The ELISA Procedure: Allow For Multiple Analytes Per Well, Highly Sensitive Readouts, and Direct Cell-Based Output

Introduction
The basic enzyme-linked immunosorbent assay (ELISA), or

enzyme immunoassay (EIA), is distinguished from other
antibody-based assays because separation of specific and
non-specific interactions occurs via serial binding to a solid
surface, usually a
The ELISA procedure
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 carryout, and since they

are designed to rapidly handle a large number of
samples in parallel, they are a very popular choice for
the evaluation of various research and diagnostic
targets..
ELISAs were first developed in the early as a

replacement for radioimmunoassays.
So They remain in wide use in their original format and

in expanded formats with modifications that allow for
multiple analytes per well, highly sensitive readouts,
and direct cell-based output.
.
Steps
1-ELISAs begin with a coating step, where the first layer,
either an antigen or an antibody, is adsorbed to a well in an
ELISA plate.
2-Since the assay uses surface binding for separation,

several washes are repeated between each ELISA step to
remove unboundmaterials.
3-During this process it is essential that excess liquid is

removed
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 is detection, where multiple layers of
antibodies can be
used to amplify signal.
Fig. 1. Typical ELISAoutput. Darker wells indicate
higher levels of analyte in the originalsample
ELISAs, by definition, take advantage of an enzymatic
label to produce a detectible signal that is directly
correlated to the binding of an antibody to an antigen.
of enzymes and enzyme substrates (covered in Section
6) are typically used for ELISAs.
Afew different types of enzymes and enzyme substrates

(covered in Section 6) are typically used for ELISAs.
Several methods for incorporating 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 the substrate 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 adhere to the use of these terms as they

apply to the detection portion of the assay as indicated
below:
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)..
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:
1_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 (Figure
6).
2-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
controlantigen.
3-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 concentration to produce a
sigmoidal curve as shown in Figure 6.
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.
Glossary
1-Adsorption -
the passive attachment of a liquid to a solid surface
creating a thin film.
2-Antigen -
substance, protein, chemical compound, or virus that is
able to elicit an immune response against which
antibodies are raised.
3-AP (Alkaline Phosphatase) -

phosphatase enzyme that removes a phosphate
group from a substrate. In ELISAs the substrate is p-
NitroPhenylPhosphate (pNPP).
⁴-Assay diluent (see also buffer) -

buffer solution in which the sample to be analyzed is
diluted in.
5-Assay sensitivity -
a measure of the ability of the ELISAto distinguish
between small changes in concentration.
6-Background -
the signal readout attributable to all reagents
excluding the analyte. Should be low.
7-Blocking
- application of reagents, generally buffers, to lower
background by binding to the potential non-specific
binding sites of antibodies and enzyme conjugates.
8-Buffer -
solutions containing compounds, generally
proteins, to reduce the non-specific binding of
antibodies; used in blocking to reduce
background.
9-Cross-reactivity -
an antibody binding to a target that is very similar
to but not the intended target analyte, i.e. a
closely related molecule with structural
similarities to the target antigen.
10-Detection limit -
the smallest quantity of analyte that can be reliably
measured by the ELISA assay; it is often set to 2
standard deviations (2 SD) above background level.
11-Dilution -
addition of a buffer to a protein solution to make it less
concentrated, used in optimizing antibody concentration
but also applied to samples to obtain readings within the
dynamic range of the assay.
12-Dynamic range –
range in the ELISA over which the absorbance reading
increases in a
linear mode and the analyte can be reliably measured.
13-Edge effect –
the result of inconsistencies in the production of
ELISAmultiwell plates or when assay conditions, such
as stacking plates, cause the outer wells to behave
differently.
As a result, unexpected values can appear in the

outer wells whichmay be out of line with
neighboring well.
This can best be controlled for by using duplicates

or triplicates for all samples, and noting any large
variations in the results for a given sample.
15-Heterophilic interference –
arises from antibodies found in the sample analyzed by
the ELISA; it is binding by these antibodies to the
detection antibody used in the assay. The
best known example of heterophilic interference is
HAMA (human anti-mouse antibody) found in some
patients where it interferes with the accurate analyte
determination, showing false positive readings.
16-HRP (Horseradish Peroxidase) –

- an enzyme that breaks down hydrogen peroxide to
water a peroxidase. Chromogenic substrates such as
TMB serve as indicators of that enzyme activity.
17-Hook effect –
caused by very high levels of antigen in the sample.

As a result specific binding of the antigen by the
antibody is insufficient to match analyte levels and
signalis lower than expected.
The best way to avoid this issue is to test

several dilutions of each sample.
17-Immunoassay –
any type of assay that uses antibodies to measure

the concentration of an analyte in a sample

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Introduction

The basic enzyme-linked immunosorbent assay (ELISA), or

The ELISA procedure

The ELISA procedure results in a colored end product

ELISAs are quick and simple to carryout, and since they

ELISAs were first developed in the early as a

So They remain in wide use in their original format and

2-Since the assay uses surface binding for separation,

3-During this process it is essential that excess liquid is

For greatest consistency specialized plate washers

ELISAs can be quite complex, including various

The most complex and varying step in the overall

Afew different types of enzymes and enzyme substrates

The final assay signal is measured with a

One aspect of ELISA terminology that often leads to

We will adhere to the use of these terms as they

HRP and AP substrates typically produce a

Detection can also occur by fluorescently

Antibodies are coupled to biotin, followed by a

Alternatively, it is possible to use unlabeled primary

tertiary step is required for detection. In this case

Known concentrations of antigen are used

This can be done directly on the graph or with

3-AP (Alkaline Phosphatase) -

⁴-Assay diluent (see also buffer) -

As a result, unexpected values can appear in the

This can best be controlled for by using duplicates

16-HRP (Horseradish Peroxidase) –

caused by very high levels of antigen in the sample.

The best way to avoid this issue is to test

any type of assay that uses antibodies to measure

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