ENZYME IMMUNOASSAY

ENZYME IMMUNOASSAY
(EIA)
Measurement of Enzyme-labelled Antigen & Antibody

HOMOGENEOUS HETEROGENOUS

Can be completed in one step, Multistep procedure,

all reagents added simultaneously reagents added sequentially

ELISA
 ELISA
ELISA is a highly sensitive and specific test used for the
detection of a variety of antigens and antibodies of
microbial and human origin.

FIRST SECOND THIRD

GENERATION GENERATION GENERATION

used infected viral cell used glycopeptides synthetic peptides are

lysate as antigen (recombinant antigens) used as antigens.

FOURTH
GENERATION

simultaneous detection
of Ag & Ab.
 ELISA - PRINCIPLE
 Antigen or antibody is adsorbed on the solid-phase which may be
polystyrene or polyvinyl microtitre plates.

 Test serum with antigen or antibody is added and incubated at 37°C for 2
hours for formation of antigen-antibody complex and unbound components
are finally removed by washing.

 The amount of bound fraction is subsequently measured by addition of

enzyme-linked anti-human globulin or specific antibody and an appropriate
substrate to produce colour and products detected by the ELISA reader.
 ELISA - REQUIREMENTS
 Patient serum
 Diagnostic kit
 Microtitre plate
 Enzyme conjugate (peroxidase-labelled purified antigen)
 Buffered substrate (contains H2O2).
 Chromogen solution (tetramethylbenzidine in dimethyl sulfoxide)
 Stopping solution (sulphuric acid)
 Washing solution
 Sample diluent
 Negative control
 Positive control
 Cut-off serum
 Conjugate diluent
 Diluents for reagents and specimen
 Microplate-ELISA reader
 ELISA - PROCEDURE
1. Add appropriate volume of diluent in each well.
2. Add appropriate volume of test serum and equal volume of
positive and negative controls into the respective wells of
microtitre plate.
3. Cover the plate with adhesive film and incubate at 37°C for
specified time.
4. Aspirate the contents and wash the wells four times by using
working wash solution. Now add appropriate volume of the
conjugate. Mix well and incubate at 37°C for specified time.
5. Aspirate the contents and wash the wells five times by using
wash solution.
6. Add appropriate volume of buffered substrate and chromogen
solutions. Incubate at 37°C for specified time.
7. Add stop solution and read absorbance bichromatically at 450
nm and 660 nm by using a microplate reader.
VIRUS SAMPLE ON SURFACE
Y Y Y
DIRECT METHOD OF ELISA
Antibody Enzyme conjugate
attached to Viral Antigen
Y Y Y
 Substrate is added,
Detecting antibody and is
Enzyme-linked secondary
converted
is added, by enzyme
andand
binds into a
antibody is added, binds
detectable form
to antigen
to detecting antibody
 Sample is added, and any
Y Y
SANDWICH
Plateantigen present
is coated METHOD
withbinds to
Y Y Y
capture
a capture OF ELISA
antibody
antibody

Y Y Y Y Y
 ELISA
RESULTS & INTERPRETATION
 Development of yellow color indicates the test serum contains
antigens to tested antibody.
ϴ Development of no color indicates negative reaction.
 Dot ELISA
 Dot ELISA is a rapid visually read microassay.
 The principle is similar to that of ELISA where enzyme is used as a
marker or label to detect the binding of antigen and antibody.
 The test is performed on a nitrocellulose membrane.
 Enzyme converts colourless substrate to a coloured product, which
indicates the presence of antigen-antibody binding.
 When the test serum is layered on to the membrane, specific antibodies if
present, will bind to corresponding dot of the antigen.
 Addition of a labeled serum antibody (conjugate) and the subsequent
development of the colour allow the detection of the presence of
antibodies based on the pattern of the antigen sites.
 Dot-ELISA can be used for the detection of antibodies as well as antigen.
 ELISA
Enzymes
Horseradish peroxidase.
Alkaline phosphatase.
β-D galactosidase,
Glucose 6-phosphate dehydrogenase (G6PD).
Acetylcholinesterase.

Substrates
Hydrogen peroxide (H2O2).
Nicotinamide adenine dinucleotide phosphate (NADP+).
2-nitrophenyl--D-galactoside.
Glucose-6 phosphate.
Acetylcholine.
 ELISA
Advantages
Highly sensitive
Specific
Rapid

Disadvantages
Requires specialized expert personnel.
Requires expressive laboratory equipment.
Enzymes require cold environment for
storage.
LAB DIAGNOSIS OF DENGUE
Dengue NS1 Ag detected by “Direct Sandwich”
principle in ELISA
NS1 antigen is generally found during Day 1 and up to
Day 9 after onset of fever.
The detection of NS1 Ag is inhibited if anti-NS1
antibodies produced.
Whereas IgM become detectable by Day 3 to Day 5
after onset of illness in primary dengue and by Day
1 to Day 2 after onset of illness in secondary
infections.
 Y
Y
Y Y
Y
Y

Y
YY

YY
YYYYY
MAC Dengue IgM ELISA
Dengue IgG ELISA
 ELISA - USES
ELISA kits have been developed for detecting:
 HIV-1 and HIV-2 antigens and antibody.
 Entamoeba histolytica antigens in faeces.
 Toxoplasma antigens in patient serum.
 Haemophilus influenza antigens in spinal fluid.
 B-haemolytic streptococcal antigens in spinal fluid.
 Hepatitis A virus in stools.
 Respiratory syncytial virus in pharyngeal secretions.
 Adenovirus antigens in nasopharyngeal specimens.
 Labile enterotoxin of Escherichia coli in stools.
 Hepatitis B surface antigen and its antibody.
 Cytomegalovirus antigen.
 Rubella IgG and IgM.
 Mycobacterium tuberculosis IgA.
 Thyroid hormones T3, T4 and TSH.
 etc.,
VIVA
1 Expand ELISA.
Ans. Enzyme-linked immunosorbent assay.
2 What is the principle of enzyme immuno assay?
3 What are different steps in the ELISA procedure?
Ans.
a Coating solid surface with antigen/antibody.
b Binding antibody/antigen from test sample.
c Binding conjugate.
d Addition of substrate.
e Observe the colour change and read the O.D. values.
4 What are various types of the ELISA?
Ans. Competitive ELISA, sandwich ELISA, indirect ELISA,
membrane bound ELISA, cassette ELISA, biotin-avidin ELISA,
protein-A ELISA, etc.
5 What are the enzymes and substrates used in the ELISA?
6 What do you understand by the first, second and third generation
ELISA?
7 What are the advantages and disadvantages of the ELISA?
THANK YOU
ELISA
Immunosorbent
Here, an absorbing material is used (e.g.
polystyrene, polyvinyl) that specifically
absorbs the antigen or antibody present in
serum

Enzyme is used to label one of the components

of immunoassay (i.e. antigen or antibody).

Substrate-chromogen system
A substrate-chromogen system is added at the
final step of ELISA.
 ENZYME IMMUNOASSAY
(EIA)
Measurement of Enzyme-labelled Antigen & Antibody

HOMOGENEOUS HETEROGENOUS

Can be completed in one step, Multistep procedure,

all reagents added simultaneously reagents added sequentially

ELISA
 ELISA
ELISA is a highly sensitive and specific test used for the
detection of a variety of antigens and antibodies of
microbial and human origin.

FIRST SECOND THIRD

GENERATION GENERATION GENERATION

used infected viral cell used glycopeptides synthetic peptides are

lysate as antigen (recombinant antigens) used as antigens.

FOURTH
GENERATION

simultaneous detection
of Ag & Ab.
 ELISA - PRINCIPLE
 Antigen or antibody is adsorbed on the solid-phase which may be
polystyrene or polyvinyl microtitre plates.

 Test serum with antigen or antibody is added and incubated at 37°C for 2
hours for formation of antigen-antibody complex and unbound components
are finally removed by washing.

 The amount of bound fraction is subsequently measured by addition of

enzyme-linked anti-human globulin or specific antibody and an appropriate
substrate to produce colour and products detected by the ELISA reader.
Y
 Microtiter Plate

Polystyrene, Polyvinyl or Polycarbonate material

 POSITIVE CONTROL
NEGATIVE CONTROL
ENZYME CONJUGATE
SUBSTRATE/CHROMOGEN
DILUTION BUFFER
 ELISA

INDIRECT SANDWICH MAC

DIRECT
Y Y Y
Y Y
Y
Y Y
  DETECTION OF ANTIGEN IN THE PATIENTS’ SERUM
DIRECT
MICROTITRE PLATE IS EMPTY
ELISA ADD THE PATIENTS’ SAMPLE CONTAINING Ag
ENZYME-LABELED PRIMARY ANTIBODIES
SUBSTRATE–CHROMOGEN SYSTEM IS ADDED

Y
  DETECTION OF ANTIBODY > ANTIGEN IN THE PATIENTS’ SERUM
NDIRECT
ELISA MICROTITRE PLATE IS PRECOATED WITH ANTIGEN
ADD THE PATIENTS’ SAMPLE CONTAIN PRIMARY Ab
ENZYME-LABELED SECONDARY ANTIBODIES
SUBSTRATE–CHROMOGEN SYSTEM IS ADDED

Y
Y
  DETECTION OF ANTIGEN IN THE PATIENTS’ SERUM
ANDWICH
ELISA MICROTITRE PLATE IS PRECOATED WITH CAPTURE Ab
ADD THE PATIENTS’ SAMPLE CONTAIN ANTIGEN
ENZYME-LABELED PRIMARY ANTIBODIES
SUBSTRATE–CHROMOGEN SYSTEM IS ADDED

Y
  DETECTION OF ANTIBODY IN THE PATIENTS’ SERUM
MICROTITRE PLATE IS PRECOATED WITH CAPTURE Ab
ADD THE PATIENTS’ SAMPLE CONTAIN PRIMARY ANTIBODY
ADD THE RECOMBINANT ANTIGEN
ENZYME-LABELED (biotin +avidin) SECONDARY ANTIBODIES
AC ELISA
SUBSTRATE–CHROMOGEN SYSTEM IS ADDED

Y
Y
Y
ELFA
 Discussion
1. What do you mean by first, second and third generation
ELISA?
 First generation ELISA: Here the antigen is prepared by
propagating viruses in mammalian cell line. It is coated on
microwell plates after disrupting and inactivating the viral
concentrate.
 Second generation ELISA: Here the antigen is prepared
either by bacterial recombinant DNA technology or
proteins which corresponds to viral com-ponent.
 Third generation ELISA: Here the synthetic peptides are
used as antigen source and they generally correspond to
viral antigenic epitopes.
 Discussion
2. What do you mean by immunosorbent in term ELISA?
 An absorbing material specific for one of the components of
the reaction, the antigen or antibody.
3. Enumerate few immunosorbents.
 Particulate:
 Cellulose
 Agarose
 Solid phase :
(Tubes or microwell)
 Polystyrene
 Polyvinyl
 Polycarbonate
 Membranes or disc:
 Polyacrylamide
 Paper
 Plastic
 Discussion

4. Name different types of ELISA.

 Indirect ELISA
 Competitive ELISA
 Sandwich ELISA(direct and indirect)

5. What do you mean by indirect ELISA?

 This is used for the detection of antibody and, there-fore,
the solid-phase is conjugated with antigen.
 Discussion
6. Describe briefly competitive ELISA.
 This method can be used for detection of antigen or
antibody. For the detection of antibodies, solid-phase is
coated with antigen.
 Test sample and enzyme-labelled specific antibody
conjugate are added at the same time for specific period.
 During incubation there is competition between
antibodies of patient serum and specific antibody
conjugate for sharing of antigen.
 Intense colour suggests the binding of specific antibody
conjugate and hence a non-reactive reaction, while lack
of colour indicates binding of patient serum antibodies
and hence a reactive reaction.
 Discussion
7. What is the advantage of competitive over indirect ELISA?
 It take less time.
 No predilution of test serum is required.

8. How many types of sandwich ELISA are known to you?

 Two types:
 Single antibody or direct sandwich ELISA
 Double antibody or indirect sandwich ELISA

9. What is the difference between EIA and radioimmunoassay

(RIA)?
 Both these tests are based on the same principle. The key
difference is that for enzyme immunoassays the antigen or
antibody is conjugated to an enzyme rather than a
radioactive isotope.
 Discussion
10. What is the function of this enzyme?
 Enzyme converts a colourless substrate into a coloured one.

11. Which enzymes are commonly used in this test?

 Horseradish peroxidase
 Alkaline phosphatase

12. What are the advantages of ELISA test?

 Highly sensitive
 Safe and economical
 Simple instrumentation required
 Many tests can be performed at the same time.
VIVA
1 Expand ELISA.
Ans. Enzyme-linked immunosorbent assay.
2 What is the principle of enzyme immuno assay?
3 What are different steps in the ELISA procedure?
Ans.
a Coating solid surface with antigen/antibody.
b Binding antibody/antigen from test sample.
c Binding conjugate.
d Addition of substrate.
e Observe the colour change and read the O.D. values.
4 What are various types of the ELISA?
Ans. Competitive ELISA, sandwich ELISA, indirect ELISA,
membrane bound ELISA, cassette ELISA, biotin-avidin ELISA,
protein-A ELISA, etc.
5 What are the enzymes and substrates used in the ELISA?
6 What do you understand by the first, second and third generation
ELISA?
7 What are the advantages and disadvantages of the ELISA?
THANK YOU