ELISA

HISTORY

 Priorto the development of the EIA/ELISA,

the only option for conducting an
immunoassay was radioimmunoassay, a
technique using radioactively-labeled
antigens or antibodies.
 Radioimmunoassay was first described in
a paper by Rosalyn Sussman Yalow and
Solomon Berson published in 1960.
Continued

 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
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.
A 96-WELL MICROTITER PLATE USED FOR ELISA
COMPONENTS OF AN ELISA
 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

 Antibody is immobilized on micro-plate wells

 Competition between in sample and labeled
enzyme for antibody binding sites
 The unbound material is washed out
 Chromogenic substrate added to develop color
 Resulting color is read in a spectrophotometer
TYPES OF ELISA

 DIRECT ELISA
 INDIRECT ELISA
 SANDWICH ELISA
 COMPETETIVE ELISA
DIRECT ELISA

The direct ELISA uses the method of

directly labeling the antibody itself.
Microwell plates are coated with a
sample containing the target antigen,
and the binding of labeled antibody is
quantitated by a colorimetric,
chemiluminescent, or fluorescent end-
point.
DIRECT ELISA
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.
INDIRECT ELISA

 The indirect ELISA utilizes an unlabeled

primary antibody in conjunction with a
labeled secondary antibody. Since the
labeled secondary antibody is directed
against all antibodies of a given species
(e.g. anti-mouse), it can be used with a
wide variety of primary antibodies (e.g.
all mouse monoclonal antibodies).
INDIRECT ELISA
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.
SANDWICH ELISA

1. Plate is coated with a capture antibody

2. Sample is added, and any antigen present
binds to capture antibody
3. Detecting antibody is added, and binds to
antigen
4. Enzyme-linked secondary antibody is added,
and binds to detecting antibody
5. Substrate is added, and is converted by
enzyme to detectable form.
COMPETITIVE ELISA

In this Unlabeled antibody is incubated in the

presence of its antigen.
 These bound antibody/antigen complexes are then
added to an antigen coated well.
 The plate is washed unbound antibody is removed.
 The secondary antibody, specific to the primary
antibody is added. This second antibody is coupled to
the enzyme.
 A substrate is added, and remaining enzymes elicit a
chromogenic or fluorescent signal.
 For competitive ELISA, the higher the original antigen
concentration, the weaker the eventual signal.
ELISA Reverse method & device
(ELISA-R m&d)
A newer technique uses an 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 chromogenous) are carried out
by dipping the ogives in microwells of standard
microplates pre-filled with reagents.
ADVANTAGES
 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 (saliva, urine), food (bulk milk, pooled
eggs) and environmental (water) samples.
 One ogive is left unsensitized to measure the non-specific
reactions of the sample.
 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.
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.
 Detector antibody not compatible with capture
antibody (for sandwich assays).
ELISA with weak signal
 Wash buffer not adequately drained after every wash
step.
 Inadequate incubation times.
 Detection reagents too dilute. Perform checkerboard
titrations.
 Enzyme conjugate defective or inhibited by contaminant.
 Substrate defective or contaminated.
 Microwell plates poorly coated.
 Loss of capture antibody during blocking/washing.
Decrease or eliminate use of Tween-20.
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 allergens in food and house dust
 Measuring "rheumatoid factors" and other autoantibody in
autoimmune diseases like lupus erythematosus
 Measuring toxins in contaminated food
 Detecting illicit drugs, e.g.,
 cocaine
 opiates
THANKS