THE COMPLETE GUIDE TO

ELISA
FOREWORD

The enzyme-linked immunosorbent assay (ELISA) is considered the gold standard of

immunoassays available today. It is a popular plate-based method for detecting and quantifying
peptides, proteins, antibodies or hormones, and is used in a wide range of both research and
applied settings, including clinical applications, such as the detection of viral infections.

We have compiled this detailed eBook – consisting of in-depth educational articles, relevant
app notes and customer testimonials – to help you understand how ELISAs work, the materials
required to perform them, and their limitations in comparison to other immunoassays, such as
western blot. We also highlight what you should consider in order to run ELISAs successfully,
and demonstrate how our solutions can help you to enhance the throughput of your lab and
become an ELISA expert.

Dr Éva Mészáros

Application Specialist

[email protected]

Anina Werner

Content Manager

[email protected]
TABLE OF CONTENTS

CHAPTER 1: What you need to know about ELISAs 2

1.1 An introduction to the different types of ELISA tests 3
1.2 ELISA vs. western blot: a comparison of two common immunoassays 13

CHAPTER 2: INTEGRA Biosciences’ ELISA solutions 22

CHAPTER 3: Application notes

3.1 Performing an ELISA with the ASSIST PLUS pipetting robot 26
3.2 Performing an ELISA with the VIAFLO 96 or 384 handheld electronic pipette 34

CHAPTER 4: Customer testimonials and product stories

4.1 VOYAGER adjustable tip spacing pipette accelerates immunology research 41
at the University of Basel
4.2 How did we manage without a 96 channel pipette? 43
4.3 Making COVID-19 ELISA testing safer and easier with convenient buffer removal 45

CHAPTER 5: Conclusion 47

CHAPTER 6: References 48
2 CHAPTER 1: What you need to know about ELISAs

CHAPTER 1:
What you need to know about ELISAs
In this chapter, we will cover the different types of ELISAs, analysis of ELISA data, and the
different reagents and equipment required to perform these tests in the lab. We will also explain
the differences and similarities between ELISAs and western blotting, and their respective pros
and cons, to allow you to make an informed decision on which technique to use for your specific
application.
 CHAPTER 1: What you need to know about ELISAs 3

1.1 An introduction to the different types of

ELISA tests
ELISA stands for 'enzyme-linked immunosorbent assay’, and ELISA tests are mainly used
in research and clinical applications, including detecting viral infections such as hepatitis B
virus (HBV), hepatitis C virus (HCV) or human immunodeficiency virus (HIV). In this article, we
will outline the different types of ELISAs, explain how to analyze the assay data, and give an
overview of the reagents and equipment required.

What is an ELISA?
Let's start with a general definition of an ELISA before looking at different assay methods:

ELISA
An ELISA test is a type of enzyme immunoassay (EIA). These highly specific and sensitive
assays are used to detect concentrations as low as 0.01 nanograms of antigen or antibody
per milliliter. When performing ELISAs, antigen-antibody complexes are immobilized to a solid
surface. An enzyme is covalently attached to one of the molecules in the complex, and the
subsequent addition of an enzyme-specific substrate results in a colored reaction product.

Different types of ELISA

The four main types of ELISAs are direct, indirect, sandwich and competitive. All of these tests
are commonly performed in 96 well plates, using the bottom of the wells as the solid surface to
immobilize the antigen-antibody complexes. The differences between the four types – and their
advantages and disadvantages – are discussed in the following sections.
4 CHAPTER 1: What you need to know about ELISAs

Direct ELISAs
The direct ELISA protocol can be used to detect antigens, and consists of four steps:

1. Plate coating: Dilute your samples using a coating buffer, then pipette them into a microwell
plate. After incubation, discard the coating solution and wash the plate with a wash
buffer. The proteins contained in your sample – including the antigen of interest – are now
immobilized to the plate.
2. Plate blocking: Dispense blocking buffer into the plate and incubate it. The blocking buffer
will bind to any remaining protein-binding sites in the coated wells, reducing subsequent
non-specific binding of antibodies to the plate. Wash your plate again.
3. Antibody incubation: Transfer enzyme-labeled antibodies to the plate, then incubate. The
antibodies will bind to the antigens of interest in wells that contain the analyte. Following
incubation, wash away unbound antibodies with a wash buffer.
4. Detection: Add an enzyme-specific substrate to the plate. The enzymes covalently attached
to the antibodies will start producing a colored reaction product. After adding a stop solution
to terminate the color development, read the absorbance of each well with a plate reader.
The signal intensity allows you to determine whether a sample contains the antigen of
interest, and at what concentration.

Substrate

Enzyme-labeled
antibody

Antigen

Direct ELISA
 CHAPTER 1: What you need to know about ELISAs 5

Indirect ELISAs
The only difference between a direct and indirect ELISA is that the antibody incubation process
is divided into two steps. After coating and blocking the plate, primary antibodies that bind to
the antigens of interest are added to the plate, which is then incubated. Following a wash step,
enzyme-conjugated secondary antibodies – which bind to the primary antibodies but not to the
target antigens – are added. After incubating the plate a second time and washing off unbound
secondary antibodies, the detection step can be performed.

Substrate

Enzyme-labeled
secondary antibody

Primary
antibody

Antigen

Indirect ELISA
6 CHAPTER 1: What you need to know about ELISAs

Direct vs. indirect ELISAs

The advantage of direct ELISAs is that they save time and reagents, as the protocol is slightly
shorter. In addition, the risk of cross-reactivity is reduced. Cross-reactivity occurs when
antibodies bind non-specifically to epitopes on molecules that are not the antigen of interest.
Because direct ELISAs use only one antibody instead of two, this risk is lower.

Despite the longer protocol and higher risk of non-specific binding due to cross-reactivity,
indirect ELISAs have many advantages too. For example, they are more sensitive, because
multiple secondary antibodies can bind to one primary antibody, amplifying the detectable
signal.

Substrate

Secondary
Substrate antibodies
Substrate

Primary
Antigen antibody

Signal amplification
(indirect ELISA)
Moreover, indirect ELISAs offer greater flexibility. A secondary antibody can be specific to
several primary antibodies, as long as they are the same type and from the same host species.
This means that the same labeled secondary antibody can be used in different indirect ELISA
applications, whereas several labeled primary antibodies are needed to perform different direct
ELISA protocols.

Another advantage of indirect ELISAs is that they can not only detect antigens, but also
antibodies. This requires coating the plate with antigens that bind to the antibodies of
interest, then adding the samples in place of the primary antibodies during the first antibody
incubation step.
 CHAPTER 1: What you need to know about ELISAs 7

Sandwich ELISAs
For sandwich ELISAs, the plate isn't coated with antigens, but with capture antibodies specific
to the antigen of interest. After blocking the remaining protein binding sites with a buffer that is
designed to bind to the sites not occupied by the capture antibodies, the samples can be added
to the wells. During incubation, the antigens of interest bind to the capture antibodies. Next, the
plate is incubated either with enzyme-labeled detection antibodies (direct sandwich ELISA) or
with primary antibodies followed by enzyme-labeled secondary antibodies (indirect sandwich
ELISA). Please note that the capture and primary antibodies for indirect sandwich ELISAs need
to be from different host species to increase specificity.

Substrate

Enzyme-labeled
secondary antibody

Primary
antibody

Antigen

Capture
antibody

Indirect sandwich ELISA

The advantage of sandwich ELISAs is that they are highly specific. The two antibodies capturing
an antigen bind to different epitopes of it, making it almost impossible for both antibodies to
bind non-specifically. Thus, sandwich ELISAs are the perfect method for complex samples. A
disadvantage of sandwich ELISAs is that it can sometimes be difficult to find two antibodies that
work well together while binding to different epitopes of the same antigen.
8 CHAPTER 1: What you need to know about ELISAs

Competitive ELISAs
In competitive ELISAs, also known as inhibition ELISAs, the amount of labeled molecules is
limited, and the sample antigens or antibodies compete with reference antigens or antibodies
to bind to these labeled molecules. This means that the analyte concentration is measured by
the detection of signal interference. Direct, indirect and sandwich ELISAs can all be adapted to
this format, but the steps and reagents of a competitive ELISA protocol can vary. To perform a
competitive direct ELISA, you could, for example, proceed as follows:

• Coat the plate with a reference antigen and block the remaining binding sites with a buffer.
• Incubate your sample that has an unknown antigen concentration with a limited amount of
labeled antibodies. If the antigen concentration in the sample is low, a large portion of the
antibodies will not be able to bind to an antigen, and vice versa.
• Add the sample-antibody mixture to the coated wells and incubate the plate. The antibodies
that couldn't bind to a sample antigen in the previous step will now bind to a reference antigen
(step 1 in the image below).
• Wash your plate. Antibodies bound to sample antigens will be washed away because they
aren't immobilized to the plate (step 2 in the image below).
• Add a substrate (step 3 in the image below) and measure the colored reaction product. The
stronger the color change, the less antigen was present in the sample, and vice versa.

The advantages and disadvantages of a competitive ELISA depend on the base ELISA (direct,
indirect, competitive) selected.

Wash away
unbound
antibodies
1) 2) 3)

Substrate

Enzyme- Enzyme- Enzyme-

Sample labeled Sample labeled labeled
Antigen antibody Antigen antibody antibody

Reference Reference Reference

Antigen Antigen Antigen

Competitive direct ELISA

 CHAPTER 1: What you need to know about ELISAs 9

How to analyze ELISA results

After looking at the working principle of the different types of ELISAs, let's now talk about data
analysis. ELISAs can either deliver qualitative, semi-quantitative or quantitative results:

• Qualitative results: Is the antigen or antibody present in a sample or not?

• Semi-quantitative results: Is the antigen or antibody concentration in sample A higher or
lower than in sample B?
• Quantitative results: What is the antigen or antibody concentration in a sample?

To obtain reliable results, you should include negative and positive controls in every ELISA
plate that you set up. Negative controls will allow you to check for false positive results caused
by non-specific binding or contamination, whereas positive controls confirm that the test is
working as intended, even if all the samples are negative.

If you want to obtain quantitative results, you also need to set up a standard curve. To do so,
serially dilute a sample with a known amount of the antigen or antibody of interest with a diluent
buffer. After running the ELISA, plot the known concentrations against the obtained absorbance
values, using curve fitting and data analysis software to find the curve that best fits your data.
This will give you an an equation that you can use to calculate the unknown antigen or antibody
concentrations in your samples. For example, if a linear plot is the curve that fits your data best,
the standard curve will show the concentration on the x-axis and the absorbance values on the
y-axis,1,2 with R 2 indicating how closely the data points fit the trendline. Values greater than 0.99
allow you to get accurate results.1,3

3.5

3
Absorbance at 450 nm

2.5

1.5

0.5

0
0 2 4 6 8 10 12 14

Concentration (ng/ml)
10 CHAPTER 1: What you need to know about ELISAs

The equation for the linear regression line of the standard curve (y = mx + b) will then allow
you to calculate the antigen or antibody concentration of your samples. As y corresponds to
the absorbance and x to the concentration, the equation for the linear regression line is
equivalent to:

Absorbance = m(concentration) + b

Solving this equation for the concentration will give you the formula:

Concentration = (Absorbance - b) / m

For example, if your equation for the linear regression line is y = 0.2497x + 0.0077 (as in the
graph above), a sample with an absorbance value of 2 would have a concentration of 7.9788:

7.9788 = (2 - 0.0077) / 0.2497

When performing quantitative ELISAs with complex samples, you should also include a spike
control. A spike control is a serial dilution of a sample with a known amount of antigen or
antibody of interest in serum instead of diluent buffer. Comparing the absorbance values of
the spike control and the standard curve will allow you to see whether proteins other than the
antigens or antibodies of interest in your sample hinder antigen-antibody binding, leading to an
underestimation of the target concentration.

Note that the negative and positive controls, standard curves, spike controls and samples
should all be run in duplicate or triplicate. This lowers the number of samples that can be
analyzed per plate, but increases assay reliability. The chance that factors such as pipetting
errors remain unnoticed is much lower when working with average absorbance values of
duplicates or triplicates during data analysis, as you would notice high deviations of the mean if
only one of the wells in the set was affected. An acceptable deviation of the mean for duplicates
is a value of 20 % or lower 2.

Reagents for ELISAs

The reagents needed for an ELISA test depend on the protocol, but generally include coating,
blocking and wash buffers, enzyme-conjugated antibodies or antigens, a substrate and a stop
solution. For the detection of common antigens and antibodies, you can usually purchase
ready-to-use ELISA kits that contain everything you need.

The most widely used reagents in these kits are:

• Coating buffer: The two most common coating buffers are PBS and bicarbonate. They
stabilize the antigen or antibody used to coat the plate during incubation.4
• Blocking buffer: To prevent non-specific binding of detection antibodies to the plate
surface, remaining protein binding sites need to be blocked. This is achieved with blocking
proteins such as bovine serum albumin (BSA), non-fat dry milk (NFDM), casein or caseinate,
normal serum or fish gelatin.5
 CHAPTER 1: What you need to know about ELISAs 11

• Wash buffer: Wash steps to remove unbound materials are mostly performed with PBS
containing a small concentration of a nonionic detergent, such as Tween ® 20.4
• Enzyme-conjugated antibodies or antigens: The antibodies or antigens required
will depend on the analyte. To label the antibodies or antigens with an enzyme, a
streptavidin-biotin bridge is generally used to link the antibodies or antigens to the
detection enzyme.6 The two most common enzymes used are horse radish peroxidase
(HRP) and alkaline phosphatase (ALP).7

Substrate
Biotinylated
enzyme

Biotinylated
antibody

Antigen

Antibody labeling

• Substrate and stop solution: The substrates and stop solutions are enzyme specific.
The substrate for HRP contains TMB (3,3′,5,5′-tetramethylbenzidine), substrate buffer and
hydrogen peroxide, and the reaction can be stopped with sulfuric acid. The substrate for ALP
is a p-nitrophenyl phosphate (pNPP) solution,4 and the reaction can be stopped with NaOH.8
12 CHAPTER 1: What you need to know about ELISAs

Equipment for ELISA

To perform an ELISA, you need pipettes to add the reagents and samples to your microwell
plate, an incubator to hold it at a constant temperature, a plate washer to remove unbound
molecules, and a plate reader for assay analysis.

If you're working in a lab with a limited budget or low throughput, there might be no plate washer
at hand. In this case, you can also use pipettes in combination with an aspiration system for the
wash steps.

On the other hand, high throughput labs might be looking for solutions to increase their
productivity. In this case, various pipetting solutions may come in handy, including adjustable
tip spacing pipettes to transfer samples between different labware formats, 96 channel pipettes
to simultaneously add reagents to every well, or small benchtop pipetting robots to automate
workflows.

Conclusion
ELISAs have been used since the 1970s, and are considered the gold standard of
immunoassays.9,10,11 We hope that this article has helped you to get an overview of the most
relevant aspects related to this important application.
 CHAPTER 1: What you need to know about ELISAs 13

1.2 ELISA vs. western blot: a comparison of two

common immunoassays
If you work in a laboratory and perform immunoassays, you have probably had to choose
between the enzyme-linked immunosorbent assay (ELISA) and western blot techniques at
some point. Both approaches have their pros and cons, so deciding which one to use can be
challenging. This article takes a closer look at these techniques, to help you select the most
appropriate method for your lab’s workflow.

What are immunoassays?

Immunoassays rely on the antibody-antigen binding mechanism that occurs naturally in the
immune system. An antibody produced by the adaptive immune response is specific to a
certain antigen only, and immunoassays take advantage of this specificity to identify a molecule
of interest – typically a protein – in a sample.

Many different types of immunoassays have been developed over the last few decades,
including the ELISA and western blot, two common methods that we will compare and contrast
in this article.

ELISA
ELISAs are highly specific and sensitive assays used to detect concentrations of as little as
0.01 nanograms of antigen or antibody per milliliter of sample.1 The four main types of ELISA –
direct, indirect, sandwich, and competitive – are all usually performed in 96 well plates, using
the bottom of the wells as a solid surface to immobilize antigen-antibody complexes. Since an
enzyme is covalently attached to one of the molecules in the complex, the subsequent addition
of an enzyme-specific substrate results in a detectable colored reaction product if the antigen
or antibody of interest is present in the sample. ELISAs can either deliver qualitative, semi-
quantitative or quantitative results.
14 CHAPTER 1: What you need to know about ELISAs

Western blot
Before delving deeper into the details and methodologies involved in western blotting, we’d like
to share the story behind its name with you.

The western blot was developed in 1981 by Walter Neal Burnette, a postdoc in the Nowinski
lab at the Fred Hutchinson Cancer Center in Seattle. The name 'western blot' alluded to
the Southern blot invented in 1975 by Ed Southern, and the northern blot invented in 1977
by James Alwine. He chose the ‘western’ and not ‘eastern’ direction descriptor, to make a
geographical reference to the location of his lab in Seattle, on the West Coast of the United
States. 2,3

Now that you know the origin of its name, let's have a look at how this technique works.

What is a western blot?

In a nutshell, the western blot (also called an immunoblot) is a technique used to detect a
specific protein, such as an antigen, in a sample consisting of a mixture of proteins. To this end,
the proteins in the sample first need to be denatured and separated by size.

After transferring the proteins to a membrane, antibodies are used to detect the protein of
interest, and the result is visualized using a colorimetric, chemiluminescence or fluorescence
analysis method. Radioactive detection is also possible, but is now only rarely used due to
health and safety risks.
 CHAPTER 1: What you need to know about ELISAs 15

For those who aren't familiar with the technique, we've explained the various parts of western
blotting in more detail below.

Steps of a western blot

We can break western blotting down into three main steps:

• Separation of proteins by size

• Transfer of proteins to a membrane
• Labeling of proteins using antibodies

The first step is achieved using gel electrophoresis. Samples are loaded into wells on top of the
gel. An electrical current is then applied to the gel, causing the proteins to travel through it. Two
factors influence how fast the proteins in the sample travel: size and charge. To separate the
proteins according to their size, they need to have a proportionally uniform charge, therefore,
they are treated with sodium dodecyl sulfate (SDS) which causes proteins to unfold into linear
chains and imparts a negative charge.4

The porous polyacrylamide composition of the gel allows smaller proteins to migrate faster
than larger ones, resulting in the formation of bands containing proteins of the same size.
The polyacrylamide concentration determines the size of pores in the web of the gel, and can
be adjusted to ensure even distribution of proteins and a satisfactory electrophoresis result.
Generally, larger proteins separate easily in a gel with a low percentage of polyacrylamide,
and smaller proteins require a gel with a high percentage of polyacrylamide. Please note that
unpolymerized acrylamide is carcinogenic and mutagenic, and should always be handled in a
biosafety cabinet using appropriate personal protective equipment.

In addition to your samples, you also need to add a molecular marker containing several
proteins with known molecular weights to the gel. This will allow you to see if electrophoresis
has been successful, and whether the subsequent transfer of the proteins to the blotting
membrane has been effective. Moreover, it will enable you to estimate the length of your
proteins of interest, as you can compare their migration path through the gel with the
distance travelled by the proteins of the molecular marker. The method is often abbreviated
as SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) because gel
electrophoresis for western blotting uses SDS and polyacrylamide.
16 CHAPTER 1: What you need to know about ELISAs

The second stage of the western blotting process involves transferring the migrated proteins to
a blotting membrane. The most commonly used technique is electrophoretic transfer, where the
gel and membrane are sandwiched between filter paper and placed between electrodes, after
which an electric field moves the proteins from the gel to the membrane.5

The third step of a western blot protocol – protein labeling – is very similar to an ELISA. It
requires you to incubate the membrane with a blocking buffer, which binds to any remaining
protein binding sites to reduce subsequent non-specific binding of antibodies to the membrane.
After washing the membrane you can opt for either a direct or indirect labeling method.

In the direct method, the membrane is incubated with labeled antibodies that bind to the
proteins of interest, before washing unbound antibodies away. The indirect method uses two
antibodies, a primary and a secondary antibody. In the first step, the membrane is incubated
with unlabeled primary antibodies that bind to the proteins of interest. After washing, a
second incubation is performed, using labeled secondary antibodies that bind to the primary
antibodies. This is followed by a final washing step to remove any unbound molecules.
 CHAPTER 1: What you need to know about ELISAs 17

Labeled secondary
antibody

Labeled Primary
antibody antibody

Protein Protein

Direct labeling Indirect labeling

There are risks and benefits to both labeling protocols. Direct binding is a shorter process,
which can save time and reagents. It also reduces the risk of non-specific antibody binding
since it only uses one antibody.

On the other hand, indirect western blotting is more sensitive because several secondary
antibodies can bind to one primary antibody, amplifying the detectable signal. The indirect
method also offers greater flexibility, as secondary antibodies can be specific to several primary
antibodies of the same type and from the same host species. This means that the same labeled
secondary antibody can be used in different indirect western blot applications, whereas several
labeled primary antibodies are needed to perform different direct protocols.

Western blot analysis

To analyze a western blot, you need to measure the signal produced by the labeled antibodies.
The detection steps and equipment differ depending on whether the antibodies are tagged with
an enzyme or a fluorophore.

Enzymatic detection
You can use either colorimetric or chemiluminescence detection methods to analyze your
western blot if your antibody is labeled with an enzyme.

Colorimetric methods
Colorimetric methods require the application of an enzyme-specific chromogenic substrate to
the membrane; the reaction catalyzed by the enzymes attached to the antibodies will produce a
colored reaction product. A stop solution is added to terminate the color development, and the
chromogenic products of the enzymatic reaction are visible as bands to the naked eye.6

A colorimetric western blot analysis can be performed quickly and cost-effectively because you
don't need any special detection equipment. It is, however, not very sensitive. Proteins in the
nanogram range are required to produce a visible band.6
18 CHAPTER 1: What you need to know about ELISAs

Chemiluminescence methods
Chemiluminescence detection methods also require an enzyme-specific substrate, but one
that is luminescent instead of colorimetric. This means that the enzymatic reaction on adding
substrate to the membrane will produce light as a by-product. The light can then be detected
using an X-ray film or a charge-coupled device camera (CCD).6

The main advantage of chemiluminescence detection is its sensitivity; it can be used to detect
and quantify as little as femtograms of the protein of interest. However, a downside of this
technique is that light is only emitted briefly, when the enzyme converts the substrate into a
product, and therefore detection needs to be performed immediately after substrate addition.7

Fluorescence detection
If your antibodies are labeled with fluorophores, you don't need to add any substrate to the
membrane. Instead, you can image the membrane directly using a fluorescence imaging
system, where a light source excites the fluorophores and the fluorescent signals emitted are
detected by wavelength-specific filters and digital camera systems.8

Tagging antibodies with fluorophores instead of enzymes has several advantages. Firstly,
tagging individual antibodies with different fluorophores allows for the simultaneous detection
of various proteins with similar molecular weights (multiplexing). Secondly, as fluorescent
signals are still detectable on membranes after months of storage at room temperature, this
method offers a lot of flexibility when it comes to analyzing assay data. Thirdly, fluorescence
detection is simple to perform because you don't need to add any substrate. The only
disadvantages of this detection method are the need for a special imaging system, and reduced
sensitivity compared to chemiluminescence detection.7
 CHAPTER 1: What you need to know about ELISAs 19

ELISA vs. western blot

Now that you understand how to conduct and analyze the two immunoassays, how should you
decide which technique to use?

Generally speaking, an ELISA is easier and faster to perform than a western blot because the
protocol is shorter. Moreover, ELISAs are better suited for high throughput labs because they
can be performed with lower sample volumes and are usually carried out in 96 well plates,
allowing for workflow automation.

When it comes to obtaining reliable quantitative data in an ELISA, a simple standard curve
– serial dilutions of a known target protein – can be set up, and its equation can be used to
calculate the antigen or antibody concentration of samples based on their absorbance values.

Performing a quantitative western blot can be more demanding. It requires the consideration of
several factors, such as overloading of samples, membrane saturation and signal saturation.
Additionally, internal loading controls like a housekeeping protein are required, data must be
normalized, and the background signal must be subtracted for quantitative analysis.

However, even though performing a western blot can be complex and time-intensive, they are
preferred over ELISAs for some applications. The first advantage of a western blot is that they
are highly specific, and are sometimes used to rule out false positives and confirm positive
ELISA results in clinical settings. This is due to the initial protein size separation which ensures
that non-specific signals from analytes – which would likely be a different size – can be easily
detected as a second band on the membrane.
20 CHAPTER 1: What you need to know about ELISAs

Secondly, a western blot generates more information about the target protein and the sample
in general. Rather than simply identifying the presence or absence of an analyte in a sample,
western blot can also determine the length of a protein and analyze sample purity. Therefore,
the slightly longer and more tedious protocol may be worthwhile if your assay requires more
extensive information.

A third advantage of western blotting – although only true if the detection antibody is labeled
with a fluorophore – is its ability to detect several proteins per sample in parallel.

Alternatives to ELISAs and western blotting

We have determined that the ELISA and western blot methodologies have their individual
benefits and disadvantages for various laboratory applications. However, these techniques
fall short when looking to perform more complex high throughput multiplexing immunoassays:
the enzyme-labeled antibodies used in ELISAs don't allow you to detect several analytes in
parallel, and fluorescent western blots are only suitable for low throughput settings. Luckily,
alternative techniques like bead-based immunoassays and protein microarrays have been
developed for just this purpose.

Bead-based immunoassays
To perform a bead-based immunoassay, you need as many bead sets as you have analytes
of interest. Each bead set is coated with capture antibodies specific for a certain analyte, and
labeled with a red fluorescent dye with a unique intensity. Different detection antibodies are
also needed for this assay. These are labeled with a green fluorophore, and bind to the analytes
of interest. When the bead sets and detection antibodies are added to a sample and incubated,
they form complexes with the analytes of interest.

Beads Detection
antibodies

Sample
 CHAPTER 1: What you need to know about ELISAs 21

These bead complexes can then be analyzed using flow cytometry, during which individual
complexes pass through a red and a green laser beam. The red laser identifies the signal
intensity of the fluorescent dye, and can consequently tell which type of analyte the bead is
supposed to bind. The green laser is then used to determine whether analytes and detection
antibodies are bound to the beads, by detecting fluorophores on the detection antibodies.9,10,11

Protein microarrays
Protein microarrays (also called protein chips) are comparable to performing a lot of
sandwich ELISAs simultaneously on a single solid surface. The solid surface – which can be
a membrane, a glass slide or the well of a microplate – is pre-spotted with different capture
antibodies specific for the analytes of interest. When incubating the solid surface with a sample,
the analytes, if present, will bind to the capture antibodies and can then be detected with
labeled antibodies. As you know that spot 1 is supposed to bind analyte A, spot 2 analyte B,
etc., the pattern generated after analysis will allow you to determine which analytes are present
in your sample.

Sample Antibody
Analysis
addition addition

Spot 1 Spot 2 Spot 3 Spot 1 Spot 2 Spot 3

Conclusion
In conclusion, ELISAs and the western blot are two immunoassays that can be used to detect
the presence of a protein in a sample. Each method has its pros and cons; ELISAs are easier,
faster to perform, and better suited for high throughput labs, the western blotting technique is
more specific, can provide additional information about the target protein and the sample under
analysis, and allows for multiplexing. The decision as to which method to use depends on the
application. Moreover, there are alternative techniques such as bead-based immunoassays
and protein microarrays that may be more suitable when performing high throughput
multiplexing assays.
22 CHAPTER 2: INTEGRA Biosciences’ ELISA solutions

CHAPTER 2:
INTEGRA Biosciences’ ELISA solutions
ELISAs are the mainstay for a wide range of studies but, unfortunately, generally involve
multiple time-consuming and tedious processing steps that require consistent pipetting – from
well-to-well and plate-to-plate – to ensure success. On top of this, the repetitive nature of this
work can also frequently result in user fatigue and handling mistakes.

Fortunately, with the right tools, you can quickly and easily ramp up your throughput, reduce
errors, increase consistency and improve the reproducibility of your results. Here, we will
demonstrate how INTEGRA’s range of liquid handling solutions can help you to save time and
elevate your ELISA workflows for more successful experiments.

Handheld electronic pipettes

The VIAFLO 96 and VIAFLO 384 handheld electronic pipettes can vastly improve
ELISA workflows by enabling the transfer of samples and reagents into 96 or 384 wells
simultaneously. All the steps needed to run the protocol can be saved as a custom program on
the instrument, which then guides the user with easy to follow prompts. This streamlines the
workflow and eliminates user variability. These instruments can also quickly fill multiple plates
using the Repeat Dispense mode, or can be used for partial plate processing, providing even
higher throughput. Crucially, the VIAFLO 96 and VIAFLO 384 offer all of these benefits while
still being as easy to use as traditional handheld pipettes.

Learn more
about
VIAFLO 96
and VIAFLO 384
 CHAPTER 2: INTEGRA Biosciences’ ELISA solutions 23

Pipetting robots
The ASSIST PLUS pipetting robot automates the ELISA process, and can be easily adapted
to the requirements of your specific application. Pipetting settings and assay set-up options
– including volumes, sample predilutions and plate layouts – can easily be modified using our
VIALAB software, making sure the system is always perfectly suited to your assay, even for
partial plate processing. Various samples and tube types – as well as multiple reagent vessels
– can be accommodated on the deck, and you can mount any of our 25 electronic pipettes
or the D-ONE single channel pipetting module onto the ASSIST PLUS. This combination of
flexibility and automated, optimized processing improves productivity, reproducibility and
consistency.

Learn more Learn more

about about
ASSIST PLUS D-ONE
24 CHAPTER 2: INTEGRA Biosciences’ ELISA solutions

Pipette tips
ELISA buffers often contain surfactants – like Tween ® 20 – which tend to interact with standard
pipette tips, forming a thin liquid film on the inner wall. This leads to inaccurate and inconsistent
pipetting results, as well as loss of precious reagents. INTEGRA’s low retention GRIPTIPS
are made from a unique polypropylene blend that offers heightened hydrophobic properties,
reducing the residual tip volumes for maximum sample recovery.

Learn more
about
GRIPTIPS
 CHAPTER 2: INTEGRA Biosciences’ ELISA solutions 25

Aspiration systems
There are many advantages to using an aspiration system to remove buffers from samples,
especially for 96 well plate formats, where there are multiple wells to process. The
VACUSIP portable aspiration system provides safer, faster and more convenient waste removal
than a multichannel pipette, is simple to operate, and comes ready to use straight out of the
box. It’s an affordable and versatile tool that helps to increase productivity for ELISA workflows,
while also protecting the wellbeing of users.

Learn more
about
VACUSIP
26 CHAPTER 3: Application Notes

CHAPTER 3:
Application notes
Our pipetting instruments are used across a broad spectrum of life sciences applications, and
we endeavor to share knowledge and experience of using our products with the wider scientific
community. To this end, we have compiled an extensive database over the years, which
contains a wide range of thorough and useful application notes. Here are some of the most
relevant app notes for ELISA protocols and workflows.

3.1 Performing an ELISA with the ASSIST PLUS

pipetting robot

Enzyme-linked immunosorbent assays with ASSIST PLUS

pipetting robot

The enzyme-linked immunosorbent assay (ELISA) is a standard method

used to detect and quantify peptides, proteins, antibodies or hormones
in a sample. It consists of multiple repetitive steps that are time
consuming and tedious to perform manually. The ASSIST PLUS
pipetting robot allows this process to be automated, which not only
increases the reproducibility of your results, but also gives you
more time to focus on your science. Any VIAFLO or
VOYAGER electronic pipette can be automated
using the ASSIST PLUS; the VOYAGER
adjustable tip spacing pipette enables
reformatting of samples from one labware
type to another in the blink of an eye. All the
steps needed to run an ELISA are saved on the
pipette as a VIALAB program – the smart and easy-to-use pipetting automation software of
the ASSIST PLUS. Simply place the labware on the deck, choose the program corresponding
to the ELISA step and let the ASSIST PLUS do the work.
 CHAPTER 3: Application Notes 27

Key benefits

• Optimal pipette settings – including tip • VOYAGER and VIAFLO electronic pipettes,

immersion depth, pipetting speeds and in combination with the ASSIST PLUS,

angles – maximize the consistency and provide unmatched pipetting ergonomics.

reproducibility of the ELISA.

• The ASSIST PLUS pipetting robot is

• The full automation capability of the perfectly adapted to handle different plate

ASSIST PLUS frees highly valuable time layouts, increasing the flexibility of your

that you can use for more important tasks. work depending on your needs.

• Repeat Dispense and Multi Aspirate steps • Various sample input tubes can be used.

can be used for fast dispense and removal The samples are easily transferred to

of reagents to speed up the process. The the assay plate using multichannel and

automatic Tip Change ensures assay adjustable tip spacing pipettes, increasing

contamination is avoided. the assay productivity while avoiding

reformatting errors.

Overview of the sandwich ELISA steps and

corresponding programs:
The ASSIST PLUS is used to perform a sandwich ELISA. The pipetting robot operates
a VOYAGER 8 channel 1250 μl electronic pipette with 1250 μl sterile, filter, low retention
GRIPTIPS. The use of low retention GRIPTIPS guarantees optimal liquid recovery when
pipetting ELISA buffers that contain surfactants, such as Tween ® 20.

Below is an example set-up for a sandwich ELISA with a standard curve and 24 samples in
triplicate. The pipetting programs are prepared with the VIALAB software. The protocol is
divided into eight programs that guide the user through the eight steps of the ELISA.
28 CHAPTER 3: Application Notes

Step-by-step procedure
1. Coat the ELISA plate
Adding the capture antibody to coat
the ELISA plate.

Place the capture antibody, prediluted in

the coating buffer, in a 10 ml polypropylene
multichannel reagent reservoir. Select and run
the first VIALAB program, 1_E_Coating. The
pipette automatically transfers 100 μl of the
capture antibody into the ELISA plate using the
Repeat Dispense mode. The plate is ready to
Figure 1: Adding the capture antibody to coat the ELISA plate.
be incubated.

2. Block the plate

Blocking the ELISA plate’s non-specific
binding sites.

Select the VIALAB program 2_E_Blocking and

set up the deck with the required labware.

The program incorporates all the necessary

pipetting steps, including removing of the
coating buffer from the plate followed by
washing three times with 200 μl of the washing Figure 2: Blocking the ELISA plate’s non-specific binding sites.
buffer. In our example, we included a 15 second
incubation time, which can be easily adjusted to your protocol using the VIALAB software. The
use of the Repeat Dispense and Multi Aspirate modes speeds up this fully automated process.
At the end of the washing steps, the user is prompted to blot the plate against clean paper
towels – the only manual step of this ELISA protocol. After confirming that this step has been
completed, the ASSIST PLUS pipetting robot continues by adding the blocking buffer into the
ELISA plate using the Repeat Dispense mode. Finally, the pipette informs the user that the
plate is ready for incubation.
 CHAPTER 3: Application Notes 29

3. Prepare your samples

Diluting your samples 1:10.

In this example, centrifuged blood samples are

stored in EDTA collection tubes placed in an
INTEGRA rack. The plasma is diluted 1:10 with
the dilution buffer.

Select and run the 3_E_Sample_Preparation

program. The pipette automatically fills the
microcentrifuge tubes with 900 μl of the Figure 3: Diluting your samples 1:10.

dilution buffer. This is followed by transfer of

the plasma samples from the EDTA tubes to
the microcentrifuge tubes, and careful and
thorough mixing. The samples are then ready
to be used.

Tips:

• Using a VOYAGER adjustable tip spacing pipette together with the ASSIST PLUS allows
automatic and error-free sample reformatting.
• The ASSIST PLUS pipetting robot ejects and loads the tips automatically, eliminating any risk
of sample cross-contamination.

4. Add your controls and samples

Adding the controls and diluted
samples to the ELISA plate.

Prepare the deck of the ASSIST PLUS.

Select and run the 4_E_Sample_Addition
program to remove the blocking buffer and
subsequently wash the plate. In our example,
each sample is added to the ELISA plate in
triplicate. The ASSIST PLUS pipetting robot
uses the Repeat Dispense mode to transfer the
triplicate samples into the plate, replacing the Figure 4: Adding the controls and diluted samples to the ELISA plate.

pipette tips before aspirating the next series of

samples.

Tip:

Each pipetting step is done in exactly the same way, ensuring the reproducibility of the assay
from row to row, and plate to plate.
30 CHAPTER 3: Application Notes

5. Add the detection antibody

Adding the diluted detection antibody
to the ELISA plate.

After incubation, prepare the deck and select

program 5_E_Detection_Antibody. The
addition of the detection antibody is performed
automatically by the ASSIST PLUS. Incubate
the plate again.

Figure 5: Adding the diluted detection antibody to the ELISA plate.

6. Add the enzyme conjugate

Adding the enzyme conjugate to the
ELISA plate.

Set up the ASSIST PLUS deck. Select program

6_E_Enzyme_Conjugate, which includes the
removal of the previous solution, the three
washing steps, and the addition of the enzyme
conjugate to the ELISA plate. The plate is ready
for incubation.

Figure 6: Adding the enzyme conjugate to the ELISA plate.

7. Add the substrate

Adding the TMB substrate and
incubating the plate until the color
develops sufficiently.

Select and run program 7_E_Substrate. The

ASSIST PLUS removes the previous buffer
then washes the plate six times before adding
the TMB substrate. Incubate the plate at
room temperature until the color is sufficiently
developed. The color of the solution changes Figure 7: Adding the TMB substrate and incubating the plate until the
color develops sufficiently.
from transparent to blue in wells where the
samples have reacted with the antibodies. The
color intensity is dependent on the sample
concentration.
 CHAPTER 3: Application Notes 31

Figure 8: Example of a sandwich ELISA plate after incubation with TMB substrate, showing positive
(blue) and negative (clear) reactions of the triplicate samples with the antibodies. The color intensity
directly depends on the sample concentration.

Tips:

• The pipetting robot automatically processes the plate, regardless of the multiple and
repetitive pipetting steps, freeing up time for you to concentrate on other tasks.
• The ASSIST PLUS tells you when to add the TMB substrate into the corresponding row of the
reservoir, preventing the photosensitive substrate from being exposed to light for too long a
period of time.

8. Stop the reaction

Adding the stop solution to the plate
before detection.

Select and run the final program, 8_E_Stop_

Solution.

The ASSIST PLUS adds the stop solution to

the sample triplicates in the plate; the color
changes from blue to yellow in the wells where
Figure 9: Adding the stop solution to the plate before detection.
the samples reacted with the antibodies. The
plate is now ready for detection.
32 CHAPTER 3: Application Notes

Figure 10: Example of a sandwich ELISA plate using a TMB substrate after addition of the stop solution,
showing positive (yellow) and negative (clear) reactions of the triplicate samples with the antibodies.

Remarks
Partial plate
If your particular ELISA
doesn’t require processing
of 96 samples, the
ASSIST PLUS is able to work
with any number of columns.
Simply adapt the VIALAB
program to fit your need.

VIALAB software
The VIALAB programs Figure 11: Partial plate and VIALAB software.

can be easily adapted to

your specific labware and
protocols.
 CHAPTER 3: Application Notes 33

Conclusion
• ELISAs can be fully automated using the ASSIST PLUS pipetting robot, offering users
increased walk-away time.

• Optimized pipetting settings and tip immersion, together with the use of low retention
GRIPTIPS, guarantee the consistency and reproducibility of the ELISAs.

• Using the ASSIST PLUS pipetting robot allows various sample tube types and multiple
reagents to be accommodated on the deck, for improved productivity and unrivaled
flexibility.

• Automatic Tip Change avoids any assay contamination while using the Repeat Dispense
and Multi Aspirate modes whenever possible speeds up the process.

• Thanks to the VIALAB software, the pipetting programs can be easily adapted to specific
protocols and labware.

For more information

and a list of materials
used, please refer to
our website.
34 CHAPTER 3: Application Notes

3.2 Performing an ELISA with the VIAFLO 96

or 384 handheld electronic pipette

Enzyme-linked immunosorbent assays with the VIAFLO 96

and VIAFLO 384 handheld electronic pipettes

ELISA (enzyme-linked immunosorbent assay) is a plate-based assay that

is commonly used to quantify and detect peptides, proteins, antibodies or
hormones. Well to well and plate to plate consistency and reproducibility
are key success factors for ELISAs, which consist of multiple pipetting steps
that are time-consuming and tedious to perform manually. Using the
VIAFLO 96 or 384 handheld electronic pipette can vastly improve ELISAs.
The 96 channel pipetting head makes the whole process faster, less
error prone and much more reproducible
compared to traditional single- or
multichannel pipettes by transferring
the samples and reagents into all
96 wells of the plate at the same time.
The VIAFLO 96 and VIAFLO 384
electronic pipettes offer clear gains in
productivity, consistency and reproducibility while
remaining as easy to use as traditional handheld pipettes.

Key benefits

• The VIAFLO 96 and 384 allow easy and • The pipetting heights are saved to avoid

rapid simultaneous transfer of all samples any crashes and scratching of the coated

for optimal productivity, reproducibility, and surface.

massive time reduction.

• For higher throughput assays, multiple

• All of the steps and settings needed to run plates can be quickly filled using the Repeat

the ELISA protocol can be saved on the Dispense mode of the VIAFLO 96 or 384.

pipette as a custom program, with easy to

follow prompts to guide the user through the
process.
 CHAPTER 3: Application Notes 35

Overview of the sandwich ELISA steps and

corresponding programs:
The following protocol shows an example set-up for performing a sandwich ELISA using the
VIAFLO 96 or 384 handheld electronic pipette together with a three position stage. In this
protocol, position A is dedicated to the GRIPTIPS box and the liquid waste reservoir, position
AB to reagent reservoirs containing the various reagents and washing solutions required, and
position B to the ELISA plate. A 96 channel pipetting head (10-300 μl) is used together with
300 μl sterile, filter, low retention GRIPTIPS. Customized VIALINK programs are provided for
performing a sandwich ELISA with the VIAFLO 96 or 384.

Note: The VIALINK programs provided can be easily adapted to any other ELISA type, e.g.
direct, indirect or competitive ELISAs, by just adding or removing steps from the protocols
supplied.

Step-by-step procedure
1. Coat the ELISA plate
Adding the capture antibody to coat
the ELISA plate.

Place the capture antibody, prediluted in the

coating buffer, into a 150 ml automation-friendly
reagent reservoir. Select and run the VIALINK
E_REAGENTS program, then simply follow the
instructions on the pipette. Pipette 100 μl of the
capture antibody into the ELISA plate and then
Figure 1: Adding the capture antibody to coat the ELISA plate.
incubate it.

Tips:

• A Z-height limit is defined to ensure an optimal tip immersion depth, preventing both air
entering into the tips during the aspiration step and the pipette tips from touching the
bottom of the plate. We also recommend setting the Tip Align support strength to 3 for this
application.
• To increase your throughput, the Repeat Dispense mode of the VIAFLO 96 or 384 electronic
pipette can be used to rapidly prepare several plates at once. The VIALINK program
E_REAGENTS_3 PL is an example of a multiple plate filling protocol.
36 CHAPTER 3: Application Notes

2. Block the plate

Blocking the ELISA plate’s non-specific
binding sites.

Select the VIALINK program E_REAGENTS

to remove the coating buffer from the plate,
then use the E_WASH program to wash the
plate three times with 200 μl of washing buffer
from a 300 ml automation-friendly reagent
reservoir. Firmly blot the plate against clean
Figure 2: Blocking the ELISA plate’s non-specific binding sites.
paper towels. To block the ELISA plate, select
the corresponding E_BLOCK program and transfer 300 μl of the blocking buffer from a 150 ml
automation-friendly reservoir into the plate before incubation.

Tips:

• All wells are treated simultaneously and in the same way. This ensures not only the well to
well consistency of the assay, but also the reproducibility from plate to plate.
• The Z-height is set 1 mm above the bottom of the plate, preventing the tips from scratching
the coated surface, which can lead to inaccurate or inconsistent assay results.

3. Add the controls and samples

Adding the diluted samples and controls
to the ELISA plate.

Select and run the E_BLOCK program to remove

the blocking buffer, then wash the plate using the
E_WASH program. In the current example, the
controls and samples are dispensed into a
96 well plate. The E_REAGENTS program allows
rapid, simultaneous transfer of all samples and
Figure 3: Adding the diluted samples and controls to the ELISA plate.
controls into the ELISA plate before incubation.

Tips:

• Adding all the samples at once ensures optimal productivity and reproducibility of the
ELISA. If your samples are stored in microcentrifuge tubes, using a VOYAGER 8 channel
300 μl adjustable tip spacing pipette allows you to quickly reformat them from the tubes to the
plate at the simple touch of a button, while reducing transcription errors when compared to a
single channel pipette.
• ELISA buffers often contain surfactants, such as Tween ® 20. Using low retention GRIPTIPS
reduces the residual volume in the tips for maximum sample recovery.
 CHAPTER 3: Application Notes 37

Pipetting buffers with Low Retention GRIPTIPS pipette tips

Figure 4: The image highlights the advantages of using low retention GRIPTIPS (left) vs. standard
GRIPTIPS (right) when pipetting buffers containing surfactants.

4. Add the detection antibody

Adding the diluted detection antibody
to the ELISA plate.

After incubation, select the E_REAGENTS

program to remove the sample buffer, then
use E_WASH to wash the plate three times.
Place the detection antibody into a 150 ml
automation-friendly reagent reservoir on
position AB. Run the E_REAGENTS program
Figure 5: Adding the diluted detection antibody to the ELISA plate.
to rapidly transfer the detection antibody into
the ELISA plate before incubation.

5. Add the enzyme conjugate

Adding the enzyme conjugate to the
ELISA plate.

Repeat step 4 to add the enzyme

conjugate. Incubate the plate.

Figure 6: Adding the enzyme conjugate to the ELISA plate.

38 CHAPTER 3: Application Notes

6. Add the substrate

Adding the TMB substrate and
incubating the plate until the color
develops sufficiently.

Remove the buffer then thoroughly wash the

plate six times before adding 100 µl of TMB
substrate to the ELISA plate. Incubate the
plate at room temperature; the color of the
solution changes from transparent to blue in Figure 7: Add the TMB substrate and incubate the plate until the
wells where the samples have reacted with the color develops sufficiently.

antibodies. The color intensity depends on the

sample concentration.

Figure 8: Example of a sandwich ELISA plate after incubation with TMB substrate; positive (blue) and
negative (clear) reaction of the samples with the antibodies. The color intensity directly correlates with
the sample concentration.
 CHAPTER 3: Application Notes 39

7. Stop the reaction

Adding the stop solution to the plate
before detection.

Use the VIALINK E_REAGENTS program

to add the stop solution to the plate; the color
changes from blue to yellow in wells where the
samples have reacted with the antibodies. The
plate is now ready for detection.

Figure 9: Adding the stop solution to the plate before detection.

Figure 10: Example of a sandwich ELISA plate after incubation with TMB substrate and addition of the
stop solution; positive (yellow) and negative (clear) reaction of the samples with the antibodies. The color
intensity directly correlates with the sample concentration.
40 CHAPTER 3: Application Notes

Remarks
Partial plate
If your particular ELISA doesn't require processing of 96 samples, the VIAFLO 96 or 384 is
able to work with any number of tips loaded, giving you the benefit of simultaneous and precise
dispensing of a smaller number of samples.

Automation
The instrument can also operate on its own, reducing user interaction, which in turn
improves ergonomics and reproducibility. This also makes the VIAFLO 96 or 384 ideal for use
in tight spaces, such as under a laminar flow cabinet.

Conclusion
• The VIAFLO 96 and VIAFLO 384 electronic pipettes allow all wells of a 96 well plate to be
treated at the same time, offering optimal consistency and reproducibility of results from
well to well and plate to plate.

• Thanks to their unique operating concept, the VIAFLO 96 and VIAFLO 384 electronic
pipettes are as easy to use as any traditional handheld pipette.

• Optimized pipetting settings ensure easy and fast sample and reagent transfers, while
avoiding the risk of damaging the coated surface of the plate.

• The VIAFLO 96 and VIAFLO 384 electronic pipettes are adaptable to your needs. Work
with a reduced number of tips for partial plate processing, or automate processing for
improved ergonomics in tight spaces, such as a laminar flow cabinet.

For more information

and a list of materials
used, please refer to
our website.
 CHAPTER 4: Customer Testimonials 41

CHAPTER 4:
Customer testimonials and product stories
Our range of innovative liquid handling products has helped many laboratories to achieve
success with their ELISA projects, improving lab throughput and meeting their exciting research
goals. But don’t just take our word for it! Here is a small selection of real-life experiences from
a few of our valued customers, along with a key product story, which illustrate why INTEGRA
Biosciences' pipetting solutions and labware are the best choice for your ELISA workflow.

4.1 VOYAGER adjustable tip spacing pipette

accelerates immunology research at the
University of Basel
The Department of Biomedicine at the University of Basel specializes in a number of key
research areas, including neurobiology, infection and immunity, cancer, stem cell biology
and regenerative medicine. Korcan Ayata is a member of the gastroenterology group – led
by Professor Doctor Jan Nies – which focuses on researching mucosal immunology and
gastroenterology. The group is using INTEGRA’s VOYAGER adjustable tip spacing electronic
pipette to increase the efficiency of their workflows and reduce pipetting errors.

The role of immune cells

The immune system plays a critical role in everyday life
and disease, and Korcan’s group is specifically interested
in the role of immune cells in inflammatory conditions,
such as inflammatory bowel diseases (IBD) and
colitis-associated colorectal cancer. He explained: “We
mainly work with human patient samples and transgenic
mouse models, looking at how monocytes enter the
tissue and differentiate into macrophages to have pro- or
anti-inflammatory effects during the disease course.
We are primarily looking for the cytokines and cytokine
receptors expressed by macrophages and epithelial cells,
and studying their interactions.”

Photo courtesy of the University of Basel

42 CHAPTER 4: Customer Testimonials

Increasing workflow efficiency

The group’s normal workflow involves extracting RNA from samples and converting it to
cDNA, before conducting qPCR, or running ELISAs for serum samples. Korcan continued:
“Most of our samples come from the Swiss IBD cohort, and we’re usually analyzing biopsies
and blood, looking at criteria such as electrolytes, minerals, trace elements or antibodies.
Our work involves transferring many samples between different well formats. Previously, we
were conducting this work with single channel pipettes, which was a laborious and error-prone
process.”

VOYAGER capabilities are second to none

The VOYAGER adjustable tip spacing electronic pipette
has proven perfect for the group’s highly manual workflow,
as the automatic adjustable tip spacing allows users to
transfer multiple samples simultaneously, reducing transfer
steps and pipetting errors. “We looked at a range of
different pipetting systems, and the VOYAGER really stood
out, as nothing compared to its adjustable tip spacing
abilities. We already had a number of single- and
multichannel VIAFLO electronic pipettes, which are well
liked in the department, and this made it easier to integrate
the VOYAGER into our workflow.”

“We mainly use the VOYAGER in qPCR setup, using Photo courtesy of the University of Basel
9 to 13 mm spacing to transfer samples from 1.5 ml tubes
to eight tube strips. After the reverse transcription reaction,
we transfer the cDNA to 384 well qPCR plates using 4 mm spacing. The repeat dispensing
function is really beneficial too, allowing us to distribute different qPCR master mix solutions
from 1.5 ml tubes to 384 well plates. We’re also using the VOYAGER in cell culture and ELISA
applications, for the addition of reagents from 1.5 and 2 ml tubes to 24, 48 and 96 well plates,
plus the transfer of samples between different plate formats. The GRIPTIP pipette tips are
the perfect partner to our VOYAGER; the tight connection between pipette and tip ensures
nothing is loose or leaks, and this means less errors are made. Overall, the VOYAGER
saves us a huge amount of time compared to using manual pipettes, and our throughput has
significantly increased since using it – it really helps to accelerate our research.”
 CHAPTER 4: Customer Testimonials 43

4.2 “How did we manage without a 96 channel

pipette?”

Researchers at the University of North Texas (UNT) discovered INTEGRA’s VIAFLO 96

electronic pipette by accident – and it has transformed their lab! The team is now using the
system to increase the throughput of their ELISA, Luminex and flow cytometry workflows, as
well as to improve the reproducibility of their results.

The Applied Physiology Laboratory at UNT

focuses on research in two key areas –
physiologic and immunologic consequences
of weight change, and using nutritional
countermeasures to maximize immune health
after exercise – and performs analysis and
testing for other research groups in related
fields. Prof Brian McFarlin explained: “We
conduct a lot of multiplexed analysis of protein
and RNA biomarkers, and this requires precise
pipetting. For one study, we were performing
50 flow cytometry preps per blood sample, and
we had over 2000 samples. The work was only
between one graduate student and myself, and
we quickly decided that we needed a solution to
reduce the amount of manual pipetting we were
carrying out!” Photo courtesy of University of North Texas

Brian continued: “We looked around the market, but the solutions on offer were fixed volume
systems, meaning we would need up to five separate platforms. I first learned about the
VIAFLO 96 multichannel pipette at a biomarker conference before it was even available and,
after seeing its ability to change modules to have different volumes, I knew it was just what I
needed. The conference’s demo model was packed up and sent to me for a trial, and it’s been in
constant use ever since!”

The VIAFLO 96 offers an electronic solution that is as easy to use as a traditional handheld
pipette, with improved reproducibility and throughput. “It’s been an absolute lifesaver for our
work; we use it for sample transfer from storage tubes to reagent plates and reagent additions,
especially for running Luminex® assays and ELISAs. Transferring the assays from handheld
pipettes on to the VIAFLO 96 was seamless; INTEGRA was able to support us by writing
protocols for specific applications and assisting in the set-up of these programs. The team
showed us how to use the VIALINK pipette management software which is very intuitive, and
now it’s easy for us to tweak a volume here or there on the protocols. We have since added
44 CHAPTER 4: Customer Testimonials

VIAFLO electronic pipettes and VOYAGER variable tip spacing pipettes – which are great for
our plate transfer work – to our collection. We also have an ASSIST that we use to automate
some of our standard curve development, and we’re now looking to purchase an
ASSIST PLUS pipetting robot for our workflow as well.”

“We now exclusively use INTEGRA pipettes for all of our liquid transfer work because of the
benefits they offer; not only has our throughput increased tremendously, saving a lot of time,
but the performance of our assays has improved. Our results are much more reproducible and
there is a reduced risk of error – it’s revolutionized our workflow,” Brian concluded.
 CHAPTER 4: Customer Testimonials 45

4.3 Making COVID-19 ELISA testing safer and

easier with convenient buffer removal

The COVID-19 pandemic led to the development of a number of novel RT-PCR-based

tests for the detection of the SARS-CoV-2 virus in patients. As the situation progressed, a
need emerged for serology tests to evaluate the immune response of patients, as well as to
assess immunity in the general population. These immunoassays became critical in allowing
movement restrictions to be eased and for preventing subsequent peaks in infections.

SARS-CoV-2 ELISA tests

Most serological tests are based on sandwich
ELISA techniques to detect antibodies against
the virus in patients that have recovered from
COVID-19. The protocols for these ELISAs
require several aspiration steps to remove
buffers after incubation and washing, and
the safest way to perform this is using an
instrument such as our VACUSIP portable
aspiration system.

Why use an aspiration system?

There are many advantages to using an
aspiration system to remove buffers from
samples, especially for 96 well plate formats,
where there are multiple wells to process.
These include:

• Safer, faster and more convenient waste VACUSIP for easy aspiration of small volumes of biological liquid
waste
removal than with a multichannel pipette
• Aspirated liquid waste goes directly into the
collection bottle, avoiding the risk of spillages
or exposure to biohazardous waste during unnecessary liquid transfer
• Pooled waste can be safely and easily autoclaved, chemically deactivated, or transferred to a
larger container
46 CHAPTER 4: Customer Testimonials

Highlights include:
• Simple to operate – consistent and
reliable processing of even small liquid
volumes via pressure-controlled hand
operation
• Ready to use – an out-of-the-box solution
with integrated silent vacuum pump and
optional rechargeable battery
• Compact design – with a small footprint
and minimal cables/tubing, ensuring it fits
onto any bench or into any safety cabinet
• Safe to use – with a hydrophobic filter
to protect against contamination, and
a fully autoclavable liquid path for easy
decontamination
• A choice of waste containers – including
single-use PP and reusable glass bottles
• Adaptable to your needs – with a large
choice of adapters, including 8 channel
adapters for 96 well plates

Why choose the VACUSIP?

The VACUSIP is a convenient and affordable tool for liquid waste removal, helping to increase
productivity for ELISA workflows. Its flexibility, ease of use and portability mean that it can be
used in a variety of other biochemical and cell-based methods, from high throughput screening
and immunofluorescence studies to cell viability and virus entry inhibition assays. Ultimately,
our VACUSIP portable aspiration system can be used by anyone, anywhere, speeding up
workflows and supporting a range of applications.
 CHAPTER 5: Conclusion 47

CHAPTER 5:
Conclusion
By now, you should have all the information you need to become an ELISA expert and run
successful experiments in your lab, whatever the application. We hope you found this eBook
helpful but, if you’d still like to learn more about this interesting topic, we have a wealth of
articles on our website. Whatever your ELISA requirements, INTEGRA Biosciences is always
available to answer your questions and provide you with the best workflow solutions.
48 CHAPTER 6: References

CHAPTER 6:
References
1.1 An introduction to the different types of ELISA tests
1. Abcam (n.d.). ELISA analysis.
https://www.abcam.com/kits/elisa-analysis

2. Abcam (n.d.). Calculating and evaluating ELISA data.

https://www.abcam.com/Protocols/calculating-and-evaluating-elisa-data

3. Elisakit (n.d.). ELISA Tips #1: Curve fitting best practices.

https://elisakit.com/index.php/elisa-tips

4. Bio-Rad Laboratories (n.d.). ELISA Basics Guide.

https://www.bio-rad-antibodies.com/static/2017/an-introduction-to-elisa/elisa-basics-
guide.pdf

5. KPL (2013). Technical Guide for ELISA.

https://issuu.com/kplinc/docs/kpl_elisa_technical_guide

6. Rockland Immunochemicals (n.d.). Tips for Biotin, Avidin, & Streptavidin.

https://www.rockland.com/resources/tips-for-biotin-avidin-and-streptavidin

7. Abcam (2018). ELISA guide – Everything you need to perform your ELISA
experiments.
https://docs.abcam.com/pdf/kits/elisa-guide.pdf

8. Interchim (n.d.). pNPP Solution (AP Substrate for ELISA).

https://www.interchim.fr/ft/6/664790.pdf

9. Engvall, E., Perlmann, P. (1971). Enzyme-linked immunosorbent assay (ELISA).

Quantitative assay of immunoglobulin G. Immunochemistry, 8(9), 871-874.
https://doi.org/10.1016/0019-2791(71)90454-x

10. Van Weemen, B. K., Schuurs, A. H. (1971). Immunoassay using antigen-enzyme

conjugates. FEBS letters, 15(3), 232-236.
https://doi.org/10.1016/0014-5793(71)80319-8

11. Alhajj, M., Farhana, A. (2022). Enzyme Linked Immunosorbent Assay.

https://www.ncbi.nlm.nih.gov/books/NBK555922
 CHAPTER 6: References 49

1.2 ELISA vs. western blot: a comparison of two common immunoassays

1. Krumm, A. (2019). Optimizing your ELISA Assays.
https://www.bmglabtech.com/en/blog/optimizing-your-elisa-assays

2. Oswald, N. (2008). Southern, northern, western (and eastern?).

https://bitesizebio.com/639/southern-northern-western-and-eastern

3. Burnette, W. N. (1981). "Western Blotting": Electrophoretic transfer of proteins

from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose
and radiographic detection with antibody and radioiodinated protein A. Analytical
biochemistry, 112(2), 195-203.
https://doi.org/10.1016/0003-2697(81)90281-5

4. Nature Education (n.d.). Western blot.

https://www.nature.com/scitable/definition/western-blot-288

5. Bio-Rad Laboratories (n.d.). Western Blotting Transfer Techniques.

https://www.bio-rad.com/en-ch/applications-technologies/western-blotting-transfer-
techniques?ID=PQEEOP70KWE7

6. Proteintech (n.d.). Choosing The Right Western Blot Detection Method.

https://www.ptglab.com/support/western-blot-protocol/choosing-the-right-western-
blot-detection-method

7. Szczesna, K. (2019). How To Choose the Right Western Blot Detection Method.
https://www.technologynetworks.com/analysis/how-to-guides/how-to-choose-the-
right-western-blot-detection-method-323714

8. Conrad, B. (2019). How do you Choose the Right Western Blot Detection Method?
https://www.enzolifesciences.com/science-center/technotes/2019/april/how-do-you-
choose-the-right-western-blot-detection-method?/

9. Martinez, L. M. (2018). Multiplex immunoassays.

https://www.sepmag.eu/blog/multiplex-immunoassays

10. Thermo Fisher Scientific (2016). Luminex™ bead-based immunoassays drive

immunoassays towards higher-content biomarker discovery.
https://www.thermofisher.com/blog/behindthebench/luminex-bead-based-
immunoassays-drive-immunoassays-towards-higher-content-biomarker-discovery/

11. BD Biosciences (n.d.). Bead-based immunoassays.

https://www.bdbiosciences.com/en-ch/learn/applications/immunoassays#Bead-
based-immunoassays
 www.integra-biosciences.com

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