ELISA

ELISA Basics Guide



Table of Contents

Chapter 1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

Chapter 2 ELISA Technology

ELISA Formats. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Direct ELISA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Indirect ELISA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Sandwich ELISA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Competition/Inhibition ELISA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Principles of Multiplex Immunoassays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Chapter 3 ELISA Detection Options

Direct Detection and Indirect Detection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Direct Detection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Indirect Detection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Chapter 4 ELISA Results

Quantitative. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Qualitative . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Semi-Quantitative. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Standard Curve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Calibration Curve Models. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Sensitivity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Chapter 5 Controls. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Chapter 6 ELISA Components and Considerations

ELISA Plates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Plate Format . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Plate Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Standard Buffers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Coating Buffers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Blocking Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

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Washing Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Antibodies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Monoclonal Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Polyclonal Antibodies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Matched Pairs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Sample Handling and Preparation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Antibody Labeling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Substrate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
TMB Core+. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
TMB Sensitive. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
TMB CORE. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
TMB SIGNAL+. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
pNPP. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Chapter 7 ELISA Optimization

Consistency between Wells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Suitability and Concentration of Antibodies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Matrix Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Coating Buffers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Blocking Buffers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Washing Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Sample Buffers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Specialist Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

Chapter 8 Protocols
Direct ELISA with Streptavidin-Biotin Detection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Indirect ELISA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Sandwich ELISA with Direct Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Sandwich ELISA with Streptavidin-Biotin Detection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Competitive ELISA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

Chapter 9 ELISA Troubleshooting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

Chapter 10 Glossary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

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 Introduction

1 Introduction

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

is distinguished from other antibody-based assays because separation of specific and
nonspecific interactions occurs via serial binding to a solid surface, usually a polystyrene
multiwell plate, and because quantitative results can be achieved. The ELISA procedure
results in an enzyme-catalyzed change in color, fluorescence, or luminescence which
correlates to the amount of analyte present in the original sample. ELISAs are quick and
simple to carry out, and since they are designed to rapidly handle a large number of
samples in parallel, they are a very popular choice for the evaluation of various research and
diagnostic targets. Figure 1 shows a typical ELISA result.

Fig. 1. Typical ELISA output. Darker wells indicate higher levels of analyte in the original sample.

ELISAs were first developed in the early 1970s as a replacement for radioimmunoassays.
They remain in wide use in their original format and in expanded formats with modifications
that allow for multiple analytes per well, highly sensitive readouts, and direct cell-based
output.

ELISAs begin with a coating step, where the first layer, either an antigen or an antibody, is
adsorbed to a well in an ELISA plate. Coating is followed by blocking and detection steps as
shown in the simple schematic diagram on page 4.

Since the assay uses surface binding for separation, several washes are repeated between
each ELISA step to remove unbound materials. During this process it is essential that
excess liquid is removed in order to prevent the dilution of the solutions added in the next
stage. For greatest consistency, specialized plate washers are used.

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Introduction 

ELISAs can be quite complex, including various intervening steps and the ability to measure
antigen concentrations in heterogeneous samples such as blood. The most complex and
varying step in the overall process is detection, where multiple layers of antibodies can be
used to amplify signal.

Coating
Antigen is adsorbed onto well in
ELISA plate in coating buffer

Remove buffer and wash plate

Blocking
A buffer containing unrelated protein is
used to block free sites in the wells

Remove buffer and wash plate

Detection
Enzyme conjugated detection antibody
binds antigen

Remove buffer and wash plate

Readout
Substrate is catalyzed by enzyme to
generate colored readout

Key
Directly conjugated
Analyte/ primary antibody
Antigen
Conjugated
Enzyme secondary antibody

Capture
antibody

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 ELISA Technology

2 ELISA Technology

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

Direct ELISA
Figure 2 illustrates the setup of direct ELISA; an antigen is immobilized in the well of an
ELISA plate. The antigen is then detected by an antibody directly conjugated to an enzyme
such as HRP.

Fig. 2. Overview of direct ELISA.

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ELISA Technology 

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

Advantages Disadvantages
Faster than other ELISA – the technique has Antigen immobilization is not specific –
fewer steps may cause higher background noise than
Less prone to error – as less reagents and indirect ELISA. Mainly because all proteins
fewer steps are required in the sample, including the target protein,
will bind to the plate
Less flexible – each target protein needs a
specific conjugated primary antibody
No signal amplification – reduces assay
sensitivity
Best for: when analyzing the immune response to an antigen.

Indirect ELISA
Figure 3 demonstrates how an indirect ELISA is set up; antigen is adsorbed to a well in an
ELISA plate. Detection is a two-step process. First, an unlabeled primary antibody binds to
the specific antigen. Second, an enzyme conjugated secondary antibody that is directed
against the host species of the primary antibody is applied.

Fig. 3. Overview of indirect ELISA.

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 ELISA Technology

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

Advantages Disadvantages
High sensitivity – more than one labeled Possibility of background noise – secondary
secondary antibody can bind the primary antibody may be cross-reactive
antibody Longer procedure than direct ELISA
Economical – fewer labeled antibodies are technique – additional incubation step for
needed secondary antibody needed
Greater flexibility – different primary
antibodies can be used with a single labeled
secondary antibody
Best for: determining total antibody concentration in samples.

Sandwich ELISA
Sandwich ELISAs require the use of matched antibody pairs (capture and detection
antibodies) as shown in Figure 4. Each antibody is therefore specific for a different and
nonoverlapping region or epitope of the antigen. It is important that matched antibody pairs
are tested specifically in sandwich ELISA to ensure that they detect different epitopes, to
achieve accurate results. The capture antibody, as its name implies, binds the antigen which
can then be detected in a direct ELISA or in an indirect ELISA configuration.

Fig. 4. Overview of direct sandwich ELISA.

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ELISA Technology 

The procedure for a sandwich ELISA firstly requires the well of an ELISA plate to be coated
with a capture antibody. The analyte or sample is then added, followed by a detection
antibody. The detection antibody can be enzyme conjugated, in which case this is referred
to as a direct sandwich ELISA. If the detection antibody used is unlabeled, a secondary
enzyme-conjugated detection antibody is required. This is known as an indirect sandwich
ELISA. The key advantage of a sandwich ELISA is its high sensitivity; it is 2-5 times more
sensitive than direct or indirect ELISAs. It also delivers high specificity, as two antibodies are
used to detect the antigen, and offers flexibility since both direct and indirect methods can
be used. The advantages bring with them a few disadvantages; if a standardized ELISA kit
or tested antibody pair is not available, antibody optimization will be required to choose a
pair of antibodies that bind to different epitopes on the antigen.

If a secondary antibody is used, it is important to ensure that the capture and detection
antibodies have different host species to avoid cross-reactivity from the secondary antibody.
The secondary antibody selected should also ideally have been tested for cross-reactivity
with IgG from the host species of the capture antibody. It is possible to use capture and
detection antibodies that have the same host species, as long as they are of different IgG
isotypes. However a secondary antibody that specifically targets the isotype of the detection
antibody would be required.

Sandwich ELISAs are particularly suited to the analysis of complex samples, since the
antigen does not need to be purified prior to the assay, yet still delivers high sensitivity and
specificity (e.g. measuring cytokine levels in an immune response).

Advantages Disadvantages
High sensitivity – 2-5 times more sensitive Antibody optimization can be difficult –
than direct or indirect ELISA cross-reactivity may occur between the
High specificity – two antibodies are capture and detection antibodies. Needs a
involved in capture and detection standardized ELISA kit or tested antibody
pair
Flexibility – both direct and indirect detection
can be used
Best for: analysis of complex samples, since the antigen does not need to be purified prior
to measurement.

Competition/Inhibition ELISA
How it works: the competition/inhibition ELISA, also known as a blocking ELISA, is perhaps
the most complex of all the ELISA techniques. However, each of the above assay types
can be adapted to a competitive format. The competitive/inhibition ELISA is predominantly
used to measure the concentration of an antigen or antibody in a sample by detecting
interference in an expected signal output. Essentially, sample antigen or antibody competes
with a reference for binding to a limited amount of labeled antibody or antigen, respectively.
The higher the sample antigen concentration, the weaker the output signal, indicating that
the signal output inversely correlates with the amount of antigen in the sample.

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 ELISA Technology

Coating
Control antigen is absorbed onto well
in ELISA plate in coating buffer

Remove liquid and wash plate

Blocking
A buffer containing unrelated protein is
used to block free sites in the wells

Remove liquid and wash plate

Prepare sample (unknown antigen) and

detection antibody mix

Sample
Add test sample mix to wells

Remove liquid and wash plate

Detection Antibody
Add enzyme conjugated secondary
detection antibody

Remove liquid and wash plate In this example the

antigen concentration in
the sample was low. The
Readout antibodies bound the
control antigen that had
Substrate is catalyzed by enzyme to
been absorbed to the
generate colored readout
plate in the coating step

Fig. 5. Competition ELISA.

An example of a competition ELISA to test for antigen based on the direct detection method
is shown in Figure 5.

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ELISA Technology 

In this example, a known antigen is used to coat a multiwell plate. Following standard
blocking and washing steps, samples containing unknown antigen are added.
Labeled detection antibody is then applied for detection using relevant substrates (e.g.
3,3’,5,5’-Tetramethylbenzidine or TMB). If there is a high concentration of antigen in the
sample, a significant reduction in signal output will be observed. In contrast, if there is very
little antigen in the sample, there will be very little reduction in the expected signal output. In
the example shown in Figure 5, there would be a reduction in signal output.

Advantages Disadvantages
Main advantage – no sample processing is Same limitations as base ELISA – as each
required and crude or impure samples can ELISA technique can be adapted to a
be used competitive format
More robust – less sensitive to sample
dilution and sample matrix effects than the
sandwich ELISA
More consistent – less variability between
duplicate samples and assays
Maximum flexibility – it can be based on
direct, indirect, or sandwich ELISA
Best for: commonly used when only one antibody is available for the antigen of interest.
It is also suitable for detecting small antigens that cannot be bound by two different
antibodies such as in the sandwich ELISA technique.

Principles of Multiplex Immunoassays

Multiplexed immunoassays are comprised of fluorescently dyed magnetic microspheres
(also called beads), each with a distinct color code or spectral address, to allow for
simultaneous detection of multiple biomolecules in a single well of a 96-well microplate.
The assays are read on dedicated flow cytometry-based instruments with two lasers and
associated optics, to identify the unique target assayed and measure the different molecules
bound to the surface of the beads. A high-speed digital signal processor efficiently manages
the fluorescence data, which is then output in a software and analyzed.

Multiplex immunoassays formats are similar to ELISA and can be a sandwich, competitive,
indirect, or direct.

Visit bio-rad.com/multipleximmunoassays to learn more on the technique.

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 ELISA Detection Options

3 ELISA Detection Options

Direct Detection and Indirect Detection

ELISAs, by definition, take advantage of an enzymatic label to produce a detectible signal
that is directly correlated to the binding of an antibody to an antigen. A few different
types of enzymes and enzyme substrates (covered in Section 6) are typically used for
ELISAs. Several methods for incorporating the enzyme step into the process can also be
applied. The final assay signal is measured with a spectrophotometric, fluorescence, or
luminescence plate reader (depending upon the substrate chosen).

One aspect of ELISA terminology that often leads to confusion is the variability in the way
the terms direct and indirect are applied. We will adhere to the use of these terms as they
apply to the detection portion of the assay as indicated below.

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

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

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 ELISA Results

4 ELISA Results

The ELISA assay yields three different types of data output:

Quantitative
ELISA data can be interpreted in comparison to a standard curve (a serial dilution of a
known, purified antigen) in order to precisely calculate the concentrations of antigen in
various samples (Figure 6).

Qualitative
ELISAs can also be used to achieve a yes or no answer indicating whether a particular
antigen is present in a sample, as compared to a blank well containing no antigen or an
unrelated control antigen. The inclusion of a single fixed level positive control can be used to
give a yes/no criteria for minimum antigen level.

Semi-quantitative
ELISAs can be used to compare the relative levels of antigen in assay samples, since the
intensity of signal will vary directly with antigen concentration.

Standard Curve
ELISA data is typically graphed with optical density vs log concentration to produce a
sigmoidal curve as shown in Figure 6. Known concentrations of antigen are used to produce
a standard curve, and then this data is used to measure the concentration of unknown
samples by comparison to the linear portion of the standard curve. This can be done directly
on the graph or with curve fitting software which is typically found on ELISA plate readers.

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ELISA Results

40,000

Fluorescence (arbitrary units)

30,000

20,000

10,000

0.1 1 10 100 1,000

Human IFNγ [ng/ml]

Fig. 6. A typical ELISA standard curve.

Calibration Curve Models

If a quantitative result is needed, the simplest way to proceed is to average the triplicate of
the standards readings and deduct the reading of the blank control sample. Next, plot the
standard curve, find the line of best fit or at least draw a point to point curve so that the
concentration of the samples can be determined. Any dilutions made need to be adjusted
for at this stage. This is generally the practical extent to which manual calculation can be
taken.

A variation is to plot the data using semi-log, log/log, log/logit and its derivatives – the 4 or 5
parameter logistic models. Using software based/automated solutions makes it possible to
consider more sophisticated graphing approaches. Using linear regression within a software
package adds several more checking possibilities; it is possible to check the R2 value
to determine overall goodness of fit. For that portion of the curve where the relationship
of concentration to readout has a linear relationship, R2 values >0.99 represent a very
good fit. Accuracy can then be further enhanced by using further standard concentrations
in that range. One aspect of the linear plot is that it compresses the data points on the
lower concentrations of the standard curve, hence making that the most accurate range
(area most likely to achieve the required R2 value). To counteract this compression a
semi-log chart can be used; here the log of the concentration value (on x-axis) is plotted
against the readout (on y-axis). This method gives an S-shaped data curve that distributes
more of the data points into the more user friendly sigmoidal pattern. The log/log (log of
concentration against log of readout) plot type manages to linearize more of the data curve.
The low to medium standard concentration range is generally linear in this model, only
the higher end of the range tends to slope off. The log/logit and its derivatives, the 4 or 5
parameter logistic models, are more sophisticated requiring more complex calculations
and estimations of max, min, EC50, and slope values. The 5 parameter model additionally
requires the asymmetry value.

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 ELISA Results

While these calibration curve models can deliver improved performance, a good starting
point would be using the log-log plot with a check on the recovery percentage (analyte
recovery from spiked samples). Alternatively, at least ‘back-fitting’ the standard curve
readout values, is frequently ‘a good enough’ approach. The simplest way to check is to
back calculate the calibration standards and check that they fall within 20% of the nominal
readout value. One caveat is not to rely on ‘good’ R2 values and find that calibration curve
model that delivers the best recovery values for the standards.

Sensitivity
ELISAs are one of the most sensitive immunoassays available. The typical detection range
for an ELISA is 0.1 to 1 fmole or 0.01 ng to 0.1 ng, with sensitivity dependent upon the
particular characteristics of the antibody-antigen interaction. In addition, some substrates
such as those yielding enhanced chemiluminescent or fluorescent signal, can be used to
improve results.

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

ELISA Basics Guide | 15

 Controls

5 Controls

The previous section covered the need for standards to obtain quantitative ELISA results. It
should be noted that standards should be acquired in sufficient amounts (or a reliable supply
needs to be found) not only for the development phase but also for the expected service life
of the assay. Additional to standards, controls should be included in ELISAs.

The most basic control is the blank sample control. Additional controls are needed to
provide a comparison to real world physiological conditions and a control mechanism for
assurance that the assay continues to provide accurate results. Hence control samples that
have had their analyte concentration validated by another method are employed. These
can be set up as positive, but also as negative controls. A control subcategory is spiked
samples; here a known amount of standard has been added to the matrix used for the
ELISA. Spiked controls can indicate assay performance by calculating percent recovery
from the ELISA readout. When recombinant proteins are used, their equivalent functionality
to the endogenous wild type versions of the protein needs to be checked.

16 | ELISA Basics Guide

 ELISA Components and Considerations

6 ELISA Components and

Considerations

ELISA Plates

Plate Format
Flat-bottomed, 96-well plates, made from polystyrene or polyvinyl chloride, are used in the
vast majority of ELISA assays. Alternatively a strip well plate can be used. This is a frame
in the size of a 96-well plate that is populated with as many 8-well or 12-well strips as the
experiment requires. Further variants are 384-well and 1536-well plates; these have the
same footprint as the traditional 96-well plates but obviously are able to process more
samples per plate. For optimum use they require automated handling and hence are near
exclusively used in high throughput screening. Some enzyme substrates, such as those
that produce fluorescent or chemiluminescent signals may require opaque plates for optimal
results.

Plate Characteristics
It is important to use plates designed for ELISAs because they are manufactured to maintain
consistency, minimizing edge effects, and providing optimal optical conditions for data
collection. It is a good idea to test plates from several manufacturers for batch-to-batch
and plate-to-plate variability, especially if an assay is being developed for commercial,
diagnostic, or quality control uses. The usual expectation is a 5% or lower variation in
common controls across 2 plates. Standard polystyrene ELISA plates fall into the low
to medium binding type, meaning that they will capture around 100–200 ng of IgG/cm2.
Modification of the polystyrene yields binding capacities of 400–500 ng of IgG/cm2, these
are commercially available as high-binding plates. Finally, antigen or antibody pre-coated
plates are commercially available although most often as part of an optimized ELISA kit with
all components included.

ELISA Basics Guide | 17


ELISA Components and Considerations

Buffers

Standard Buffers
Several different buffers are used during an ELISA: one for coating, another for blocking,
another for washing, and perhaps another for sample and antibody dilution. Buffers can
be produced in house or sourced from a variety of commercial antibody and reagent
suppliers. Basic ELISA buffer recipes can be found on our ELISA protocols page at
bio-rad-antibodies.com/elisa-protocols.

Coating Buffers
Coating is the first step in any ELISA and is the process where a suitably diluted antigen or
antibody is incubated until adsorbed to the surface of the well. Adsorption occurs passively
as the result of hydrophobic interactions between the amino acids side chains on the
antibody or antigen used for coating, and the plastic surface. It is dependent upon time,
temperature, and the pH of the coating buffer, as well as the concentration of the coating
agent.

Typical coating conditions involve adding 50-100 µl of coating buffer, containing antigen or
antibody at a concentration of 1-10 µg/ml, and incubating overnight at 4°C or for 1-3 hours
at 37°C. Alternative temperatures, times, buffers, and coating agent concentrations can be
used and should be optimized for your experimental setup. During coating, it is important to
maintain a moist environment in the well to minimize evaporation; plate sealers are generally
used to achieve this in a repeatable and constant fashion.

It is best to test a range of concentrations of coating agent since higher concentrations of

antibody/antigen may actually have a negative effect on coating, leading to oversaturation of
the wells, which can inhibit antibody binding due to steric hindrance.

Conversely, when crude antigen or antibody preparations are used for coating, it is
possible that the effective antigen/antibody concentration may be low and outcompeted by
contaminating proteins, making the specific assay signal too low to be useful. In this case a
sandwich assay is more suitable.

Coating buffers stabilize the antigen or antibody which is used to coat the ELISA multiwell
plate, maximizing adsorption to the plate and optimizing interactions with the detection
antibody. It is imperative that no other proteins are included in the coating buffer as these
will compete with the antigen for binding to the plate.

The two most common coating buffers are bicarbonate buffer at pH 9.6 or PBS; basic
buffer recipes can be found on our ELISA protocols page.

Blocking Buffers
Blocking is often necessary to prevent the nonspecific binding of detection antibodies to
the multiwell plate surface itself. There are two main types of blocking agents, proteins and
detergents. Proteins are classified as permanent blocking agents and hence added after
the capture antibody has adsorbed to the well surface. Detergents only block temporarily,

18 | ELISA Basics Guide

 ELISA Components and Considerations

meaning their blocking function disappears during washing steps. As there is no ideal
universal blocking buffer, blocking is a compromise between achieving the desired sensitivity
and reduced background. Starting with a buffer, that contains an unrelated protein or a
protein derivative that does not react with any of the antibodies being used in the detection
step, is a recommended starting point for finding an effective blocking buffer.
It is important to note that if a protein is used in the blocking reagent, that detergent should
not be included before or during the blocking step as this will prevent effective blocking by
the protein.

When a plate is fully blocked, assay sensitivity will be enhanced since additional nonspecific
signal will be reduced. The most basic blocking buffer contains 1% BSA or milk proteins
dissolved in PBS. Usually 150 μl of blocking buffer is added to the well to incubate for one
hour at 37°C in order to fully block the plate.

Washing Buffers
Since the ELISA uses surface binding for separation, wash steps are repeated between
each step to remove unbound materials. The wash steps are a critical part of the process
and entail filling the wells entirely with buffer, usually PBS, with a small concentration of a
nonionic detergent such as Tween-20.

Washing is typically repeated 3-5 times between each step in the ELISA to thoroughly
remove unbound material. Usually the wash solution is only briefly retained on the plate.
Excess wash solution must be removed in the final wash step to prevent the dilution of the
reagents added in the subsequent stage. This is accomplished most simply by tapping the
washed plate upside down on an absorbent paper to remove excess liquid or by careful
aspiration. However, plate washers can provide a quicker and more effective method for
performing these steps. It is crucial not to let the plate dry out.

Antibodies
The antibodies used in ELISA assays can be monoclonal, polyclonal, or a combination of
both. Each antibody type offers distinct advantages in the development of ELISAs, so it is
important to appreciate the differences between them and how these can be used to obtain
an advantage during ELISA development.

The interaction between antibodies and their antigens is described in three ways: specificity,
affinity, and avidity. During ELISA development, these factors influence the amount of
optimization of, e.g. antibody concentration and buffers, required (see ELISA Optimization
Section).

Specificity is an indication of whether an antibody binds solely to a unique epitope from a

single antigen in a single species, or whether it binds to similar epitopes present on several
molecules from a few different species. Cross-reactivity is the opposite of specificity.

Affinity describes the strength of binding of an antibody to a single epitope. Since binding
is reversible, affinity determines how much antigen is bound by an antibody, how quickly

ELISA Basics Guide | 19


ELISA Components and Considerations

binding occurs, and for how long the binding lasts. High affinity antibodies are the best
choice for all types of immunoassay because they rapidly produce the greatest number of
stable immune complexes and therefore provide the most sensitive detection.

Avidity is a more complex term that accounts for the total stability of the antibody-antigen
interaction. It is based upon affinity, but is also influenced by the valency of the antibody,
or total number of antigen binding sites. Thus, avidity varies with isotype and whether
the antibody is intact or fragmented. There is also a contribution made by the spatial
arrangement of the whole complex.

Monoclonal Antibodies
Monoclonal antibodies are homogeneous by definition, with specificity for a single
epitope or small region of a protein. As a result, they are less likely to interact with closely-
related proteins and are not generally expected to trigger nonspecific signals in a given
immunoassay.

Monoclonal antibodies can be used for all antibody-containing steps in all types of ELISAs.
They are commonly used in sets as matched pairs in sandwich ELISAs, but can be used
for capture or detection, in conjunction with a polyclonal antibody to enhance signal or to
provide a greater chance of capturing antigen from a complex solution.

Polyclonal Antibodies
Polyclonal antibodies are complex antibody pools which represent a collection of
specificities to various epitopes found in a single antigen. Some epitopes predominate or
there may be wide representation of the epitopes available in any given antigen. Polyclonals
can vary significantly from batch-to-batch, and must be tested and validated thoroughly.

As a result of their heterogeneity and the wide representation of epitopes present, polyclonal
antibodies can be powerful tools for the thorough detection of an antigen, often yielding
higher signal levels. It is also rare that they will fail to bind due to a single blocked antibody
binding site, antigen configuration change, or misfolding. However, polyclonal antibodies
are also more likely to share one or more epitopes with closely-related proteins, resulting in
higher nonspecific signal. One solution to reduce this problem is to use affinity purified or
cross-absorbed polyclonal antibodies.

Sometimes the detection method for an ELISA is switched from direct to indirect detection,
and thus from a monoclonal to a polyclonal, in order to increase assay sensitivity due to
higher levels of polyclonal antibody binding to the target antigen.

Polyclonal antibodies bring an additional aspect to ELISAs. They can be used as capture
and detection antibodies. Antibodies from the same polyclonal batch can both capture the
analyte and subsequently also detect it, in a biotin conjugated format.

20 | ELISA Basics Guide

 Matched Pairs

Matched Pairs
Matched pairs are the basis of many sandwich ELISAs, either in kits or for in house assay
set up. The name refers to sets of antibodies which are known to be capable of detecting
different epitopes on the same protein antigen, so they can be used together for the capture
and detection of a single antigen in a sandwich ELISA or related immunoassay. Matched
pairs can consist of two monoclonal antibodies, two polyclonal antibodies, or a combination
of both. If a monoclonal and polyclonal antibody are used together, the monoclonal antibody
should be used as the coating antibody. If it is used as the detection antibody, its epitope
may be masked by a component clone of the polyclonal antibody, resulting in reduced or
lost signal.

ELISA Basics Guide | 21

 ELISA Components and Considerations

Sample Handling and Preparation

A wide variety of samples can be tested in an ELISA and the choice of assay conditions will
depend upon the complexity of the sample and the expected amount of antigen present.

Samples are usually considered to be homogeneous or heterogeneous, depending on their

complexity. This is essentially equivalent to a purified antigen vs. a crude unpurified mixture.
In the simplest case, ELISA samples are diluted in PBS, wash buffer, or other specialty
buffers, and applied in a final volume of 100 μl. Blood presents special challenges due to
the proteins present that can disrupt the assay results. Since sera can contain antibodies,
there can also be unexpected cross-reactivity. Hence, special treatments and buffers are
sometimes needed for the dilution of blood samples in order to obtain optimal results.

It is possible to use the samples to coat the wells themselves, as in a direct ELISA, or to
capture and quantitate the antigen samples using a sandwich assay if a matched pair is
available. A complex, heterogeneous protein mixture would be less suitable for coating a
plate for direct ELISA detection unless the protein of interest is over-expressed and thus the
majority of protein present in the sample.

It is important to test all samples in duplicate or triplicate in conjunction with a known

standard to ensure the accuracy of results and for quantitation. If possible, it is better to
test several dilutions of a sample to make sure the final results fall within the linear portion
of the standard curve. This is because highly concentrated samples can underestimate
concentration, while highly diluted samples can overestimate it. This will avoid the Hook
effect, observed when very high levels of antigen are present in the sample, leading to
reduced specific binding of the antigen, that is insufficient to match analyte levels and
showing lower signal intensity than expected.

Antibody Labeling
It is generally advantageous to standardize the detection antibodies and source them from
a commercial supplier, for consistency and convenience. In certain cases, e.g. direct ELISA
it may be impossible to obtain a labeled detection antibody; in these cases the chosen
antibody needs to be labeled.

If the antibody is purified and in 10-50 mM amine-free buffer (e.g. HEPES, MES, MOPS
and phosphate) at a pH range of 6.5-8.5, it can be quickly and conveniently labeled with
Horseradish Peroxidase (HRP) using Bio-Rad’s LYNX Rapid HRP Antibody Conjugation Kit
(#LNK001P) or to alkaline phosphatase with the LYNX Rapid Alkaline Phosphatase Antibody
Conjugation Kit (#LNK0011AP).

Substrate
The final step in an ELISA is the enzyme catalyzed reaction to obtain a colored end product
that can be read in a spectrophotometer as absorbance values, representing the analyte
concentration. Bio-Rad supplies a range of substrates for HRP and AP enzyme based
detection systems.

22 | ELISA Basics Guide

ELISA Components and Considerations

Visit bio-rad.com/en-us/product/elisa-enzyme-antibody-conjugates to browse

Bio-Rad’s selection of highly specific, affinity-purified HRP- and AP- conjugated antibodies,
optimized for microtitration enzyme immunoassays.

TMB Core+
TMB Core+ (#BUF062) is a high-performance TMB (3,3´,5,5´- tetramethylbenzidine) solution,
recommended for use in ELISAs as a substrate for HRP. TMB Core+ contains TMB,
substrate buffer and hydrogen peroxide and has been optimized for increased sensitivity,
minimal background and rapid development. It produces a deep blue color read at 655 nm.
The reaction may be stopped with sulfuric acid, resulting in a yellow color read at 450 nm.

TMB Sensitive
TMB Sensitive (#BUF066) offers greater sensitivity than other TMB reagents offered by
Bio-Rad.

TMB CORE
TMB CORE (#BUF056) is a high performance substrate for HRP that contains TMB,
substrate buffer and hydrogen peroxide in a safe, ready-to-use solution.

TMB SIGNAL+
TMB SIGNAL+ (#BUF054) has been optimized to enable increased sensitivity and enhanced
stability. It also provides minimal background and rapid development times.

pNPP
pNPP (#BUF044) is a high-performance p-NitroPhenyl Phosphate (pNPP) solution
formulated to increase pNPP activity and stability. It is ready-to-use and recommended for
ELISAs as a substrate for AP for kinetic and endpoint tests.

ELISA Basics Guide | 23

 ELISA Optimization

7 ELISA Optimization

Consistency between Wells

One of the most important aspects of any assay is consistency and standardization of
conditions, as this will affect the reproducibility and accuracy of your results. In the initial
stages of assay development, it is important to test a range of parameters, usually by
completing a checkerboard dilution series to test various conditions in systematic manner.
In addition, buffers, temperature, and humidity must be kept constant between and within
experiments in order to produce standardized results.

In a typical ELISA, multiwell plates, multichannel pipets, and plate washers provide for more
consistent and faster results, as well as higher throughput. It is very important to make
sure that all pipettors used in ELISAs are properly calibrated on a regular basis, to prevent
significant variation in the results. Furthermore, it is good practice to observe the level of the
liquid in the pipet tip and the wells while following the procedure, to make sure no sample
is far out of line with the others. This is particularly important when multichannel pipets are
used, as sometimes the tips in the end rows do not always attach fully to the pipettes.

Use Bio-Rad’s ImmunoWash Microplate Washer for accurate and reproducible plate
washing. Create custom wash routines and control needle position. FDA-approved.
Visit bio-rad.com/en-us/product/model-1575-immunowash-microplate-washer, for
more information.

Suitability and Concentration of Antibodies

Using the assumption that a matched pair of antibodies is available to the analyte and
that the aim is to establish a sandwich ELISA, the initial task is to determine working
concentrations for the antibodies.

Prepare capture antibody dilutions in coating buffer at 0.5, 1, 2, and 5 μg/ml. Then follow
standard procedure for a sandwich ELISA which can be found on our ELISA protocols page
(bio-rad-antibodies.com/elisa-protocol), distributing the capture antibody as shown in
Figure 7.

At the stage where sample addition would occur, add a high and low concentration of your
analyte that reflects the expected working range. Finally include a blank, again following the
layout in Figure 7 and standard sandwich ELISA protocol until the detection stage.

At the detection stage, prepare detection antibody dilutions at 1:200, 1:1,000, 1:5,000, and
1:25,000 in buffer, adding it as detailed in Figure 7. Including a high and low concentration
of the analyte helps to determine the dynamic range. The low concentration analyte
indicates the sensitivity of the assay. The blank will indicate nonspecific binding.

24 | ELISA Basics Guide

ELISA Optimization

Capture Antibody A
Detection
Antibody
5 µg/ml 2 µg/ml 1 µg/ml 0.5 µg/ml

H L 0 H L 0 H L 0 H L 0
1:200
H L 0 H L 0 H L 0 H L 0
H L 0 H L 0 H L 0 H L 0
1:1,000
H L 0 H L 0 H L 0 H L 0
H L 0 H L 0 H L 0 H L 0
1:5,000
H L 0 H L 0 H L 0 H L 0
H L 0 H L 0 H L 0 H L 0
1:25,000
H L 0 H L 0 H L 0 H L 0

Fig. 7. Antibody concentration optimization plate layout.

Find the set with the maximum signal-to-noise ratio/largest difference between low and high
analyte concentrations; these are the antibody concentrations for further optimization.

Should the blank sample show excessive readings, above 0.2 absorbance units, these
key components need to be checked for optimization: ELISA plate type and the blocking
and washing buffers. If the background readings are appropriate but the sensitivity is
not high enough further experimentation with matrix conditions, buffers, and incubation
timings should be carried out. However, if no improvement is possible different antibody
combinations need to be generated.

Bio-Plex assay preparation is made easy, when using Bio-Rad

96-well plate template Post-It Pad.

Request your free copy at bio-rad.com/GetPlateTemplate

Matrix Effects
Matrix effects occur most often in plasma and serum samples where a series of
components can cause interference. These can be cross-reactive or nonspecific interactions
to substances in the matrix or breakdown products that develop during the sample
handling process. It is possible to reduce matrix effects by dilution of the sample; this
should be verified by analyte recovery from spiked samples. Specialized buffers for sample
dilution, coating, blocking, and washing can ameliorate matrix effects and provide constant
performance.

ELISA Basics Guide | 25

 ELISA Optimization

Coating Buffers
Specialist coating buffers are available which have been optimized for ELISA, such as
#BUF030 which has been developed to stabilize the adsorbed protein, preserving the
antigenic regions and allowing greater binding reactivity in order to enhance the specific
signal.

AbGuard Plate Stabilizer (#BUF063) simultaneously stabilizes and preserves microwell plates
coated with proteins or other biomolecules and blocks any free binding sites. It preserves
the biological activity of bound molecules and prevents degradation, denaturation and
leaching. It also blocks free binding sites without creating any interference.

Blocking Buffers
A wide range of sophisticated commercial blocking buffers are available, some of which
are BSA-based (#BUF032), but also contain preservatives to create a stable long-term
environment. Another option is to use a high performance commercial blocking buffer,
such as ELISA Ultrablock (#BUF033), which is an optimized formulation that includes small
protein fragments which thoroughly block less accessible surfaces of the multiwell dish.
Ultrablock is recommended for use on mammalian samples, particularly, human, pig, and
cow, as there is less chance of cross-reactivity. When maximal blocking strength is required,
or for most sandwich ELISAs, preparations such as ELISA SynBlock (#BUF034), an inert,
synthetic blocking formula that reduces nonspecific interference.

Washing Buffers
For greatest consistency, specialized plate washers are used to add and remove the wash
liquid. Specialized wash buffers are also available, such as #BUF031, which contains an
optimized formulation of pH stabilizers, salts, and detergents that reduce background noise
and enhance specific signal.

Sample Buffers
One type of specialty sample buffer used for sample dilutions is HISPEC assay diluent,
#BUF049. This buffer is used to dilute a range of sample types or for the dilution of
detection antibodies. It reduces cross-reactivity, nonspecific binding, and matrix effects.

#BUF037 is a specialized buffer that is recommended for use in sandwich ELISA assays
with samples containing plasma, serum, or cell culture supernatant. This buffer contains
goat serum proteins which reduce the difference between the sample matrix and the diluent
used to generate the standard curve. It also contains a proprietary chelating agent that
blocks interference from complement and thrombin in plasma and serum. It is easy-to-use
and applied by pipetting 50-100 μl directly onto the plate before adding the sample. Since
this buffer contains proteins derived from goat, it is not recommended for ELISAs that use
anti-goat secondary antibodies for detection, as this will result in nonspecific signal.

26 | ELISA Basics Guide

ELISA Optimization

Specialist Buffers
Some specialist buffers are multifunctional, allowing for simultaneous coating, stabilization,
and blocking. One such example is AbGuard Plate Stabilizer (#BUF063), which preserves
the biological activity of adsorbed molecules and prevents degradation, denaturation,
and leaching of the coating material. As a result, this buffer can shorten the length of time
required for the assay and will increase efficiency by extending the life of coated plates.

Another option is Block Ace (#BUF029), a buffer that can be used for blocking, sample
dilution, and washing for ELISAs and western blots. It also improves ELISA results by
reducing background and producing sharper standard curves.

ELISA Basics Guide | 27

 Protocols

8 Protocols

Direct ELISA with Streptavidin-Biotin Detection

Reagents

1. Coating buffer

Na2CO2, 1.5 g
NaHCO3, 2.93 g
Distilled water, 1 liter, pH to 9.6
For an alternative coating buffer use ELISA coating buffer (#BUF030)

2. Blocking buffer

Phosphate buffered saline (PBS) (#BUF036A) containing 1% w/v BSA

For an alternative blocking buffer, use either ELISA BSA Block (#BUF032), ELISA
Ultrablock (#BUF033), or ELISA SynBlock (#BUF034)

3. Wash buffer

PBS containing 0.05% v/v Tween-20

For an alternative wash buffer use ELISA wash buffer (#BUF031)

Recommended Substrates and Stop Solutions

TMB Core+ (#BUF062), for use with HRP-conjugated antibodies. Stop with 2M H2SO4.
pNPP (#BUF044), for use with alkaline phosphatase-conjugated antibodies. Stop with 1M
NaOH.

Method

1. Coat microtiter plate wells with 100 µl of the appropriate coating antigen/analyte, at a
concentration of between 1-10 µg/ml in coating buffer. Cover the plate and incubate
overnight at 4°C. Wash the plate 3 times in wash buffer.

28 | ELISA Basics Guide

Protocols

2. Add 150 µl of blocking solution to each well. Incubate for 60 minutes at 37°C. Wash
4 times in wash buffer.

3. Add 100 µl of biotin-conjugated detection antibody (appropriately diluted in wash

buffer) to each well. Incubate for 1 hour at 37°C. Wash 3 times in wash buffer.

4. Add 100 µl of enzyme-conjugated streptavidin (appropriately diluted in wash buffer)

to each well. Incubate for 60 minutes at 37°C. Wash 3 times in wash buffer.

5. Add 100 µl of the appropriate substrate solution to each well. Incubate at room
temperature (and in the dark if required) for 30 minutes, or until desired color change
is attained.

6. Read absorbance values immediately at the appropriate wavelength or add 50 µl of

“stop solution”. Gently tap plate to ensure thorough mixing. Measure absorbance
within 30 minutes.

Indirect ELISA

Reagents

1. Coating buffer

Na2CO2, 1.5 g
NaHCO3, 2.93 g
Distilled water, 1 liter, pH to 9.6
For an alternative coating buffer, use ELISA coating buffer (#BUF030)

2. Blocking buffer

Phosphate buffered saline (PBS) containing 1% w/v BSA

For an alternative blocking buffer, use either ELISA BSA Block (#BUF032), ELISA
Ultrablock (#BUF033), or ELISA SynBlock (#BUF034)

3. Wash buffer

PBS containing 0.05% v/v Tween-20

For an alternative wash buffer use ELISA wash buffer (#BUF031)

Recommended Substrates and Stop Solutions

TMB Core+ (#BUF062), for use with HRP-conjugated antibodies. Stop with 2M H2SO4.
pNPP (#BUF044), for use with alkaline phosphatase-conjugated antibodies. Stop with 1M
NaOH.

ELISA Basics Guide | 29

 Protocols

Method

1. Coat microtiter plate wells with 100 µl of the antigen/analyte solution, at a

concentration of between 1-10 µg/ml in coating buffer. Cover the plate and incubate
overnight at 4°C. Wash the plate 3 times in wash buffer.

2. Add 150 µl of blocking solution to each well. Incubate for 1 hour at 37°C. Wash 4
times in wash buffer.

3. Add 100 µl of unconjugated detection antibody (appropriately diluted in wash buffer)

to each well. Incubate for 1 hour at 37°C. Wash 3 times in wash buffer.

4. Add 100 µl enzyme-conjugated secondary antibody (appropriately diluted in wash

buffer) to each well. Incubate for 1 hour at 37°C. Wash 3 times in wash buffer.

5. Add 100 µl of the appropriate substrate solution to each well. Incubate at room
temperature (and in the dark if required) for 30 minutes, or until desired color change
is attained.

6. Read absorbance values immediately at the appropriate wavelength or add 50 µl of

“stop solution”. Gently tap plate to ensure thorough mixing. Measure absorbance
within 30 minutes.

Sandwich ELISA with Direct Detection

Reagents

1. Coating buffer

Na2CO2, 1.5 g
NaHCO3, 2.93 g
Distilled water, 1 liter, pH to 9.6
For an alternative coating buffer use ELISA coating buffer (#BUF030)

2. Blocking buffer

Phosphate buffered saline (PBS) containing 1% w/v BSA

For an alternative blocking buffer, use either ELISA BSA Block (#BUF032), ELISA
Ultrablock (#BUF033), or ELISA SynBlock (#BUF034)

3. Wash buffer

PBS containing 0.05% v/v Tween-20

For an alternative wash buffer use ELISA wash buffer (#BUF031)

30 | ELISA Basics Guide

Protocols

Recommended Substrates and Stop Solutions

TMB Core+ (#BUF062), for use with HRP-conjugated antibodies. Stop with 2M H2SO4.
pNPP (#BUF044), for use with alkaline phosphatase-conjugated antibodies. Stop with 1M
NaOH.

Method

1. Coat microtiter plate wells with 100 µl of the appropriate coating antibody, at a
concentration between 1-10 µg/ml in coating buffer. Cover the plate and incubate
overnight at 4°C. Wash the plate 3 times in wash buffer.

2. Add 150 µl of blocking solution to each well. Incubate for 1 hour at 37°C. Wash 4
times in wash buffer.

3. Dilute samples and standards in wash buffer and add 100 µl of suitably diluted
samples and standards to the relevant wells. Samples or standards should preferably
be run in triplicate. Incubate for 90 minutes at 37°C. Wash 3 times in wash buffer.

4. Add 100 µl of appropriately diluted enzyme-conjugated detection antibody to each

well. Incubate for 1 hour at 37°C. Wash 3 times in wash buffer.

5. Add 100 µl of appropriate substrate solution to each well. Incubate at room

temperature (and in the dark if required) for 30 minutes, or until desired color change
is attained.

6. Read absorbance values immediately at the appropriate wavelength or add 50 µl of

“stop solution”. Gently tap plate to ensure thorough mixing. Measure absorbance
within 30 minutes.

Sandwich ELISA with Streptavidin-Biotin Detection

Reagents
1. Coating buffer

Na2CO2, 1.5 g
NaHCO3, 2.93 g
Distilled water, 1 liter, pH to 9.6
For an alternative coating buffer use ELISA coating buffer (#BUF030)

2. Blocking buffer

Phosphate buffered saline (PBS) containing 1% w/v BSA

For an alternative blocking buffer, use either ELISA BSA Block (#BUF032), ELISA
Ultrablock (#BUF033), or ELISA SynBlock (#BUF034)

ELISA Basics Guide | 31

 Protocols

3. Wash buffer

PBS containing 0.05% v/v Tween-20

For an alternative wash buffer use ELISA wash buffer (#BUF031)

Recommended Substrates and Stop Solutions

TMB Core+ (#BUF062), for use with HRP-conjugated antibodies. Stop with 2M H2SO4.
pNPP (#BUF044), for use with alkaline phosphatase-conjugated antibodies. Stop with 1M
NaOH.

Method

1. Coat microtiter plate wells with 100 μl of the appropriate coating antibody, at a
concentration between 1-10 μg/ml in coating buffer. Cover the plate and incubate
overnight at 4°C. Wash the plate 3 times in wash buffer.

2. Add 150 μl of blocking solution to each well. Incubate for 1 hour at 37°C. Wash 4
times in wash buffer.

3. Add 100 μl of suitably diluted samples to the relevant wells. Ensure that appropriately
diluted standards are included (dilute samples and standards in wash buffer).
Samples or standards should preferably be run in triplicate. Incubate for 90 minutes
at 37°C or overnight at 4°C. Wash 3 times in wash buffer.

4. Add 100 μl of biotin-conjugated detection antibody (appropriately diluted in wash

buffer) to each well. Incubate for 1 hour at 37°C. Wash 3 times in wash buffer.

5. Add 100 μl of enzyme-conjugated streptavidin (appropriately diluted in wash buffer)

to each well. Incubate for 1 hour at 37°C. Wash 3 times in wash buffer.

6. Add 100 μl of the appropriate substrate solution to each well. Incubate at room
temperature (and in the dark if required) for 30 minutes, or until desired color change
is attained.

7. Read absorbance values immediately at the appropriate wavelength or add 50 μl of

“stop solution”. Gently tap plate to ensure thorough mixing. Measure absorbance
within 30 minutes.

Competitive ELISA

Reagents

1. Coating buffer

Na2CO2, 1.5 g
NaHCO3, 2.93 g

32 | ELISA Basics Guide

Protocols

Distilled water, 1 liter, pH to 9.6

For an alternative coating buffer use ELISA coating buffer (#BUF030)

2. Blocking buffer

Phosphate buffered saline (PBS) containing 1% w/v BSA

For an alternative blocking buffer, use either ELISA BSA Block (#BUF032), ELISA
Ultrablock (#BUF033), or ELISA SynBlock (#BUF034)

3. Wash buffer

PBS containing 0.05% v/v Tween-20

For an alternative wash buffer use ELISA wash buffer (#BUF031)

Recommended Substrates and Stop Solutions

TMB Core+ (#BUF062), for use with HRP-conjugated antibodies. Stop with 2M H2SO4.
pNPP (#BUF044), for use with alkaline phosphatase-conjugated antibodies. Stop with 1M
NaOH

Method

1. Coat microtiter plate wells with 100 µl of the antigen solution, at a concentration of
between 1-10 µg/ml in coating buffer. Cover the plate and incubate overnight at 4°C.
Wash the plate 3 times in wash buffer.

2. Add 150 µl of blocking solution to each well. Incubate for 1 hour at 37°C. Wash 4
times in wash buffer.

3. Prepare the antigen antibody mixture by adding 50 µl of antigen to 50 µl of antibody

for each well in the assay (use a range of antigen concentrations appropriately
diluted in wash buffer). Incubate for 1 hour at 37°C.

4. Add 100 µl of the mixture to each well. Incubate for 1 hour at 37°C. Wash 3 times in
wash buffer.

5. Add 100 µl enzyme-conjugated secondary antibody (appropriately diluted in wash

buffer) to each well. Incubate for 1 hour at 37°C. Wash 3 times in wash buffer.

6. Add 100 µl of the appropriate substrate solution to each well. Incubate at room
temperature (and in the dark if required) for 30 minutes, or until desired color change
is attained.

7. Read absorbance values immediately at the appropriate wavelength or add 50 µl of

“stop solution”. Gently tap plate to ensure thorough mixing. Measure absorbance
within 30 minutes.

ELISA Basics Guide | 33

 ELISA Troubleshooting

9 ELISA Troubleshooting
Problem Possible Causes Action/Solution
No signal Assay set up incorrectly or used Review protocol. Repeat assay using a
incorrect reagents positive control

Incorrect secondary antibody used Retrace steps. Repeat assay using the
correct secondary antibody

Not enough antibody used Increase concentration of the primary and/or

secondary antibody

Detection reagent too old or Use fresh detection reagents

contaminated

Antigen not coated properly Try longer coating times, different coating
buffers, or avidin plates with biotinylated
antigen

Plate reader has the wrong settings Check plate reader for wavelength, filters,
gain etc

Antibody stored at 4°C for several Use a fresh aliquot of antibody that has been
weeks or subjected to repeated stored at -20°C or below
freeze/thaw cycles
Weak signal Insufficient amount of antigen was Use more antigen for coating or very coating
coated to microtiter plate buffer

Not enough antibody used Increase concentration of the primary and/

or secondary antibody. Optimize antibody
concentrations for your assay

Detection reagent too old, Use fresh detection reagents at the correct
contaminated, or used at the wrong pH
pH
Detection reagent too dilute Use a higher concentration of detection
reagent

Plate reader has the wrong settings Check plate reader for wavelength, filters,
gain etc

Incubation temperature too low Optimize the incubation temperature for

your assay. Reagents should be at room
temperature before beginning the assay

34 | ELISA Basics Guide

ELISA Troubleshooting

High Too much antibody used Reduce the concentration of the primary and/
background or secondary antibody. Optimize antibody
signal concentrations for your assay
Nonspecific antibody binding Use a suitable blocking buffer or use an
affinity-purified antibody

Too much detection reagent used Repeat assay with a higher dilution of
detection reagent

Too few washing cycles Increase the number of washing cycles

Contaminated blocking agent Use fresh blocking agent

Wrong concentration of blocking Check the concentration of blocking agent in

agent the recommended protocol

Presence of blocking buffer Wash off blocking buffer before adding

interferes with antibody binding antibody

Reaction not stopped Use stop solution to prevent overdevelopment

Plate left too long before reading Start taking measurements shortly after the
addition of detection reagent

Wrong settings on the plate reader Check settings and adjust as needed

Insufficient amount of Tween in the Optimize the concentration of Tween in

buffers the blocking and wash buffers for your
experiment using 0.05% Tween as a starting
point
Incubation with substrate carried out Perform substrate incubation in the dark
in the light
Incubation temperature too high Optimize the incubation temperature for your
assay
Plates stacked during incubations Avoid stacking plates
leading to uneven temperature
distribution
Pipetting errors Calibrate pipets so that they dispense the
correct volumes

Reagents were not mixed properly Before pipetting solutions into wells, make
sure all reagents and samples have been
thoroughly mixed

Inconsistent washing of wells Take precautions to reduce variability of

washes, ensuring all liquid is removed from
each well of the plate after each wash step.
Consider using a plate washer to make wash
steps more efficient

ELISA Basics Guide | 35

 ELISA Troubleshooting

Uneven evaporation of solution from Always incubate with a lid on the plate
wells during incubation

Interference caused by dirt on the Clean the plate carefully and reread
bottom of the plate

Slow color Incubation temperature is wrong Ensure plates and reagents are kept at room
development temperature

Contaminated solutions Make fresh solutions

Detection reagent too old, Use fresh detection reagents at the correct
contaminated or used at the wrong pH
pH

Technical Support
For further help and expert advice, contact our scientist staffed technical support
department.

bio-rad-antibodies.com/contact

36 | ELISA Basics Guide

 Glossary

10 Glossary
Adsorption - the passive attachment of a liquid to a solid surface creating a thin film.

Antigen - substance, protein, chemical compound, or virus that is able to elicit an immune
response against which antibodies are raised.

AP (Alkaline Phosphatase) - phosphatase enzyme that removes a phosphate group from a

substrate. In ELISAs the substrate is p-NitroPhenyl Phosphate (pNPP).

Assay diluent (see also buffer) - buffer solution in which the sample to be analyzed is
diluted in.

Assay sensitivity - a measure of the ability of the ELISA to distinguish between small
changes in concentration.

Background - the signal readout attributable to all reagents excluding the analyte. Should
be low.

Blocking - application of reagents, generally buffers, to lower background by binding to the

potential nonspecific binding sites of antibodies and enzyme conjugates.

Buffer - solutions containing compounds, generally proteins, to reduce the nonspecific

binding of antibodies; used in blocking to reduce background.

Cross-reactivity - an antibody binding to a target that is very similar to but not the intended
target analyte, i.e. a closely related molecule with structural similarities to the target antigen.

Detection limit - the smallest quantity of analyte that can be reliably measured by the ELISA
assay; it is often set to 2 standard deviations (2 SD) above background level.

Dilution - addition of a buffer to a protein solution to make it less concentrated, used in

optimizing antibody concentration but also applied to samples to obtain readings within the
dynamic range of the assay.

Dynamic range - range in the ELISA over which the absorbance reading increases in a
linear mode and the analyte can be reliably measured.

Edge effect - the result of inconsistencies in the production of ELISA multiwell plates or
when assay conditions, such as stacking plates, cause the outer wells to behave differently.
As a result, unexpected values can appear in the outer wells which may be out of line with
neighboring well. This can best be controlled for by using duplicates or triplicates for all
samples, and noting any large variations in the results for a given sample.

Heterophilic interference - arises from antibodies found in the sample analyzed by the
ELISA; it is binding by these antibodies to the detection antibody used in the assay. The
best known example of heterophilic interference is HAMA (human anti-mouse antibody)
found in some patients where it interferes with the accurate analyte determination, showing
false positive readings.

ELISA Basics Guide | 37

Glossary

HRP (Horseradish Peroxidase) - an enzyme that breaks down hydrogen peroxide to water
– a peroxidase. Chromogenic substrates such as TMB serve as indicators of that enzyme
activity.

Hook effect - caused by very high levels of antigen in the sample. As a result specific
binding of the antigen by the antibody is insufficient to match analyte levels and signal is
lower than expected. The best way to avoid this issue is to test several dilutions of each
sample.

Immunoassay - any type of assay that uses antibodies to measure the concentration of an
analyte in a sample.

Interference - effects on the immunoassay that interfere with the accurate measurement of
the analyte (e.g. matrix effect, heterophilic interference).

Matrix effect - the effect compounds in the sample have on the measurement of the
analyte.

pNPP (para-Nitrophenylphosphate) - colorimetric substrate for alkaline phosphatase, it

precipitates as a yellow substance.

Protein stabilizers - reagents that promote maintenance of the native structure of proteins
during adsorbtion to the assay surface.

Substrate - compound such as pNPP and TMB that is used to measure the analyte in an
immunoassay.

TMB - a colorimetric substrate for horseradish peroxidase, turning blue upon completion of
the enzymatic reaction.

38 | ELISA Basics Guide

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39 | ELISA Basics Guide ELISA Basics Guide | 39

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