ELISA

A BIOLOGY INVESTIGATORY PROJECT

THIS PROJECT LOOKS AT THE TECHNIQUE

CALLED ELISA TEST

NAME:Shaurya pratap singh

CLASS: XII B
ROLL NUMBER:

Signature of subject Teacher

Signature(Examiner)
 BONAFIDE CERTIFICATE

This is to certify that Shaurya pratap

singh,a student of class XII B has
successfully completed the reasearch
project titled “ELISA TEST” in partial
fulfillment of his investigatory project
in Biology during the year 2022-2023

Internal Examiner

External Examiner
 ACKNOWLEDGEMENT

I would like to acknowledge all the kind

hearts that have featured the project
along side with me.First I would like to
thank my parents how have fed me with
immense knowledge. I would also be
grateful to my Principal and Biology
teacher who has been keen in my
development and my friends who have
encouraged me throughout.
Finally I thank the Lord almighty for
having given a wonderful surrounding
and the opportunity to gain more
knowledge.
 TABLE OF CONTENT
Introduction
Principle
History
ELISA Procedure
Types of ELISA
Direct,
Indirect,
Sandwich,
Competitive ELISAs
ELISA Detection Options - Direct & Indirect
Direct ELISA detection
Indirect ELISA detection
ELISA Results
Quantitative
Qualitative
Semi-quantitative
Standard curve
Calibration curve models

ELISA Sensitivity
Applications
Introduction to ELISA
The basic enzyme-linked immunosorbent assay
(ELISA), or enzymeimmunoassay (EIA), is
distinguished from other antibody-based assays
because separation of specific and non-specific
interactions occurs viaserial binding to a solid
surface, usually a polystyrene multiwell plate, and
because quantitative results can be achieve.The
ELISA procedure results in a colored end product
which correlates To the amount of analyte present
in the original sample.ELISAs are quick and
simple to carry out, and since they are designed
to rapidly handle a large number of samples in
parallel, they are a very popularchoice for the
evaluation of various research and diagnostic
targets.ELISAs were first developed in the early
1970s as a replacement for radioimmunoassays.
They remain in wide use in their original format
andin expanded formats with modifications that
allow for multiple analytes perwell, highly
sensitive readouts, and direct cell-based output.

Principle
As an analytical biochemistry assay and a "wet
lab" technique, ELISA involvesdetection of an
analyte (i.e., the specific substance whose
presence is being quantitatively or qualitatively
analyzed) in a liquid sample by a method that
continues to use liquid reagents during the
analysis (i.e., controlled sequence of biochemical
reactions that will generate a signal which can be
easily quantified and interpreted as a measure of
the amount of analyte in the sample)that stays
liquid and remains inside a reaction chamber or
well needed to keep the reactants contained. This
is in opposition to "dry lab" techniques that use
dry strips. Even if the sample is liquid (e.g., a
measured small drop), the final detection step in
"dry" analysis involves reading of a dried strip by
methods such as reflectometry and does not need
a reaction containment chamber to prevent spill
over or mixing between samples.
As a heterogenous assay, ELISA separates some
component of the analytical reaction mixture by
adsorbing certain components onto a solid phase
which is physically immobilized. In ELISA, a liquid
sample is added onto a stationary solid phase
with special binding properties and is followed by
multiple liquid reagents that are sequentially
added, incubated, and washed, followed by some
optical change (e.g., colour development by the
product of an enzymatic reaction) in the final
liquid in the well from which the quantity of the
analyteis measured. The quantitative "reading" is
usually based on detection of intensity of
transmitted light by spectrophotometry, which
involves quantitation of transmission of some
specific wavelength of light through the liquid (as
well as the transparent bottom of the well in the
multiple-well plate format).
The sensitivity of detection depends on
amplification of the signal during the analytic
reactions. Since enzyme reactions are very well
known amplification processes, the signal is
generated by enzymes which are linked to the
detection reagents in fixed proportions to allow
accurate quantification,and thus the name
"enzyme-linked." 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
(the stationary "solid phase"/"solid substrate"
here as opposed to solid microparticle/beads that
can be washed away), 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.

History
Before the development of the ELISA, the only option
for conducting an immunoassay was
radioimmunoassay, a technique using radioactively
labeled antigens or antibodies. In radioimmunoassay,
the radioactivity provides the signal, which indicates
whether a specific antigen or antibody is present in the
sample. Radioimmunoassay was first described in a
scientific paper by RosalynSussman Yalow and
Solomon Berson published in 1960.As radioactivity
poses a potential health threat, a safer alternative was
sought. A suitable alternative to radioimmunoassay
would substitute an on radioactive signal in place of
the radioactive signal. When enzymes (such as horse
radish peroxidase) react with appropriate substrates
(suchas ABTS or TMB), a change in color occurs, which
is used as a signal.However, the signal has to be
associated with the presence of antibody or antigen,
which is why the enzyme has to be linked to an
appropriate antibody.This linking process was
independently developed by Stratis Avrameas and
G.B. Pierce. Since it is necessary to remove any
unbound antibody or antigen by washing, the antibody
or antigen has to be fixed to the surface of the
container;i.e., the immunosorbent must be prepared. A
technique to accomplish this was published by Wide
and Jerker Porath in 1966.In 1971, Peter Perlmann and
Eva Engvall at Stockholm University in Sweden, and
Anton Schuurs and Bauke van Weemen in the
Netherland sin dependently published papers that
synthesized this knowledge into methodsto perform
EIA/ELISA.
Traditional ELISA typically involves chromogenic
reporters andsubstrates that produce some kind of
observable color change to indicate the presence of
antigen or analyte. Newer ELISA-like techniques use
fluorogenic, electro chemiluminescent, and quantitative
PCR reporters to create quantifiable signals. These
new reporters can have various advantages,including
higher sensitivities and multiplexing. In technical
terms, newer assays of this type are not strictly
ELISAs, as they are not "enzyme-linked", but are
instead linked to some nonenzymatic reporter.
However, given that thegeneral principles in these
assays are largely similar, they are often grouped inthe
same category as ELISAs.In 2012, an ultrasensitive
enzyme-based ELISA test using nanoparticles as
achromogenic reporter was able to give a naked-eye
colour signal, from thedetection of mere attograms of
analyte. A blue color appears for positive resultsand
red colour for negative. Note that this detection only
can confirm the presence or the absence of analyte not
the actual concentration.

ELISA Procedure
ELISAs begin with a coating step, where the first layer,
either an antigen oran antibody, is adsorbed to a well in
an ELISA plate.
Coating is followed by blocking and detection steps
Since the assay uses surface binding for separation,
several washes arerepeated between each ELISA step
to remove unbound materials. During this process it is
essential that excess liquid is removed in order to
prevent the dilution of the solutions added in the next
stage. For greatest consistency specialized plate
washers are used.ELISAs can be quite complex,
including various intervening steps and the ability to
measure protein concentrations in heterogeneous
samples such as blood. The most complex and varying
step in the overall process isdetection, where multiple
layers of antibodies can be used to amplify signal.
ELISA STEPS
Types of ELISA

ELISA Formats
The first step in an ELISA experiment is the
immobilization of the antigenin a sample to the wall of
the wells of a microtiter plate. This can be achieved by
direct adsorption to the plate’s surface or by using a
“capture antibody”. The capture antibody has to be
specific to the target antigen and is mainly used in a
specific ELISA type called “sandwich ELISA”. After
immobilization, a detection antibody is added, which
binds to the adsorbed antigen thereby leading to the
formation of an antigen-antibody complex.The detection
antibody is either directly conjugated to an enzyme,
such as horse radish peroxidase (HRP), or provides a
binding site for a labeled secondary antibody. In
general, ELISAs can be grouped into the four
maincategories: direct, indirect, sandwich, and
competitive ELISAs.

Direct ELISA

Direct ELISA detection is much faster than other ELISA

techniques as fewer steps are required. The assay is
also less prone to error since fewer reagents and steps
are needed, i.e. no potentially cross-reacting secondary
antibody needed. Although there are some
disadvantages to this method. As the antigen
immobilization is not specific, higher background noise
may be observed in comparison to indirect ELISA (see
below). This is primarily because all proteins in the
sample, including the target protein, will bind tothe
plate. Direct ELISA is less flexible since a specific
conjugated primaryantibody is needed for each target
protein. As no secondary antibody is usedthere is no
signal amplification, which reduces assay sensitivity.
Finally,the direct ELISA technique is typically used
when the immune response toan antigen needs to be
analyzed.

Indirect ELISA

The indirect ELISA method has high sensitivity since

more than one labeled secondary antibody can bind the
primary antibody; it is more economical than the direct
ELISA as fewer labeled antibodies are needed.
IndirectELISA delivers greater flexibility since different
primary antibodies can be used with a single labeled
secondary antibody. Among its disadvantages is the
possibility of cross-reactivity of secondary antibody to
the adsorbed antigen, which could increase background
noise. Also, indirect ELISA assays take longer to run
than direct ELISAs since an additional incubationstep
for the secondary antibody is required. The indirect
ELISA is most suitable for determining total antibody
concentration in samples.

Sandwich ELISA
Sandwich ELISAs require the use of matched antibody
pairs (capture anddetection antibodies). Each antibody
is therefore specific for a different and non-overlapping
region or epitope of the antigen.It is important that
matched antibody pairs are tested specifically in
sandwich ELISA to ensure that they detect different
epitopes, to achieve accurate results. The capture
antibody, as its name implies, binds the antigen that can
then be detected in a direct ELISA or in an indirect
ELISA configuration.
The procedure for a sandwich ELISA firstly requires the
well of an ELISA plate to be coated with a capture
antibody. The analyte or sample is then added, followed
by a detection antibody. The detection antibody can be
enzyme conjugated, in which case this is referred to as
a direct sandwich ELISA. If the detection antibody used
is unlabeled, a secondary enzyme-conjugated detection
antibody is required. This is known as an
indirectsandwich ELISA. The key advantage of a
sandwich ELISA is its high
sensitivity; it is 2-5 times more sensitive than direct or
indirect ELISAs.Sandwich ELISA also delivers high
specificity as two antibodies are used to detect the
antigen. It offers flexibility since both direct and indirect
methods can be used. The advantages bring with them a
few disadvantages;if a standardized ELISA kit or tested
antibody pair is not available, antibody optimization has
to be worked out since it is important to reduce cross-
reactivity between the capture and detection antibodies.
Sandwich ELISAs are particularly suited to the analysis
of complex samples, since the antigen does not need to
be purified prior to the assay yet still delivers high
sensitivity and specificity (e.g. measuring cytokine
levels in an immuneresponse).

Competition/Inhibition ELISA

How it works: the competition/inhibition ELISA, also

known as a blocking ELISA, is perhaps the most
complex of all the ELISA techniques. However,each of
the above assay types can be adapted to a competitive
format. The competitive/inhibition ELISA is
predominantly used to measure the concentration of an
antigen or antibody in a sample by detecting
interference in an expected signal output. Essentially,
sample antigen orantibody competes with a reference
for binding to a limited amount of labeled antibody or
antigen, respectively. The higher the sample antigen
concentration, the weaker the output signal, indicating
that the signal output inversely correlates with the
amount of antigen in the sample.
An example of a competition ELISA to test for antigen
based on the direct detection method\.In this example, a
known antigen is used to coat a multiwell
plate.Following standard blocking and washing steps,
samples containing unknown antigen are added.
Labeled detection antibody is then applied for
detection using relevant substrates (e.g. 3,3’,5,5’
-Tetramethylbenzidine orTMB). If there is a high
concentration of antigen in the sample, a significant
reduction in signal output will be observed. In contrast,
if there is very little antigen in the sample, there will be
very little reduction in the expected signal output, there
would be a reduction in signal output.

ELISA Detection Options - Direct & Indirect

ELISAs, by definition, take advantage of an enzymatic

label to produce a detectible signal that is directly
correlated to the binding of an antibody toan antigen. A
few different types of enzymes and enzyme substrates
are typically used for ELISAs. Several methods for in
corporating the enzyme step into the process can also
be applied. The final assay signal is measured with a
spectrophotometric or fluorescent plate reader
(depending upon thesubstrate chosen).
One aspect of ELISA terminology that often leads to
confusion is the variability in the way the terms direct
and indirect are applied. We will ad here to the use of
these terms as they apply to the detection portion of the
assay as indicated below:

Direct ELISA detection

Antibodies are directly labeled with alkaline
phosphatase (AP) or HRP; this is the most common
ELISA detection strategy. HRP and AP substrates
typically produce a colorimetric output that is read by
a spectrophotometer.Detection can also occur by
fluorescently-labeled antibodies; here the assay is
usually termed a fluorescence-linked immunosorbent
assay (FLISA).

Indirect ELISA detection

Antibodies are coupled to biotin, followed by a
streptavidin-conjugated enzyme step. Alternatively, it is
possible to use unlabeled primary antibodies followed
by enzyme-coupled or biotinylated
Secondary antibodies. If the secondary antibody is
biotinylated, then a tertiary step is required for
detection. In this case treatment with the streptavidin-
enzyme conjugate is followed by an appropriate
substrate.

ELISA Results
The ELISA assay yields three different types of data
output:

Quantitative

ELISA data can be interpreted in comparison to a

standard curve (a serial dilution of a known, purified
antigen) 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.

Standard curve
ELISA data is typically graphed with optical density vs
log concentrationto produce a sigmoidal curve as.
Known concentrations of antigen are used to produce a
standard curve and then this data is used tomeasure
the concentration of unknown samples by comparison
to the linear portion of the standard curve. This can be
done directly on the graph or withcurve fitting software
which is typically found on ELISA plate readers.

Calibration curve models

If a quantitative result is needed, the simplest way to
proceed is to average the triplicate of the standards
readings and deduct the reading of the blank control
sample. Next, plot the standard curve, find the line of
best fit or atleast draw a point to point curve so that the
concentration of the sample scan be determined. Any
dilutions made need to be adjusted for at this stage.This
is generally the practical extent to which manual
calculation can be taken.
A variation is to plot the data using semi-log, log/log,
log/log it and its derivatives - the 4 or 5 parameter
logistic models. Using software based/automated
solutions makes it possible to consider more
sophisticated graphing approaches. Using linear
regression within a software package adds several
more checking possibilities; it is possible to check the
R2 value to determine overall goodness of fit. For that
portion of the curve where the relationship of
concentration to read out has a linear relationship, R2
values>0.99 represent a very good fit. Accuracy can
then be further enhanced byusing further standard
concentrations in that range.
One aspect of the linear plot is that it compresses the
data points on the lower concentrations of the standard
curve, hence making that the most accurate range
(area most likely to achieve the required R2 value). To
counter act this compression a semi-log chart can be
used; here the log of the concentration value (on x-
axis) is plotted against the readout (on y-axis).This
method gives an S-shaped data curve that distributes
more of the data points into the more user friendly
sigmoidal pattern.

The log/log (log of concentration against log of readout)

plot type manages to linearize more of the data curve.
The low to medium standard concentration range is
generally linear in this model, only the higher end ofthe
range tends to slope off. The log/logit and its
derivatives, the 4 or 5 parameter logistic models, are
more sophisticated requiring more complex
calculations and estimations of max, min, EC50, and
slope values. The 5 parameter model additionally
requires the asymmetry value.
While these calibration curve models can deliver
improved performance, a good starting point would be
using the log-log plot with acheck on the recovery
percentage (analyte recovery from spiked samples).
Alternatively, at least ‘back - fitting’ the standard curve
readout values, isfrequently ‘a good enough’ approach.
The simplest way to check is to back calculate the
calibration standards and check that they fall within
20% of the nominal readout value. One caveat is not to
rely on ‘good’ R2 values and find that calibration curve
model that delivers the best recovery valuesfor the
standards.

ELISA 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.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 improveresults.
As mentioned earlier, indirect detection will
produce higher levels of signaland should
therefore be more sensitive. However, it can
also cause higher background signal thus
reducing net specific signal levels.

Applications
Because the ELISA can be performed to evaluate either
the presence of antigen or the presence of antibody in a
sample, it is a useful tool for determining serum
antibody concentrations (such as with the HIV test or
West Nile virus). It has also found applications in the
food industry in detecting potential food allergens, such
as milk , peanuts, walnuts, almonds, and eggs and as
serological blood test for coeliac disease. ELISA can
also be used in toxicology as a rapid presumptive
screen for certain classes of drugs.
The ELISA was the first screening test widely used for
HIV because of its highsensitivity. In an ELISA, a
person's serum is diluted 400 times and applied toa
plate to which HIV antigens are attached. If antibodies
to HIV are present inthe serum, they may bind to these
HIV antigens. The plate is then washed toremove 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 determining the
"cut-off" point between a positive and a negative result.
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 signalare "negative"
.Dr Dennis E Bidwell and Alister Voller created the
ELISA test to detect variouskind of diseases, such as
dengue, malaria, Chagas disease, Johne's disease,
andothers. ELISA tests also are used as in in vitro
diagnostics in medical laboratories. The other uses of
ELISA include:
detection of Mycobacterium antibodies in
tuberculosis
detection of rotavirus in feces
detection of hepatitis B markers in serum
detection of hepatitis C markers in serum
detection of enterotoxin of E. coli in feces
detection of HIV antibodies in blood samples

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