ELISA methods have become widely used across various fields of biology, from healthcare and food safety to cancer research and vaccine development. Whether you need to measure gluten in beer or neutralizing antibodies in serum, there is an ELISA available to provide answers to your scientific questions. Despite being first introduced over 50 years ago, ELISAs continue to be an essential tool for many researchers.
In this eBook, you’ll learn about:
- The history of the ELISA
- Working with ELISAs
- ELISA applications
- Lab automation for high-throughput ELISAs
- And more
ELISA 101
ELISA EXPLAINED:
from basics to
practical application
2
Contents
What is an ELISA .....................................................................3
History of the ELISA ..........................................................3
Direct ELISA ........................................................................ 4
Indirect ELISA ..................................................................... 4
Competitive ELISA ............................................................4
Sandwich ELISA .................................................................4
Working with ELISAs .............................................................. 5
Steps to run a sandwich ELISA assay .......................... 5
Work?ow of an ELISA protocol ......................................7
How to set up an ELISA and
perform basic analysis .....................................................8
How to con?gure an ELISA
endpoint protocol .............................................................8
ELISA applications .................................................................9
Food and beverage .......................................................... 9
Gluten quanti?cation in beer ...................................9
Determine a?atoxin in cannabis ..............................9
Protein quantitation ........................................................ 10
Chemiluminescent VEGF ELISA .................................. 10
Quantitate Interleukin-8 ................................................ 10
Lab automation for high-throughput ELISAs .................. 11
2
Introduction
ELISA methods have become widely used across various ?elds of biology, from healthcare and food
safety to cancer research and vaccine development. Whether you need to measure gluten in beer
or neutralizing antibodies in serum, there is an ELISA available to provide answers to your scienti?c
questions. Despite being ?rst introduced over 50 years ago, ELISAs continue to be an essential tool
for many researchers. This e-book provides an introduction to ELISAs and demonstrates how you
can e?ectively use them in your own research.
3
What is an ELISA?
There are four major
types of ELISAs
• Direct
• Indirect
• Competitive
• Sandwich
An ELISA (enzyme-linked immunosorbent assay) is a method used to
quantitatively detect an antigen within a sample. An antigen is a toxin or other
foreign substance, for example a flu virus or environmental contaminant,
that causes the vertebrate immune system to mount a defensive response.
The range of potential antigens is vast, so ELISAs are used in many areas
of research and testing to detect and quantify antigens in a wide variety
of sample types. Cell lysates, blood samples, food items, and more can be
analyzed for specific substances of interest using ELISAs.
There are four major types of ELISAs: direct, indirect, competitive and
sandwich. Each type is described below with a diagram illustrating how
the analytes and antibodies are used in the process.
Read Blog: The history of ELISA from creation to COVID-19 research
History of the ELISA
1971—Eva Engvall and Peter
Perlman (independently) invent
a method that revolutionized
medicine called the ELISA test.
The method uses antibodies
to seek out the presence of
hormones or viruses.
3,4
1941—Albert H. Coons
and his colleagues are the
?rst to label antibodies with
a ?uorescent dye, and use it
to identify antigens in tissue
sections. This method is known
today as immuno?uorescence.
1
1960—Radioimmunoassays
are described in a scienti?c
paper by Rosalyn Sussman and
Soloman Berson. However, due
to radioactivity posing potential
health issues, researchers were
in need of a safer alternative.
2
Explore the origin of the ELISA and how it has evolved over the years to contribute to some of the most significant
scientific breakthroughs of our time.
References
1. Coons, A. H. The beginnings of immuno?uorescence. J. Immunol. 87, 499–503 (1961).
2. Yalow, Rosalyn and Berson, Solomon. “Immunoassay of endogenous plasma insulin in man.” The Journal of Clinical Investigation. 1960;39: 1157–75.
3. Perlmann, Peter et al. “Enzyme-linked immunosorbent assay (ELISA) quantitative assay of immunoglobulin G.” Immunochemistry. 1971;8 (9): 871–4.
4. Schuurs, A. “Immunoassay using antigen—enzyme conjugates.” FEBS Letters. 1971;15 (3): 232–236.
4
Antigen
Primary
antibody
conjugate
Antigen
Primary
antibody
Secondary
antibody
conjugate
Antigen
Primary
antibody
conjugate
Inhibitor
antigen
Antigen
Primary
antibody
Secondary
antibody
Direct ELISA
In a direct ELISA, the antigen is bound to the bottom of
the microplate well, and then it is bound by an antibody
that is specific to the antigen and also conjugated to an
enzyme or other molecule that enables detection.
Indirect ELISA
In an indirect ELISA, the antigen is bound to the bottom
of the microplate well, then an antibody specific to the
antigen is added. A secondary antibody, conjugated to
an enzyme or other detection molecule, is then bound
to the first antibody.
Competitive ELISA
In a competitive ELISA, a reference antigen is bound to
the bottom of microplate wells. Sample plus antibody
are added to the wells, and if there is antigen present
in the sample, it competes with reference antigen for
binding to the antibody. Unbound material is washed
away. The more antigen was in the sample, the less
antibody ends up bound to the bottom of the wells by
the reference antigen, and the lower the signal.
Sandwich ELISA
For the sandwich ELISA, two antibodies specific
to two different epitopes on the target antigen are
used. The capture antibody is bound to the bottom
of the microplate well and binds one epitope of the
antigen. The detection antibody binds to the antigen
at a different epitope and is conjugated to an enzyme
that enables detection. (If the detection antibody is
unconjugated, then a secondary enzyme-conjugated
detection antibody is required).
5
Steps to run a sandwich ELISA
Most sandwich ELISAs are run in microplates, with the bottom of the
plate wells serving as the solid surface to which antibodies and other
reagents bind. A microplate washer is used to wash away non-specific
material in the wells, and an absorbance ELISA microplate reader
detects the color change produced when target antigen is present.
Plate reader software is used to plot standard curves and calculate results.
The illustration below shows a workflow for a typical sandwich ELISA:
Speak to a specialist
Working with ELISAs
1 2 3 4
5 6
7
8
Antibody
binds to well
SpectraMax®
Microplate Reader
SoftMax® Pro
Software
Add detection
antibody
Add
substrate
Add
sample
Wash
microplate
Wash
microplate
Read
microplate
Calculate
results
AquaMax
Microplate Washer
StakMax Microplate
Handling System
Automation &
Customization
GxP Compliance
Solutions
COMPLIANCE
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96- and 384-well
Microplates
Discuss your solution
Interested in
automating your
ELISA work?ow?
6
• Extended dynamic range at
the lower and upper limits
• Stable signal for
readout ?exibility
• No compromise on sensitivity
• One-wash, 90-minute protocol
A typical ELISA protocol can be time-consuming, with multiple incubation and
wash steps. CatchPoint SimpleStep ELISA kits give users a 90-minute ELISA
work?ow, a nearly two-thirds reduction in time compared to conventional
ELISAs. Abcam's SimpleStep ELISAs reduce the time to results, but CatchPoint
additionally o?ers better dynamic range and a more stable signal, along with
sensitivity and a fast one-wash protocol.
Step 1
Capture antibody binds to ELISA plate wells.
Step 2
Add sample to well – antigen within the sample binds to
the capture antibody.
Step 3
Wash microplate – Unbound material is washed away,
leaving only the antigen of interest.
Step 4
Add detection antibody – Enzyme-conjugated detection
antibody binds to a second site on the antigen
of interest.
Step 5
Wash microplate – Unbound antibodies are washed
away, leaving only those specific for the target
of interest.
Step 6
Add substrate – Substrate is converted by the enzyme
on the detection antibody, producing a color change.
Step 7
Read plate – The microplate reader detects the colored
reaction product and outputs optical density (OD) values.
Step 8
Calculate results – The amount of antigen in each
sample is calculated and analyzed.
While an ELISA is easy to set up, the assay procedure is time-consuming and labor-intensive. Laboratory automation
for high-throughput plate-based assay work?ows can help with providing walkaway time, increasing throughput,
e?ectiveness and e?ciency of the assay procedure, and reproducibility.
7
Work?ow of an ELISA protocol
The workflow of a typical sandwich ELISA protocol has multiple reagent addition, incubation, and wash steps. Here
we’ve highlighted each step and the instrumentation and tools needed to conduct an ELISA including a microplate
washer, absorbance ELISA plate reader and software.
WASH MICROPLATE
Unbound material is washed away,
leaving only the antigen of interest
and minimizing the potential for
high background signal.
ELISA Plate
Most ELISAs are run in 96- or 384- well mi-
croplates, a 96-well plate being the most
common. The bottom of the microplate wells
serve as the solid surface to which antibod-
ies and other reagents attach. Microplates
are often included in an ELISA kit.
ADD SAMPLE
Sample is added to the well, and
antigen within the sample binds
to the capture antibody.
Enzyme-conjugated detection
antibody binds to a second site on
the antigen of interest, providing
the means to detect the antigen.
ADD DETECTION ANTIBODY
Substrate is converted by the enzyme on
the detection antibody, producing a color
change, with intensity proportional to the
amount of antigen present.
Depending on the enzyme and substrate
used, the readout can also be ?uorescent
or luminescent.
ADD SUBSTRATE
06
The microplate reader detects the
colored reaction product and outputs
optical density (OD) values that
indicate how much light is absorbed
by the contents of each well.
READ PLATE
Unbound antibodies are washed
away, leaving only those speci?c for
the target of interest and again
minimizing the potential for
background signal.
WASH MICROPLATE
ELISA Plate Reader
A microplate reader detects the color change
produced when target antigen is present. It
does so by measuring how much of the light
passed through the wells of the microplate is
absorbed by the material within the wells.
The more antigen is present, the higher the
absorbance value.
Watch video >
General ELISA protocol
A typical ELISA protocol has multiple reagent addition, incubation and wash steps. The ELISA infographic shows how a typical sandwich
ELISA is run. Each step breaks down the assay process and highlights the instrumentation needed to conduct the ELISA assay including
a microplate washer, reader and software.
ELISA Assay Protocol Infographic
Want faster ELISA results?
CatchPoint SimpleStep ELISA kits
produce results in just 90-minutes!
Learn more >
CALCULATE RESULTS
The amount of antigen in each
sample is calculated, and di?erent
samples—for example, cells
subjected to di?erent treatment
conditions—can be compared.
ELISA Plate Reader Software
Software is used to plot standard curves and
calculate results from the absorbance values
provided by the microplate reader.
A standard curve is run so that the amount of
antigen in each sample can be accurately
calculated. A precon?gured protocol, helps
save time by calculating results automatically.
Watch video >
ANTIBODY BINDS TO WELLS
First, the capture antibody is
bound to the bottom of the
microplate well.
01
02
03
04
05
07
08
ELISA Plate Washer
A microplate washer is used to wash away
non-speci?c material in the wells.
Watch video >
AquaMax Microplate Washer
Aspiration and dispensing of 96- and
384-wells occur simultaneously in all wells
leading to high-precision assays and faster
microplate processing without mechanical
plate indexing or quadrant processing.
Watch video >
WASH MICROPLATE
Unbound material is washed away,
leaving only the antigen of interest
and minimizing the potential for
high background signal.
ELISA Plate
Most ELISAs are run in 96- or 384- well mi-
croplates, a 96-well plate being the most
common. The bottom of the microplate wells
serve as the solid surface to which antibod-
ies and other reagents attach. Microplates
are often included in an ELISA kit.
ADD SAMPLE
Sample is added to the well, and
antigen within the sample binds
to the capture antibody.
Enzyme-conjugated detection
antibody binds to a second site on
the antigen of interest, providing
the means to detect the antigen.
ADD DETECTION ANTIBODY
Substrate is converted by the enzyme on
the detection antibody, producing a color
change, with intensity proportional to the
amount of antigen present.
Depending on the enzyme and substrate
used, the readout can also be ?uorescent
or luminescent.
ADD SUBSTRATE
06
The microplate reader detects the
colored reaction product and outputs
optical density (OD) values that
indicate how much light is absorbed
by the contents of each well.
READ PLATE
Unbound antibodies are washed
away, leaving only those speci?c for
the target of interest and again
minimizing the potential for
background signal.
WASH MICROPLATE
ELISA Plate Reader
A microplate reader detects the color change
produced when target antigen is present. It
does so by measuring how much of the light
passed through the wells of the microplate is
absorbed by the material within the wells.
The more antigen is present, the higher the
absorbance value.
Watch video >
General ELISA protocol
A typical ELISA protocol has multiple reagent addition, incubation and wash steps. The ELISA infographic shows how a typical sandwich
ELISA is run. Each step breaks down the assay process and highlights the instrumentation needed to conduct the ELISA assay including
a microplate washer, reader and software.
ELISA Assay Protocol Infographic
Want faster ELISA results?
CatchPoint SimpleStep ELISA kits
produce results in just 90-minutes!
Learn more >
CALCULATE RESULTS
The amount of antigen in each
sample is calculated, and di?erent
samples—for example, cells
subjected to di?erent treatment
conditions—can be compared.
ELISA Plate Reader Software
Software is used to plot standard curves and
calculate results from the absorbance values
provided by the microplate reader.
A standard curve is run so that the amount of
antigen in each sample can be accurately
calculated. A precon?gured protocol, helps
save time by calculating results automatically.
Watch video >
ANTIBODY BINDS TO WELLS
First, the capture antibody is
bound to the bottom of the
microplate well.
01
02
03
04
05
07
08
ELISA Plate Washer
A microplate washer is used to wash away
non-speci?c material in the wells.
Watch video >
AquaMax Microplate Washer
Aspiration and dispensing of 96- and
384-wells occur simultaneously in all wells
leading to high-precision assays and faster
microplate processing without mechanical
plate indexing or quadrant processing.
Watch video >
WASH MICROPLATE
Unbound material is washed away,
leaving only the antigen of interest
and minimizing the potential for
high background signal.
ELISA Plate
Most ELISAs are run in 96- or 384- well mi-
croplates, a 96-well plate being the most
common. The bottom of the microplate wells
serve as the solid surface to which antibod-
ies and other reagents attach. Microplates
are often included in an ELISA kit.
ADD SAMPLE
Sample is added to the well, and
antigen within the sample binds
to the capture antibody.
Enzyme-conjugated detection
antibody binds to a second site on
the antigen of interest, providing
the means to detect the antigen.
ADD DETECTION ANTIBODY
Substrate is converted by the enzyme on
the detection antibody, producing a color
change, with intensity proportional to the
amount of antigen present.
Depending on the enzyme and substrate
used, the readout can also be ?uorescent
or luminescent.
ADD SUBSTRATE
06
The microplate reader detects the
colored reaction product and outputs
optical density (OD) values that
indicate how much light is absorbed
by the contents of each well.
READ PLATE
Unbound antibodies are washed
away, leaving only those speci?c for
the target of interest and again
minimizing the potential for
background signal.
WASH MICROPLATE
ELISA Plate Reader
A microplate reader detects the color change
produced when target antigen is present. It
does so by measuring how much of the light
passed through the wells of the microplate is
absorbed by the material within the wells.
The more antigen is present, the higher the
absorbance value.
Watch video >
General ELISA protocol
A typical ELISA protocol has multiple reagent addition, incubation and wash steps. The ELISA infographic shows how a typical sandwich
ELISA is run. Each step breaks down the assay process and highlights the instrumentation needed to conduct the ELISA assay including
a microplate washer, reader and software.
ELISA Assay Protocol Infographic
Want faster ELISA results?
CatchPoint SimpleStep ELISA kits
produce results in just 90-minutes!
Learn more >
CALCULATE RESULTS
The amount of antigen in each
sample is calculated, and di?erent
samples—for example, cells
subjected to di?erent treatment
conditions—can be compared.
ELISA Plate Reader Software
Software is used to plot standard curves and
calculate results from the absorbance values
provided by the microplate reader.
A standard curve is run so that the amount of
antigen in each sample can be accurately
calculated. A precon?gured protocol, helps
save time by calculating results automatically.
Watch video >
ANTIBODY BINDS TO WELLS
First, the capture antibody is
bound to the bottom of the
microplate well.
01
02
03
04
05
07
08
ELISA Plate Washer
A microplate washer is used to wash away
non-speci?c material in the wells.
Watch video >
AquaMax Microplate Washer
Aspiration and dispensing of 96- and
384-wells occur simultaneously in all wells
leading to high-precision assays and faster
microplate processing without mechanical
plate indexing or quadrant processing.
Watch video >
8
How to set up an ELISA and perform basic analysis
Part 1 of a four-part webinar series to introduce SoftMax® Pro 7.1.1 GxP Acquisition and Analysis Software and show how
easy it is to run your assays and get results both quickly and easily.
After a short introduction, you will see a live
demonstration of how to use SoftMax Pro GxP Software
and the SpectraMax® iD3 Multi-Mode Microplate Reader
to set up and acquire data using an example ELISA. We
will explain the software settings necessary to acquire
data before discussing the basics of analysis. We will use
an actual data set to introduce data reduction, creating a
template and generating a standard curve.
View on-demand webinar
How to con?gure an ELISA endpoint protocol
This video provides a step-by-step instruction on how
to configure an ELISA endpoint protocol in SoftMax
Pro Software. Using the template feature in a protocol
enables rapid, automated data analysis. Curve fitting
provides accurate visualization of results.
View on-demand tutorial
Read Application Note: Faster ELISA results
with CatchPoint SimpleStep ELISA kits
9
ELISA applications
Enzyme-linked immunosorbent assays are a commonly used analytical technique performed in many research and
biotech labs. Below is a collection of application notes, research and technology related to signi?cant ELISA assays
and applications.
Food and beverage
To ensure quality, authenticity and safety, manufacturers of foods and beverages need to perform a wide range of tests
on their products. Many of these tests are based around ELISA, which are used in areas as diverse as checking for the
presence of bacterial endotoxins, mycotoxins, various allergens, growth promoter hormones and antibiotics, and for
meat species identification. Other absorbance tests can also be used to quantify levels of substances within foods –
such as sugars, phenols, and acids.
Learn more
Gluten quantitation in beer
Gliadin levels in six commercially available beers were tested to determine
gluten levels with the RIDASCREEN Gliadin competitive ELISA.
Read Application Note: Measuring gluten in
beer using an ASBC-approved ELISA method
Detect a?atoxin in cannabis
Here, we demonstrate how the AgraQuant Total Aflatoxin 4/40 ELISA kit can
be used together with the SpectraMax® ABS Plus Microplate Reader and
SpectraMax® iD5 Multi-Mode Microplate Reader to test for levels of aflatoxins
B1, B2, G1, and G2 in one of the most common cannabis products, the flower.
Read Application Note: Determine total
a?atoxin in cannabis using an ELISA method
10
Protein quantitation
Rapid, accurate, and frequent molecular testing
is of critical importance in navigating the
SARS-CoV-2 (COVID-19) pandemic. To advance
our understanding of the relationship between
infection and immunity, it is critical to test not only
for infection, but also for antibodies.
Read Application Note: Qualitative
measurement of SARS-CoV-2 spike
and nucleocapsid IgG antibodies in
serum samples
Chemiluminescent VEGF ELISA
Vascular endothelial growth factors (VEGFs)
are a family of secreted polypeptides that
have been implicated in mammalian vascular
development and in disease processes
involving abnormal blood vessel growth.
Read Application Note:
Chemiluminescent VEGF
ELISA Using the SpectraMax L
Microplate Luminometer
Fluorescence-based detection
Read Application Note:
Faster ELISA results with
CatchPoint SimpleStep
ELISA kits
See how CatchPoint SimpleStep
ELISA kits give users a 90-minute
ELISA workflow, a nearly two-thirds
reduction in the time required
to achieve results compared to
conventional ELISAs.
Calibrator concentration (ng/mL)
Average OD
Add Stop solution,
measure signals on a plate reader
Conventional ELISA Procedure SimpleStep ELISA Procedure
260 min
Add substrate,
develop signals for 10–15 minutes
Wash wells 3–5 times
Add detection antibody, incubate for 1 hour
Wash wells 3–5 times
Add samples, incubate for 1 hour
Wash wells 3–5 times
Block wells, incubate for 30 minutes
Wash wells 3–5 times
Coat wells with capture antibody,
incubate for 1 hour
Wash wells 3–5 times
Warm reagents & plate at room temperature
Measure signals on a plate reader
90 min
Add samples, add capture and detection
antibodies, incubate for 1 hour
Add substrate,
develop signals for 10 minutes or longer
11
Lab automation for high-throughput ELISA assays
ELISA (Enzyme-linked Immunosorbent Assay) is one of the most popular quantitative methods to detect a target antigen
such as a toxin or foreign substance within a sample. The assay is easy to set up and the range of potential analytes is
vast, but the assay procedure is time-consuming and labor-intensive.
As your lab’s needs expand, automating every step of an intricate work?ow provides exponential value. From increased
walk-away time and throughput to reduced human error and improved lab safety, automation can keep routine work on
track so that scientists can focus on more critical assay development challenges.
Learn more
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