20
Phosphate buffered saline(PBS, pH 7.4) 1000ml
Tween -20
0.5ml
Used to wash the plates in order to remove
unbound reagents following incubation and as a
diluent for samples and conjugates.

Blocking buffer:
Blocking buffer:
after primary adsorption to solid
phase, other reagents are added in
blocking buffer to prevent non
specific binding to the plate
Eg: casein,BSA(bovine serum
albumin), Gelatin, Lactalbumin
hydrolysate etc
Gelatin and BSA are most widely used.

ENZYMES
Enzymes used in ELISA test Should be
stable,
highly reactive,
cheap,
safe
easily prepared and
assayable
Most commonly used are horse radish
peroxidase (HRPO) and Alkaline
phosphatase
Low molecular weight ,covalently binds
toAB

Substrate and chromogen

Substrate:
for the enzymes to act
Most commonly used is hydrogen
peroxidase
Chromogen
Which are initially colorless but yield
colors when enzymes degrade the
substrate
Most commonly used are OPD and ODD.

Stopping solution
Used to stop enzyme reaction
after suitable color development
after a certain time
Usually strong acids or alkalis are
used
Most commonly used in 1M Sulphuric
acid

Substrate

Enzyme

Chromogen

Stopping
solution

Absorbance
wavelength

p-Nitro
phenyl
phosphate

Alkaline
phosphatase

Tetra
methyl
benzadine
(TMA)

0.1M
EDTA

450nm

H2O2

Horse
radish
peroxidase

TMA

1M H2SO4

450nm

H2O2

Horse
radish
peroxidase

ABST

1M H2SO4

450nm

H2O2

Horse
radish
peroxidase

OPD
ODD

1M H2SO4

490nm

ABST:2.2 Azino di -3 ethyle benzathiozoline 6Sulphonic acid

OPD: ortho phenylene diamine hydrochloride
ODD: ortho dianisidine di hydrochloride

Standards
The inference in ELISA is absolutely based on
differences in the Optical densities of known
positives, known negatives and the sample
under question
Hence, accurate titration of known positives
is very critical in carrying out ELISA
Different types of standards commonly used
in ELISA are

Strong positives
Moderate positives
Weak positives
Negative

Types of ELISA

Direct
Indirect
Sandwich
Competitive
Dot
Avidin-biotin

Direct ELISA
(For detection of Antigen)
Procedure
1. Coating of the unknown antigen
on to the solid surface (PLATE)
using a suitable coating buffer
2. Incubation of plates at 37 c for
3hrs or at 4 c for overnight.
3. washing in wash buffer
4. Addition of known antibody
conjugated with Enzyme
5. Incubation at 37 c for 1 hr and
later washing thrice in wash
buffer.
6. Add the substrate and
chromogen and read in ELISA
reader
Note: Presently not used and has
been replaced by newer methods as
this will lead to lot of false positives
due to cross reactivity.

Indirect ELISA (For detection of

Antibody)
1. Coating of the specific antigen on to
the solid surface (plate) using a
suitable coating buffer

2.

Incubation of plates at 37 c for 3hrs

or at 4 c for overnight &.washing in
wash buffer

4.

Addition of suspected serum sample

(After suitable dilution in blocking
buffer) on to the Ag coated Elisa plate.

5. Incubation at 37 c/ 1 hr and later

washing thrice in wash buffer.
6. Addition of secondary antibody
conjugated with HRPO enzyme
7. Incubation at 37 c/ 1 hr and wash 3x.

6.

8.

Addition of substrate/chromogen in all

the wells , incubation in dark for 20
mins
Stopping of reaction and plate read

Avidin-biotin ELISA
Strong and specific binding of avidin
to biotin
Biotin with low molecular weight
easily bound to protien
Avidin small protien found in egg,
specifically bind to biotin is the
principle of this techinique

Avidin Biotin ELISA (for Ab detection)

Passive adsorption of antigen in defined buffer to the
microtitre plate by incubation
Wash
Addition and incubation with antibody (test sera) directed
against antigen
Wash
Addition and incubation with anti-bovine IgG biotin conjugate
Wash
Addition and incubation with Avidin HRPO conjugate
Wash
Addition and incubation with substrate and chromogen

Reaction is stopped and the plate is read at specified

absorbance.

Applications of Avidin Biotin ELISA

Biotin labelled hormone can be used to detect hormone
receptor.
Biotin labelled toxin can be used to detect toxin receptor
Biotin labelled viral DNA can be used to localise viral
genome
Can be applied to all test systems
IMMUNOCHEMISTRY,
ELECTRONMICROSCOPY
WESTERN BLOTS
ELISA , HYBRIDOMA SCREENING
FLOW CYTOMETRY
DNA in situ HYBRIDIZATION

Competitive ELISA
(antibody detection)

1.

Coating of the specific antigen on to the solid surface (plate) using a suitable coating buffer

2.

Incubation of plates at 37 c for 3hrs or at 4 c for overnight &.washing in wash buffer

3.

a) Addition of suspected serum sample (After suitable dilution in blocking buffer) on to the Ag coated Elisa
plate.
b)addition of standard known Mab (monoclonal antibody) against the coated antigen
Both a and b compete for the coated antigen, if the serum contains antibodies will have more avidity to the
coated antigen and binds faster than Mab which has only one epitpoe and less avidity)

5.

Incubation at 37 c/ 1 hr and later washing thrice in wash buffer.

6.

Addition of secondary antibody specific against Mab conjugated with HRPO enzyme

7.

Incubation at 37 c/ 1 hr and wash 3x.

6.

Addition of substrate/chromogen in all the wells , incubation in dark for 20 mins Stopping of reaction and
plate read

Inference
Since the secondary Ab is against Mab color is developed in those wells where Mab has bound to the antigen and
not where serum Abs have bound hence
More color means moe Mab and less serum Ab
Less color means less Mab attachment more serum antibodies

NO COLOUR:POSITIVE
COLOUR: NEGATIVE

Competitive ELISA
(antibody detection)
NO COLOUR: POSITIVE
COLOUR: NEGATIVE

Sandwich ELISA (for

detection of Ag)
Measures specific antigen
Wells are coated with a specific antibody
(capture antibody)
Antigen to be tested is added to each well
Detection antibody from different species
Formation of antibody antigen-antibody
layers
Intensity of colour is related directly to
the amount of bound antigen

Sandwich ELISA (for

detection of Ag)

1.

Coating of the monoclonal antibody /antibody on to the

solid surface (PLATE) using a suitable coating buffer

2.

Incubatrion of plates at 37 c for 3hrs or at 4 c for

overnight &.washing in wash buffer

3.

Addition of suspected sample (Ag)(After suitable

dilution in blocking buffer) on to the Ab coated Elisa
plate.

4.

Incubation at 37 c/ 1 hr and later washing thrice in wash

buffer.

5.

Addition of specific secondary antibody Ag gets

sandwiched between tow antibodies

6.

Addition of HRPO enzyme labeled antibody

7.

Incubation at 37 c/ 1 hr and wash 3x.

8.

Addition of substrate/chromogen in all the wells ,

incubation in dark for 20 mins

9.

Stopping of reaction and plate read

Sandwich ELISA
Used to detect circulating virus in
blood
Example; cats with feline leukemia
Modification of this technique is :labelled
antigen ELISA used to detect antibodies
This is favoured in manufactured diagnostic
kits

Dot ELISA
A modified ELISA performed
on Nitrocellulose membrane
adsorbed with a known
antibody and detects the Ag
in the given sample.
Unknown samples are directly
applied on to the membrane.
The Ag Ab reaction is
detected secondary antibody
labeled with an enzyme which
goes on react with the
substrate changing the color
of the chromogen.

Liquid phase Blocking

ELISA
Aimed to quantify protective Abs following

Vaccination.
Ag-Ab reactions are carried out in liquid medium
If the homologous Abs present in serum, blocks the
Ag from being
detected by GP serum.
Procedure
1.A series of 2 fold dilutions of test serum are mixed
with an equal volume of a fixed dose of virus in low
binding perplex plates allowed to react overnight
art 4 c.
2.Next day free antigens (not blocked by the
antibodies in the test serum) are trapped to the
wells of the ELISA plates coated with type specific
antibodies

INTERPRETATION

Applications of ELISA
To identify the causative agent

i.e, Typing

Discrimination of the disease agents Sub

typing
To identify the presence of specific
antibodies
To quantify the antibodies or antigen,
hormones, enzymes etc
Monitoring responses to vaccination
Sero surveillance of diseases

Applications of ELISA
Differentiation of infected and
vaccinated animals (DIVA)
Eg: Avian Influenza.
Infected : H5N1
Vaccinated : H5N2
FMD

Advantages of ELISA
Technique is Simple
Large no of samples can be
screened in a short period of time
Results can be kept up to 6 months
which helps future references
Introduction of Mabs has increased
the specificity of the test.
Antigens are inactivated and hence
not harmful.

Advantages of ELISA
Different pathogenic diseases
HIV,RUBELLA, CHOLERA SYPHLIS
,TETANUS etc.
Brucellosis, tuberculosis,
salmonellosis.
Infectious bovine rhinotrachitis

Disadvantages of ELISA
The reaction is irreversible or permanent, reagents,
plates cant be re used.
Inadequacies of pure strains, enzymes, reagents may
reduce specificity or sensitivity of the test
The reagents/reader are costly and the test as such
becomes costlier

Cross reactions may give false positives

Results are purely based on the accuracy of titration of
known standards
Many reagents/steps are involved if a simple error in any
one of them -the entire test becomes invalid.

IAH AND VB
INDIRECT ELISA:

Leptospirosis
Swine fever
Jhones disease
IB,NDV,Mycoplasma
A-B ELISA:
Brucellosis
IBR
C-ELISA;
Avian influneza

THANK YOU