Introduction, history...........to new techniques.

1. ELISA
(Enzyme-linked immunosorbent
assay )
ANITA SINGH
MEDICAL ONCOLOGY

2.  HISTORY
 Prior to the development of the EIA/ELISA, the only option for conducting
an immunoassay was radioimmunoassay, a technique using radioactively-
labeled antigens or antibodies.
 Avramais (1966, 1969) and Pierce (1967) developed methods to chemically
link antibodies to biological enzymes whose activities produce a measurable
signal with solutions containing appropriate substrates.
 This signal has to be associated with the presence of antibody or antigen .
 COMPONENTS OF ELISA
 Antibody: IgG fraction of serum.
 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.

3.  INTRODUCTION TO ELISA
A 96 - well microtiter plate
being used for ELISA.
 A test that uses antibodies and color
change to identify a substance.
 ELISA is a popular format of a
"wet-lab" type analytic 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
"immuno" assays.

4.  “Wet lab" analytic biochemistry assay, ELISA involves detection of an "analyte"
in a liquid sample by a method that continues to use liquid reagents during the
"analysis“.
 The basic principle of an ELISA is to use an enzyme to detect the Ag-Ab binding
(antigen- antibody binding). The enzyme converts a colorless substrate
(chromogen) to a colored product, indicating the presence of Ag:Ab binding.
 PRINCIPLE

5. Different antigen in sample
substrate
Colored
product
Primary antibody
Secondary antibody

6.  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 (will
latch onto) 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,
etc.) 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.
 HOW DOES ELISA WORK ?

7.  TYPES OF ELISA
 INDIRECT ELISA
 DIRECT ELISA
 SANDWICH ELISA
 COMPETETIVE ELISA
NON -COMPETETIVE
ELISA

8.  INDIRECT ELISA
 Antigen is added to plate.
 Added Blocking buffer.
 Suitable primary antibody is added.
 Secondary antibody- HRPO is then
added which recognizes and binds to
primary antibody.
 TMB substrate is added, is converted
to detectable form.
 Under standard condition ,the enzyme activity measured is proportional to
amount of specific antibody in the original serum.

9.  ADVANTAGES OF INDIRECT DETECTION
 Wide variety of labeled secondary antibodies are available commercially.
 Versatile, since many primary antibodies can be made in one species and the
Same labeled secondary antibody can be used for detection.
 Immunoreactivity of the primary antibody is not affected by labeling.
 Sensitivity is increased because each primary antibody contains several
epitopes that can be bound by the labeled secondary antibody, allowing for
signal amplification.
 DISADVANTAGES OF INDIRECT DETECTION
 Cross-reactivity may occur with the secondary antibody, resulting in
nonspecific signal.
 An extra incubation step is required in the procedure.

10.  DIRECT ELISA
1. Apply a sample of known antigen to
a surface.
2. Enzyme linked primary antibody is
applied to the plate.
3. Washed, After this wash, only the
antibody-antigen complexes remain
attached.
4. Apply a substrate which is
converted by the enzyme to elicit a
chromogenic signal.
 Under standard condition ,the enzyme
activity measured is proportional to
amount of specific antibody in the
original serum.

11.  ADVANTAGES OF DIRECT DETECTION
 Quick methodology since only one antibody is used.
 Cross-reactivity of secondary antibody is eliminated.
 DISADVANTAGES OF DIRECT DETECTION
 Immunoreactivity of the primary antibody may be reduced as a result of
labeling.
 Labeling of every primary antibody is time-consuming and expensive.
 No flexibility in choice of primary antibody label from one experiment to
another.
 Little signal amplification.

12.  SANDWICH ELISA
1. a. Plate is coated with suitable antibody.
b. Blocking buffer is added.
2. Sample is added to plate so antigen is bounded by capture antibody.
3. A suitable biotin labeled detection antibody is added to plate.
4. Enzyme HRPO is added and binds the biotin labeled detection antibody.
5. TMB substrate is added and converted by HRPO to colored product.
Under standard condition ,the enzyme activity measured is proportional to the Amount of
specific antigen in the original serum.

13.  MATERIALS NEEDED FOR ELISA KIT
 ELISA Plate
 Positive control
 Negative control
 Dilution Buffer
 Conjugate
 TMB Substrate
 Stop Solution

14. Wash
3X
Wash
4X
Wash
4X
Wash
4X
A EDCB
Alkaline phosphatase
substrate is added
and developed colour
is read at 405 nm
wavelength to
measure plasma
cencentration
Wells are
coated with
0.2 μg
primary
antibody
Diluted
plasma
is added to
coated wells,
which bind
to antibodies
0.1 μg of
biotinylated
(biotin = – )
antihuman
secondary
antibody
Incubated
overnight at 4˚C
Incubated at room temperature (24˚C)
2h 2h 1h
 PROCEDURE OF ELISA
Add 1.2000
dilution of
streptavidin
conjugate to
alkaline
phosphatase
( E)

15.  FINAL PLATE OF ELISA

16. COMPETETIVE ELISACOMPETETIVE ELISA
 COMPETETIVE ELISA
Solid phase coated with antibody
Add unknown amount of unlabeled
antigen and known amount of labeled
antigen
Free and labeled antigen are captured
Color formation by oxidation of substrate
into a colored compound
Under standard condition ,the enzyme activity measured is proportional to the
proportion of labeled antigen in the mixture of labeled and unlabled antigen.

17.  ADVANTAGES:
 Suitable for complex (crude or impure) samples, since
the antigen does not require purification prior to
measurement.
 DISADVANTAGES
 Each antigen may require a different method to couple it to
the enzyme.

18.  COMPARISON BETWEEN VARIOUS TYPES OF ELISA
Direct ELISA Indirect ELISA Sandwich ELISA Competitive ELISA

19. The ELISA assay yields three different types of data output:
 Quantitative: ELISA data can be interpreted in comparison to a standard
curve in order to precisely calculate the concentrations of
antigen in various samples.
 Qualitative: ELISAs can also be used to achieve a yes or no answer
indicating whether a particular antigen is present in a sample,
as compared to a blank well containing no antigen or an
unrelated control antigen.
 Semi-quantitative: ELISAs can be used to compare the relative levels of antigen
in assay samples, since the intensity of signal will vary
directly with antigen concentration.
 ELISA RESULTS

20.  ELISA data is typically graphed with Optical density Vs Log concentration to produce
a sigmoidal curve.
 Known concentrations of antigen are used to produce a standard curve and then this
data is used to measure the concentration of unknown samples by comparison to the
linear portion of the standard curve.
 This can be done directly on the graph or with curve fitting software which is
typically found on ELISA plate readers.

21.  SENSITIVITY
ELISAs are one of the most sensitive immunoassays
available. The typical detection range for an ELISA is 0.1 to 1 fmole
or 0.01 ng to 0.1 ng, with sensitivity dependent upon the particular
characteristics of the antibody – antigen interaction.
In addition, some substrates such as those yielding
enhanced chemiluminescent or fluorescent signal, can be used to
improve results.
As mentioned earlier, indirect detection will produce
higher levels of signal and should therefore be more sensitive.
However, it can also cause higher background signal thus reducing
net specific signal levels.

22.  PRECAUTIONS
 Negative control with strong signal
The excessive background signal can be caused by inadequate rinsing of plates, reagents not
sufficiently diluted, inadequate blocking of plates or non-specific binding of enzyme
conjugate. The appearance of color in negative control wells may also indicate cross-reactivity
of secondary antibody with components in the antigen sample.
 Positive control with no signal
 Microwell plates not coated properly.
 Reagents applied in wrong order or step omitted.
 Secondary antibody not matched to the species of primary antibody.
 Enzyme conjugate defective or inhibited by contaminant.
 ELISA with weak signal
 Wash buffer not adequately drained after every wash step.
 Inadequate incubation times.
 Enzyme conjugate defective or inhibited by contaminant.
 Substrate defective or contaminated.
 Microwell plates poorly coated.
 Loss of capture antibody during blocking/washing.

23.  PRACTICAL ASPECTS
 Solid phases used in ELISA include cross-linked dextran or
polyacrylamide beeds, filter paper(cellulose) disc or polypropylene
tubes and disposable polystyrene microtitre plates.
 Antigen or antibodies attached to solid phase by passive absorption.
 Conjugate used must contain a highly reactive antibody coupled to
an enzyme with a highly turnover number(alkaline phosphatase and
horseradish peroxide are commonly used enzymes).

24.  APPLICATIONS
 Screening donated blood for evidence of viral contamination by
 HIV-1 and HIV-2 (presence of anti-HIV antibodies)
 Hepatitis C (presence of antibodies)
 Hepatitis B (testing for both antibodies and a viral antigen)
 Measuring hormone levels
 HCG (as a test for pregnancy)
 LH (determining the time of ovulation)
 TSH, T3 and T4 (for thyroid function)
 Detecting infections
 Sexually-transmitted agents like HIV, syphilis and chlamydia
 Hepatitis B and C
 Toxoplasma gondii
 Detecting illicit drugs.
 Detecting allergens in food and house dust

25. A new technique uses a solid phase made up of an immunosorbent polystyrene
rod with 4-12 protruding ogives. The entire device is immersed in a test tube
containing the collected sample and the following steps (washing, incubation in
conjugate and incubation in chromigeneous) are carried out by dipping the ogives
in microwells of standard microplated prefilled with reagents.
Advantages of this technique:
 The ogives can each be sensitized to a different reagent, allowing the
simultaneous detection of different antibodies and different antigens
for multi-target assays.
 The sample volume can be increased to improve the test sensitivity in
clinical, food and environmental samples.
 The use of laboratory supplies for dispensing sample
aliquots, washing solution and reagents in microwells is not
required, facilitating ready-to-use lab kits and on-site kits.
 A NEW TECHNIQUE:- REVERSE ELISA