 a sensitive biochemical or immunochemical assay method that

uses analyte (antigen or antibody) and color change to detect

and identify a substance and its quantification

 also called solid-phase enzyme immunoassay as it employs an

enzyme linked antigen or antibody as a marker for the detection
of specific protein

 an enzyme conjugated with an antibody reacts with a colorless

substrate to generate a colored reaction product

 a number of enzymes labels have been employed for ELISA,

including alkaline phosphatase (AP), the most commonly used
horseradish peroxidase (HRP), and β-galactosidase
 a plate-based assay technique designed for detecting and
quantifying substances such as peptides, proteins, antibodies
and hormones

 the antigen, which is recognized by specific antibody (immuno),

must be immobilized on a solid surface (sorbent) and then
complexed with an antibody that is linked to an enzyme
(enzyme-linked)

 the most crucial element of the detection strategy is a highly

specific antibody-antigen interaction

 the choice of substrate depends upon the required assay

sensitivity and the instrumentation available for signal-detection
(spectrophotometer, fluorometer or luminometer).
 It is this binding and immobilization of reagents that makes
ELISAs so easy to design and perform. Having the ELISA
reactants immobilized to the microplate surface makes it easy
to separate bound from non-bound material during the assay.

 This ability to wash away nonspecifically bound materials

makes the ELISA a powerful tool for measuring specific
analytes within a crude preparation.
 the analyte is also called the ligand because it will specifically
bind or ligate to a detection reagent, thus ELISA falls under the
bigger category of ligand binding assays

 the ligand-specific binding reagent is "immobilized," i.e.,

usually coated and dried onto the transparent bottom and
sometimes also side wall of a well which is usually constructed
as a multiple-well plate known as the "ELISA plate."

 Conventionally, like other forms of immunoassays, the

specificity of antigen-antibody type reaction is used because it
is easy to raise an antibody specifically against an antigen in
bulk as a reagent.

 Alternatively, if the analyte itself is an antibody, its target

antigen can be used as the binding reagent.
Antigen
a toxin or other
foreign
substance
which induces
an immune
response in the Antibody (Ab) or
body, especially immunoglobulin (Ig)
the production a large, Y-shaped
of antibodies. protein produced
mainly by plasma
cells that is used by
the immune system to
4 types of ELISA on the basis of binding neutralize pathogens
structure between Antibody and Antigen such as pathogenic
bacteria and viruses
> Direct ELISA
> Indirect ELISA
> Sandwich ELISA
> Competitive ELISA
General
Procedure
of ELISA
Four Most Common ELISA Types
  the most powerful ELISA assay format is the sandwich assay.
This type of capture assay is called a “sandwich” assay because
the analyte to be measured is bound between two primary
antibodies – the capture antibody and the detection antibody.
 sandwich format is used because it is so sensitive and robust

Direct vs. Sandwich assays). The antigen of interest is immobilized by direct adsorption to the
assay plate or by first attaching a capture antibody to the plate surface. Detection of the antigen
can then be performed using an enzyme-conjugated primary antibody (direct detection) or a
matched set of unlabeled primary and conjugated secondary antibodies (indirect detection).
Sandwich ELISA
Sandwich ELISA
Direct ELISA
Direct vs. Indirect Detection ELISA Strategies

it is the detection step (as either direct or indirect detection) that largely
determines the sensitivity of an ELISA.

Direct Detection Method

 uses a labeled primary antibody that
reacts directly with the antigen.
 can be performed with antigen that is
directly immobilized on assay plate
 not widely used in ELISA, but is quite
common for immunohistochemical
staining of tissues and cells

Indirect Detection Method

 uses a labeled secondary antibody for
detection and is the most popular
format for ELISA. The secondary
antibody has specificity for the
primary antibody.
Direct ELISA Detection
Advantages • Quick because only one antibody and fewer
steps are used.
• Cross-reactivity of secondary antibody is
eliminated
Disadvantages • Immunoreactivity of the primary antibody
might be adversely affected by labeling with
enzymes or tags.
• Labeling primary antibodies for each specific
ELISA system is time-consuming and
expensive.
• No flexibility in choice of primary antibody
label from one experiment to another.
• Minimal signal amplification
Indirect ELISA Detection
Advantages • A wide variety of labeled secondary antibodies
are available commercially.
• Versatile - many primary antibodies can be made
in one species and the same labeled secondary
antibody can be used for detection.
• Maximum immunoreactivity of the primary
antibody is retained because it is not labeled.
• Sensitivity is increased because each primary
antibody contains several epitopes that can be
bound by the labeled secondary antibody,
allowing for signal amplification.
• Different visualization markers can be used with
the same primary antibody.
Disadvantages • Cross-reactivity might occur with the secondary
antibody, resulting in nonspecific signal.
• An extra incubation step is required in the
procedure.
Sandwich ELISA
typically requires the use of matched antibody pairs, where each antibody
is specific for a different, non-overlapping part (epitope) of the antigen
molecule. A first antibody (capture antibody) is coated to the wells. The
sample solution is then added to the well. A second antibody (detection
antibody) follows this step in order to measure the concentration of the
sample. This type of ELISA has the following advantages:
• High specificity: the antigen/analyte is specifically captured and
detected
• Suitable for complex (or crude/impure) samples: the antigen does
not require purification prior to measurement
• Flexibility and sensitivity: both direct or indirect detection methods
can be used

Competitive ELISA
also known as inhibition ELISA, is a surface/plate based assay,
where the plate is coated with capture antibodies reactive to the
molecule of interest. The sample (containing native molecule of
interest) and enzyme conjugated recombinant protein (the
competing molecule) are added to the coated wells.
 Summary of Key Steps in Different ELISA Types

Indirect Direct Sandwich Competitive

Capture Ab X X √ X
Coating
Antigen Coating √ √ X √
Blocking √ √ √ √
Sample X X √ √
(Antigen)
Incubation
Primary Ab √ √ √ √
Incubation
Secondary Ab √ X √ √
Incubation
Substrate Prep √ √ √ √
Signal Detection √ √ √ √
Data Analysis √ √ √ √
How does ELISA work?
There are variations of ELISA test, but the most basic type consists
of an antibody attached to the solid surface. This antibody has
affinity to the substance of interest.

For instance: 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 present in the test sample the less enzyme
linked HCG will bind.
How Does ELISA work?

The ELISA was the first screening test widely used for HIV because of its high
sensitivity.

In an ELISA, a person's serum is diluted 400 times and applied to a plate to

which HIV antigens are attached. If antibodies to HIV are present in the
serum, they may bind to these HIV antigens. The plate is then washed to
remove all other components of the serum. A specially prepared "secondary
antibody" - - an antibody that binds to other antibodies - - is then applied to
the plate, followed by another wash. This secondary antibody is chemically
linked in advance to an enzyme.

Thus, the plate will contain enzyme in proportion to the amount of

secondary antibody bound to the plate. A substrate for the enzyme is
applied, and catalysis by the enzyme leads to a change in color or
fluorescence.

ELISA results are reported as a number; the most controversial aspect of this
test is knowing the "cut-off" point between a positive and a negative result.
 How Does ELISA work?
A cut-off point may be determined
by comparing it with a known
standard.

If an ELISA test is used for drug

screening at workplace, a cut-off
concentration, 50 ng/ml, for
example, is established, and a
sample containing the standard
concentration of analyte will be
prepared.

Unknowns that generate a

stronger signal than the known
sample are "positive." Those that
generate weaker signal are
"negative".
Detection Strategies for ELISA

The final stage in all ELISA systems is a detection step. Unless a

radioactive or fluorescent tag was used, this involves the
introduction of an enzyme substrate.

The enzyme converts the substrate to a detectable product. If an

ELISA has been constructed and developed properly, then the
intensity of signal produced when the substrate is added will be
directly proportional to the amount of antigen captured in the
plate and bound by the detection reagents.

Enzyme-conjugated antibodies (especially HRP) offer the most

flexibility in detection and documentation methods for ELISA
because of the variety of substrates available for chromogenic,
chemifluorescent and chemiluminescent imaging.
Common Applications of ELISA
Dr Dennis E Bidwell and Alister Voller created the ELISA test to
detect various kind of diseases, such as dengue, malaria, Chagas
disease, Johne's disease, and others. ELISA tests also are used as in
in vitro diagnostics in medical laboratories. The other uses of ELISA
include the detection of:

• Mycobacterium antibodies in tuberculosis

• rotavirus in feces
• hepatitis B markers in serum
• hepatitis C markers in serum
• enterotoxin of E. coli in feces
• HIV antibodies in blood samples
• levels of hormones
• STDs, toxoplamosis
• illicit drugs
• food and environmental allergens
ELISA data is graphed with
optical density (or fluorescence)
vs concentration to produce a
sigmoidal curve as shown below.

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.

In fact, it is the relatively long

linear region of the curve that
makes the ELISA results
accurate and reproducible. The
unknown concentration can be
determined directly on the graph
or with curve fitting software
which is typically found on
ELISA plate readers.
ELISA commercial kits have Optical Density (OD) values for the controls or
references.
So you can tell from the OD values of the controls if everything worked well.
it is a good hint if the OD values of your measured samples are within the
range of the standard curve.