Objectives:

1.
2.
3.
4.

Screening antisera or hybridoma supernatants for specific antibodies.

To detect the presence of an antibody in a sample and to use it as a diagnostic tool in
medicine.
To detect potential food allergens.
To be used in toxicology as a rapid presumptive screen for certain classes of drugs.

Principle:
The Indirect-ELISA utilizes an unlabeled primary antibody in conjunction with a labele secondary
antibody. Since the labeled secondary antibody is directed against all antibodies of a given species, it
can be used with a wide variety of primary antibodies.

Different Stages Of Indirect ELISA:

Coating ELISA Plates :
Coating is achieved through passive adsorption of the antigen to the assay microplate. This process
occurs though hydrophobic interactions between the micro titer plate and non-polar protein residues.
Although individual proteins may require specific conditions or pretreatment for optimal binding, the
most common method for coating plates involves adding a 2-10 g/ml solution of protein dissolved in
an alkaline buffer such as phosphate-buffered saline (ph 7.4) or carbonate-bicarbonate buffer (ph
9.4).The buffer contains no other proteins that might compete with the target antigen for attachment
to the micro titer plate. Antigens ,which are protein in nature will attach passively to the micro titer
well plate during incubation in incubator at 37 0C.

Washing step:

After incubation any excess antigen is removed by washing steps by flooding and emptying the wells
with neutral phosphate buffered saline ( PBS ) or deionized water. Washing steps are necessary to
remove nonbound reagents and decrease background, thereby increasing the signal: noise ratio.
Insufficient washing will allow high background, while excessive washing might result in decreased
sensitivity caused by elution of the antigen from the well.

Add blocking buffer :

The binding capacity of microplate wells is typically higher than the amount of protein coated in each
well and the residual binding capacity of the plate is blocked in this step. The ideal blocking buffer will
bind to all potential sites of nonspecific interaction, eliminating background altogether, without altering
or obscuring the epitope for antibody binding. The blocking buffer is effective if it improves the
sensitivity of an assay by reducing background signal and improving the signal-to-noise ratio. Tween
20 (0.05%) by itself is more effective at blocking than any protein tested, but because the
combination of protein and Tween 20 may be more effective than Tween 20 alone in some cases,
bovine serum albumin (BSA; 0.25%) is included in the blocking buffer. Coated plates can be used
immediately or dried and stored at 4C for later use, depending on the stability of the coated protein.

Add primary antibody :

This step involves the addition of detecting antibodies
(test sample) being directed against the
coated antigen. The antibody is usually diluted in blocking buffer to prevent non specific attachment of
protein in the antiserum on the solid phase. The antibody present in the serum which are specific to
the antigen, binds the coated antigen on incubation.

Washing step :
Excess antibody or unbound antibodies are removed by washing step and is followed by addition of
blocking solution.

Add secondary antibody (antibody enzyme conjugate):

The next step is the addition of secondary antibody, diluted in blocking buffer directed against the
primary antibody. Followed by incubation to the achieve the binding of the enzyme-conjugated
secondary antibody. The choice of antibody enzyme conjugate is determined by the goals of the
assay. If it is necessary to detect all antibodies that bind to antigen, conjugates prepared with
antibodies specific for Ig and light chains should be used. Alternatively, protein A or protein G
enzyme conjugates may be preferable when screening monoclonal antibodies. Such antibodies are
produced against immunoglobulins (Igs) of species in which the detecting antibodies are produced
and are termed anti-species conjugates. Thus, if detecting antibodies are produced in rabbits, the
enzyme-labeled antibodies would have to be anti-rabbit Igs in nature. This allow greater flexibility in
use of anti-species conjugates in that different specificities of conjugate can be used to detect
particular Igs binding in the assay. For example, the anti-species conjugate could be anti- IgM, igg1,
igg2 and so on. The enzyme can be linked to a protein such as streptavidin if the primary antibody
is biotin labeled. The most commonly used enzyme labels horseradish peroxidase (HRP) and alkaline
phosphatase (AP). Other enzymes have been used as well, but they have not gained widespread

acceptance because of limited substrate options. These include -galactosidase, acetylcholinesterase

and catalase.

Washing step:
Unbound antibody enzyme conjugate is washed away after incubation phase.

Adding substrate :
Substrates are critical for the detection and visualization steps of an ELISA. The step involves the
addition of suitable substrate solution for the particular enzyme conjugated to the antibodies. The
objective is to allow development of color reaction through enzyme catalysis. A large selection of
substrates is available for performing the ELISA with an HRP or AP conjugate. TMB (3, 3, 5, 5tetramethyl benzidine) is the most commonly used substrate for the enzyme horseradish peroxidase
(HRP).The substrates of alkaline phosphatase (AP) , 4-methylumbelliferyl phosphate (MUP) and
PNPP (p-Nitro phenyl-phosphate) are nontoxic and relatively stable. Solutions of p-nitro-phenyl
phosphate (NPP) are stable for months at 4C, while solutions of 4-methylumbelliferyl phosphate
(MUP) can be kept for months at room temperature without any significant spontaneous
hydrolysis. The biggest disadvantage if NPP is used as a substrate is that, the yellow color of the
nitro phenyl product is relatively difficult to detect visually. Using the substrate MUP instead of
NPP can greatly enhance the sensitivity of the assay. The fluorogenic system using MUP is 10 to
100 times faster than the chromogenic system using NPP, and appears to be as sensitive as an
enhanced chromogenic assay in which alkaline phosphatase generates NAD+ from NADP. The
disadvantage of using fluorogenic substrates is that they require a microplate fluorometer costing
twice as much as a high quality micro titer plate spectrophotometer.
The choice of substrate depends upon the required assay sensitivity and the instrumentation available
for signal-detection (spectrophotometer, fluorometer or luminometer).

Stop solution :
The reaction is allowed to progress for a defined period after which the reaction is stopped by altering
the ph of the system. Stop Solution is a used to terminate the enzyme substrate reaction for ELISA
applications after attaining the desired color intensity which is an indication of analyte level. For e.g.
The TMB substrate reacts with immobilized horseradish peroxidase (HRP) conjugated secondary
antibodies to produce a blue solution. Reaction may be stopped by 0.2 M sulphuric acid which offers a
yellow end product read at 450 nm. AP stop solution (0.5M NaOH) does not change the yellow color or
the absorbance of the chromogen, and so the absorbance is read at 405 nm to 420 nm.

Quantification:
Specially designed spectrophotometers are available which reads through the micro titer wells either
singly or in rows. Several ELISA plate readers are available, with increasing levels of sophistication.
Some of these provide a measurement of optical density while some tabulate data and apply statistical
analysis. Compatibility with a small computer, and availability of a suitable program to process the
results and transform the optical density readings into concentrations of protein are important
additional things to look for when selecting an instrument. Most ELISA readers can be set to measure

the absorbance of the colors produced by the action of antibody- conjugated enzymes on their
respective substrates the microplate reader works by shining a particular type of light at each of the
samples in the micro well plate. Common detection modes for microplate assays are absorbance,
fluorescence intensity, luminescence, time-resolved fluorescence and fluorescence polarization. A light
source illuminates the sample using a specific wavelength (selected by an optical filter, or a
monochromator), and a light detector located on the other side of the well measures how much of the
initial (100%) light is transmitted through the sample, the amount of transmitted light will typically be
related to the concentration of the molecule of interest. This is called absorption detection. The range
of application of fluorescence intensity detection is much broader than when using absorbance
detection, but instrumentation is usually more expensive. Microplate readers feed the absorbance or
fluorescence measures into a computer program that analyses the particular information being
collected.

Assay optimization :
Serial dilution titration analyses are performed to determine optimal concentrations of reagents to be
used in Elisas. All three reactants in ELISA, a solid-phase coating reagent, a secondary reagent that
binds the primary reagent, and an enzyme-conjugated tertiary developing reagent that binds to the
secondary reagent are serially diluted and analyzed by a criss-cross matrix analysis. Once the optimal
concentrations of reagents to be used under particular assay conditions are determined, these
variables are kept constant from experiment to experiment.

Assay validation :
ELISA kits that are commercially available which are used for diagnostic purposes in the detection of
specific antigen or antibody in the serum sample. For e.g.,ovarian cancer antigen (CA-125) enzyme
immunoassay test kit is intended for use as a monitoring and screening test for serum CA-125 level.
An elevated serum CA-125 level can indicate ovarian cancer and suggests the need for further clinical
management, also determining serum CA-125 concentration may be useful in monitoring patients
with diagnosed ovarian cancer.
Materials provided with the test kits includes antibody coated micro titer plate with 96 wells, enzyme
conjugate reagent, substrate solution, stop solution, wash buffer concentrate, sample diluents,
reference standards, positive and negative controls.
ELISA results are reported as a number and the most controversial aspect of this test is determining
the "cut-off" point between a positive and negative result. A cut-off point may be determined by
comparing the ELISA plate reader value with a known reference standard. If an ELISA test is used
for drug screening at workplace, a cut-off concentration, 50 ng/ml, for example, is established, and a
sample will be prepared which contains the standard concentration of analyte. Unknowns that
generate a signal that is stronger than the known sample are "positive" and those that generate
weaker signal are "negative."

Blocking Buffers for Western Blotting and

ELISA
Protein Biology Resource Library

Pierce Protein Methods

Before using antibodies to detect proteins that have been dotted or transferred to
a membrane, the remaining binding surface must be blocked to prevent the
nonspecific binding of the antibodies. Otherwise, the antibodies or other detection
reagents will bind to any remaining sites that initially served to immobilize the
proteins of interest. In principle, any protein that does not have binding affinity for
the target or probe components in the assay can be used for blocking. In practice,
however, certain proteins perform better than others because they bind to the
membrane or other immobilization surface more consistently or because they
somehow stabilize the function of other system components. In fact, no single
protein or mixture of proteins works best for all Western blot experiments, and
empirical testing is necessary to obtain the best possible results for a given
combination of specific antibodies, membrane type and substrate system.

Learn more

Overview of Western Blotting

Watch this video on blocking Western blot membranes

Purpose and Function of Blocking Steps

The membrane supports used in Western blotting have a high affinity for proteins. Therefore, after the
transfer of the proteins from the gel, it is important to block the remaining surface of the membrane to
prevent nonspecific binding of the detection antibodies during subsequent steps. A variety of blocking
buffers ranging from milk or normal serum to highly purified proteins have been used to block free sites on
a membrane. The blocking buffer should improve the sensitivity of the assay by reducing background
interference and improving the signal to noise ratio. The ideal blocking buffer will bind to all potential sites
of nonspecific interaction, eliminating background altogether without altering or obscuring the epitope for
antibody binding.
The proper choice of blocker for a given blot depends on the antigen itself and on the type of detection
label used. For example, in applications where alkaline phosphatase conjugates are used, a blocking
buffer in TBS should be selected because PBS interferes with alkaline phosphatase. For true optimization
of the blocking step for a particular immunoassay, empirical testing is essential. Many factors, including
various protein:protein interactions unique to a given set of immunoassay reagents, can influence
nonspecific binding. The most important parameter when selecting a blocker is the signal:noise ratio,
measured as the signal obtained with a sample containing the target analyte, as compared to that
obtained with a sample without the target analyte. Using inadequate amounts of blocker will result in
excessive background staining and a reduced signal:noise ratio. Using excessive concentrations of
blocker may mask antibody:antigen interactions or inhibit the marker enzyme, again causing a reduction
of the signal:noise ratio. When developing any new immunoassay, it is important to test several different
blockers for the highest signal:noise ratio in the assay. No single blocking agent is ideal for every
occasion since each antibody-antigen pair has unique characteristics.

Overview of ELISA
Protein Biology Resource Library

Pierce Protein Methods

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

designed for detecting and quantifying substances such as peptides, proteins,
antibodies and hormones. Other names, such as enzyme immunoassay (EIA), are
also used to describe the same technology. In an ELISA, an antigen must be
immobilized to a solid surface and then complexed with an antibody that is linked
to an enzyme. Detection is accomplished by assessing the conjugated enzyme
activity via incubation with a substrate to produce a measureable product. The
most crucial element of the detection strategy is a highly specific antibody-antigen
interaction.

Page contents

Introduction

ELISA Formats (direct, sandwich, etc.)

Direct vs. Indirect Detection ELISA Strategies

Other ELISA Formats (competitive, ELISPOT, etc.)

Complete, Ready-to-Use ELISA Kits

Selecting and Coating ELISA Plates

Pre-Coated ELISA Plates

Antibodies and Probes for ELISA

Blocking Buffers and Wash Buffers

Detection Strategies for ELISA

View products

Browse ELISA Products

Find ELISA Kits by Target

Introduction
ELISAs are typically performed in 96-well (or 384-well) polystyrene plates, which will passively bind
antibodies and proteins. It is this binding and immobilization of reagents that makes ELISAs so easy to
design and perform. Having the reactants of the ELISA immobilized to the microplate surface makes it
easy to separate bound from nonbound material during the assay. This ability to wash away
nonspecifically bound materials makes the ELISA a powerful tool for measuring specific analytes within a
crude preparation.
A detection enzyme or other tag can be linked directly to the primary antibody or introduced through a
secondary antibody that recognizes the primary antibody. It also can be linked to a protein such as
streptavidin if the primary antibody is biotin labeled. The most commonly used enzyme labels are
horseradish peroxidase (HRP) and alkaline phosphatase (AP). Other enzymes have been used as well,
but they have not gained widespread acceptance because of limited substrate options. These include galactosidase, acetylcholinesterase and catalase. A large selection of substrates is available for
performing the ELISA with an HRP or AP conjugate. The choice of substrate depends upon the required
assay sensitivity and the instrumentation available for signal-detection (spectrophotometer, fluorometer or
luminometer).

ELISA Formats
ELISAs can be performed with a number of modifications to the basic procedure. The key step,
immobilization of the antigen of interest, can be accomplished by direct adsorption to the assay plate or
indirectly via a capture antibody that has been attached to the plate. The antigen is then detected either
directly (labeled primary antibody) or indirectly (labeled secondary antibody). The most powerful ELISA
assay format is the sandwich assay. This type of capture assay is called a sandwich assay because the

analyte to be measured is bound between two primary antibodies the capture antibody and the
detection antibody. The sandwich format is used because it is sensitive and robust.

Diagram of common ELISA formats (direct vs. sandwich assays)

Common ELISA formats. In the assay, the antigen of interest is immobilized by direct adsorption to the assay plate or by first
attaching a capture antibody to the plate surface. Detection of the antigen can then be performed using an enzymeconjugated primary antibody (direct detection) or a matched set of unlabeled primary and conjugated secondary antibodies
(indirect detection).

Direct vs. Indirect Detection ELISA Strategies

Among the standard assay formats discussed and illustrated above, where differences in
both capture and detection were the concern, it is important to differentiate between the particular
strategies that exist specifically for the detection step. However an antigen is captured to the plate (by
direct adsorption to the surface or through a pre-coated "capture" antibody, as in a sandwich ELISA), it is
the detection step (as either direct or indirect detection) that largely determines the sensitivity of an
ELISA.

Watch this video about ELISA detection and signal-amplification strategies

2:31

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English
The direct detection method uses a labeled primary antibody that reacts directly with the antigen. Direct
detection can be performed with antigen that is directly immobilized on the assay plate or with the capture
assay format. Direct detection is not widely used in ELISA but is quite common for immunohistochemical
staining of tissues and cells.
The indirect detection method uses a labeled secondary antibody for detection and is the most popular
format for ELISA. The secondary antibody has specificity for the primary antibody. In a sandwich ELISA, it
is critical that the secondary antibody be specific for the detection primary antibody only (and not the
capture antibody) or the assay will not be specific for the antigen. Generally, this is achieved by using
capture and primary antibodies from different host species (e.g., mouse IgG and rabbit IgG, respectively).
For sandwich assays, it is beneficial to use secondary antibodies that have been cross-adsorbed to
remove any antibodies that have affinity for the capture antibody.

Comparison of Direct and Indirect ELISA Detection Methods

Direct ELISA Detection

Advantages

Disadvantage
s

Quick because only one antibody and fewer steps are used.

Cross-reactivity of secondary antibody is eliminated.

Immunoreactivity of the primary antibody might be adversely affected by labeling wit

enzymes or tags.

Labeling primary antibodies for each specific ELISA system is time-consuming and
expensive.

No flexibility in choice of primary antibody label from one experiment to another.

Minimal signal amplification.

Indirect ELISA Detection

Advantages

A wide variety of labeled secondary antibodies are available commercially.

Versatile because many primary antibodies can be made in one species and the same
labeled secondary antibody can be used for detection.

Maximum immunoreactivity of the primary antibody is retained because it is not

labeled.

Sensitivity is increased because each primary antibody contains several epitopes that
can be bound by the labeled secondary antibody, allowing for signal amplification.

Different visualization markers can be used with the same primary antibody.

Indirect ELISA Detection

Disadvantage
s

Cross-reactivity might occur with the secondary antibody, resulting in nonspecific

signal.
An extra incubation step is required in the procedure.

Fluorescent tags and other alternatives to enzyme-based detection can be used for plate-based assays.
Despite not involving reporter-enzymes, these methods are also generally referred to as a type of ELISA.
Likewise, wherever detectable probes and specific protein binding interactions can be used in a platebased method, these assays are often called ELISAs despite not involving antibodies.

Other ELISA Formats

Besides the standard direct and sandwich formats described above, several other styles of ELISA exist:
Competitive ELISA is a strategy that is commonly used when the antigen is small and has only one
epitope, or antibody binding site. One variation of this method consists of labeling purified antigen instead
of the antibody. Unlabeled antigen from samples and the labeled antigen compete for binding to the
capture antibody. A decrease in signal from purified antigen indicates the presence of the antigen in
samples when compared to assay wells with labeled antigen alone.

Watch this video about competitive ELISA methods

ELISPOT (enzyme-linked immunospot assay) refers to ELISA-like capture and measurement of proteins
secreted by cells that are plated in PVDF-membrane-backed microplate wells. It is a "sandwich" assay in
which the proteins are captured locally as they are secreted by the plated cells, and detection is with a
precipitating substrate. ELISPOT is like a Western blot in that the result is spots on a membrane surface.
In-cell ELISA is performed with cells that are plated and cultured overnight in standard microplates. After
the cultured cells are fixed, permeabilized and blocked, target proteins are detected with antibodies. This
is an indirect assay, not a sandwich assay. The secondary antibodies are either fluorescent (for direct
measurement by a fluorescent plate reader or microscope) or enzyme-conjugated (for detection with a
soluble substrate using a plate reader).

ELISA is nearly always performed using 96-well or 384-well polystyrene plates and samples in solution
(i.e., biological fluids, culture media or cell lysates). This is the platform discussed in the remainder of this
article.

Complete, Ready-to-Use ELISA Kits

In addition to the individual components and general principles of ELISA discussed in the remainder of
this article, ready-to-use sandwich ELISA kits are commercially available for detection of hundreds of
specific cytokines, neurobiology analytes and phosphorylated proteins that are common targets of
research interest.
For many targets, two kit types are available:
ELISA Kits contain pre-coated antibody-plates, detection antibodies, buffers, diluents, standards,

and substrates.
Antibody Pair Kits contain only matched antibodies and standard (no plates or detection

reagents).
Learn more

ELISA Development and Optimization

Performing and Evaluating Spike and Recovery and Linearity of Dilution for ELISA

Factors Affecting Signal Generation in ELISA

ELISA Protocols

View products

Search All ELISA Kits by Target

Ready-to-Use ELISA Kits

Antibody Pair Kits

Guide to ELISA Kit Package Formats

ELISA Reagents and Accessories

Assay Development Technical Handbook

The revised Assay Development Technical Handbook is an essential resource for any laboratory using
enzyme-linked immunosorbent assay (ELISA) and related plate-based assay methods. The handbook
describes the essential techniques and tools for designing and optimizing ELISA Assays. Featured
products include coated microplates, standards, blockers, buffers, probe-labeling reagents, secondary
antibodies and detection substrates.
Contents include: Introduction to ELISA, Selecting an ELISA Plate, Thermo Scientific Pierce
Microplates, Thermo Scientific Pierce Coated Microplates, Blocking and Washing, Blocking and Washing
Reagents, Detection Probes, Antibody Labeling, Choosing a Substrate, Bulk and Custom Offerings, and
Recommended Reading.
Download the Assay Development Technical Handbook

Selecting and Coating ELISA Plates

When developing a new ELISA for a specific antigen, the first step is to optimize the plate-coating
conditions for the antigen or capture antibody. Begin by choosing an assay microplate (not tissue culture
treated plates) with a minimum protein-binding capacity of 400 ng/cm. It is also important that the CV
value (coefficient of variation) of the protein binding be low (<5% is preferred) so that there is limited
deviation in values that should be identical in the assay results between wells and plates. The choice of
plate color depends upon the signal being detected. Clear polystyrene flat bottom plates are used for
colorimetric signals while black or white opaque plates are used for fluorescent and chemiluminescent

signals. Visually inspect plates before use as imperfections or scratches in the plastic will cause
aberrations when acquiring data from the developed assay.
Plate coating is achieved through passive adsorption of the protein to the plastic of the assay microplate.
This process occurs though hydrophobic interactions between the plastic and non-polar protein residues.
Although individual proteins may require specific conditions or pretreatment for optimal binding, the most
common method for coating plates involves adding a 2-10 g/ml solution of protein dissolved in an
alkaline buffer such as phosphate-buffered saline (pH 7.4) or carbonate-bicarbonate buffer (pH 9.4). The
plate is left to incubate for several hours to overnight at 4-37C. Typically, after removing the coating
solution, blocking buffer is added to ensure that all remaining available binding surfaces of the plastic well
are covered (see subsequent discussion). Coated plates can be used immediately or dried and stored at
4C for later use, depending on the stability of the coated protein.
It is important to note that optimal coating conditions can vary with each protein. With the exception of
competition ELISAs, the plates are coated with more capture protein than can actually be bound during
the assay in order to facilitate the largest working range of detection possible. Some proteins, especially
antibodies, are best coated on plates at a concentration lower than the maximum binding capacity in order
to prevent nonspecific binding in later steps by a phenomenon called "hooking". Hooking results from
proteins getting trapped between the coating proteins which prevents effective washing and removal of
non bound proteins. When hooking nonspecifically traps detection primary and secondary antibodies,
high background signal results lowering the signal to noise ratio and thus sensitivity of an assay.
View products

ELISA Labware and Accessories

BupH Carbonate-Bicarbonate Buffer Packs (pH 9.4)

Pre-coated ELISA Plates

For antibodies and proteins, coating plates by passive adsorption usually works well. However, problems
can arise from passive adsorption, including improper orientation, denaturation, poor immobilization
efficiency and binding of contaminants along with the target molecule. Antibodies can be attached to a
microplate through the Fc region using Protein A, G, or A/G coated plates, which orients them properly
and preserves their antigen binding capability. Fusion proteins can be attached to a microplate in the
proper orientation using glutathione, metal-chelate, or capture-antibody coated plates. Peptides and other
small molecules, which typically do not bind effectively by passive adsorption, can be biotinylated and

attached with high efficiency to a streptavidin or NeutrAvidin Protein coated plate. Biotinylated antibodies
also can be immobilized on plates precoated with biotin-binding proteins. Using pre-coated plates in this
manner physically separates the antigen or capture antibody from the surface of the plate as protection
from its denaturing effects.
View products

Browse all Coated Plates

Antibodies and Probes for ELISA

Either monoclonal or polyclonal antibodies can be used as the capture and detection antibodies in
sandwich ELISA systems. Monoclonals have an inherent monospecificity toward a single epitope that
allows fine detection and quantitation of small differences in antigen. A polyclonal is often used as the
capture antibody to pull down as much of the antigen as possible. Then a monoclonal is used as the
detecting antibody in the sandwich assay to provide improved specificity.
An important consideration in designing a sandwich ELISA is that the capture and detection antibodies
must recognize two different non-overlapping epitopes. When the antigen binds to the capture antibody,
the epitope recognized by the detection antibody must not be obscured or altered. Capture and detection
antibodies that do not interfere with one another and can bind simultaneously are called "matched pairs"
and are suitable for developing a sandwich ELISA. Many primary antibody suppliers provide information
about epitopes and indicate pairs of antibodies that have been validated in ELISA as matched pairs.
Another design consideration in choosing antibodies is cost. A polyclonal antibody is generally less
expensive (~5 fold) to produce than a monoclonal. The specificity gained by using monoclonals for both
the capture and detecting antibody must be weighed against the cost and time required for producing two
monoclonal antibodies. Preparing a self-sandwich ELISA assay, where the same antibody is used for
the capture and detection, can limit the dynamic range and sensitivity of the final ELISA.
Learn more

Overview of Detection Probes

View products

Primary Antibodies

Secondary Antibodies and Conjugates

Secondary Antibody Selection Guide

Blocking Buffers and Wash Buffers

The binding capacity of microplate wells is typically higher than the amount of protein coated in each well.
The remaining surface area must be blocked to prevent antibodies or other proteins from adsorbing to the
plate during subsequent steps. A blocking buffer is a solution of irrelevant protein, mixture of proteins, or
other compound that passively adsorbs to all remaining binding surfaces of the plate. The blocking buffer
is effective if it improves the sensitivity of an assay by reducing background signal and improving the
signal-to-noise ratio. The ideal blocking buffer will bind to all potential sites of nonspecific interaction,
eliminating background altogether, without altering or obscuring the epitope for antibody binding.
When developing any new ELISA, it is important to test several different blockers for the highest
signal:noise ratio in the assay. Many factors can influence nonspecific binding, including various
protein:protein interactions unique to the samples and antibodies involved. The most important parameter
when selecting a blocker is the signal:noise ratio, which is measured as the signal obtained with a sample
containing the target analyte as compared to that obtained with a sample without the target analyte. Using
inadequate amounts of blocker will result in excessive background and a reduced signal:noise ratio. Using
excessive concentrations of blocker may mask antibody-antigen interactions or inhibit the enzyme, again
causing a reduction of the signal:noise ratio. No single blocking agent is ideal for every occasion and
empirical testing is essential for true optimization of the blocking step.
In addition to blocking, it is essential to perform thorough washes between each step of the ELISA.
Washing steps are necessary to remove nonbound reagents and decrease background, thereby
increasing the signal:noise ratio. Insufficient washing will allow high background, while excessive washing
might result in decreased sensitivity caused by elution of the antibody and/or antigen from the well.
Washing is performed in a physiologic buffer such as Tris-buffered saline (TBS) or phosphate-buffered
saline (PBS) without any additives. Usually, a detergent such as 0.05% Tween-20 is added to the buffer to
help remove nonspecifically bound material. Another common technique is to use a dilute solution of the
blocking buffer along with some added detergent. Including the blocking agent and adding a detergent in
wash buffers helps to minimize background in the assay. For best results, use high-purity detergents to
prevent introduction of impurities that will interfere with the assay such enzyme inhibitors or peroxides.
Learn more

Blocking Buffers

View products

Blocking Buffers for ELISA

Detection Strategies for ELISA

The final stage in all ELISA systems is a detection step. Unless a radioactive or fluorescent tag was used,
this involves the introduction of an enzyme substrate.The enzyme converts the substrate to a detectable
product. If an ELISA has been constructed and developed properly, then the intensity of signal produced
when the substrate is added will by directly proportional to the amount of antigen captured in the plate and
bound by the detection reagents. Enzyme-conjugated antibodies (especially those involving horseradish
peroxidase, HRP) offer the most flexibility in detection and documentation methods for ELISA because of
the variety of substrates available for chromogenic, chemifluorescent and chemiluminescent imaging.
Though not as sensitive as fluorescent or chemiluminescent substrates, chromogenic ELISA substrates
allow direct visualization and enable kinetic studies to be performed. Furthermore, chromogenic ELISA
substrates are detected with standard absorbance plate readers common to many laboratories.
Fluorescent ELISA substrates are not as common and require a fluorometer that produces the correct
excitation beam to cause signal emission to be generated from the fluorescent tag. Though best used with
a luminometer plate reader, chemiluminescent substrates can be detected by various means including
digital camera systems. Once draw back of using chemiluminescent substrates for ELISA is the signal
intensity can vary more with than other substrates. For assays requiring many plates to be read, this can
present a problem if the signal begins to decay before plates are read. For this reason, it is important to
make sure the assay has been optimized with the substrate in order to ovoid misinterpreting signal-fade in
a sample as low antigen abundance.

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ELISA Test: Principle, Materials required, Procedure and

Results

ELISA Test: Principle, Materials required,

Procedure and Results

Tankeshwar Acharya April 10, 2012 ELISA Test: Principle, Materials required, Procedure and Results2015-0423T04:00:36+00:00Filed under Immunology, serology tagged in ELISA No Comment
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Enzyme-Linked Immunosorbent Assays (ELISAs) are the most widely used type of
immunoassay. ELISA is a rapid test used for detecting or quantifying antibody or antigen against
viruses, bacteria and other materials.
ELISA is so named because the test technique involves the use of an enzyme system and
immunosorbent.
ELISA method for measuring Antigen (Ag) Antibody (Ab) reaction is becoming increasingly used in
the detection of antigen (infectious agent) or antibody for its simplicity and sensitivity. It is as
sensitive as radioimmunoassay (RIA) and requires only microlitre quantities of test reagents. It
has now been widely applied in detection of a variety of antibody and antigens such as
hormones, toxins, and viruses.
Some Salient Features
1. ELISA test has high sensitivity and specificity.
2. The result of quantitative ELISA tests can be read visually
3. A large number of tests can be done at one time.
ELISAs are designed specifically for screening large numbers of specimens at a time,
making them suitable for use in surveillance and centralized blood transfusion services
4. Reagents used for ELISA are stable and can be distributed in district and rural laboratories
but as ELISAs require sophisticated equipment and skilled technicians to perform the
tests, their use is limited to certain circumstances.
Materials needed in ELISA Testing
1. Pipettes, Washer System, ELISA Plate Reader: Readers, washers and pipette are available
as manual or automated system. One of the main factors affecting equipment selection is
the number and types of test samples being run.
A.

ELISA Readers: Readers need to have appropriate filter (650 nm and 450 nm).

B.

Pipette: Are available as fixed as well as adjustable volume as well as single channel and
multi-channel.

C.

Washing system: It can be manual system that washes one row or column at a time or
semi automated systems that wash one strip or plate at a time or fully automated systems
that can process multiple plates

2. Reagents needed for the testing- Concluded in the kit (Coated plates, Sample diluents,
Controls, Wash Concentrate, Conjugate, Substrate, Stop solution)
A.

Coated plates: The 96-well plates are made of polystyrene and are coated with either
inactivated antigen or antibody. The function of the plate has to hold the immobilized either
antigen or antibody. Antigen or antibody present in the sample will bind to the plate. This
coating acts as the binding site for the antibodies or antigens in the sample.

B.

Controls: Negative and positive controls are provided in each kit. The controls help to
normalize or standardize each plate. Controls are also used to validate the assay and to
calculate sample results. Controls might be pre-diluted and ready to use. (Please refer to kit
for specific instructions).

C.

Conjugates: ELISA conjugates are enzyme labeled antibodies that react specifically to
plate bound sample analytes. Unbound conjugates are washed away after incubation and
before the addition of substrate.

D.

Wash Concentrate: It acts as a buffered solution containing detergent to wash unbound

material from the plate. (Not all test kits have wash concentrate; in that case distilled water
can be used for washing; please refer to kit insert for specific instructions)

E.

Stop solution: It stops the enzyme substrate reaction and color development.

Principle
Most ELISA methods developed for the detection of antigen or antibody consist of use of
corresponding antibody or antigen in question which is firmly fixed on solid phase, such as plastic
surface of polyvinyl plate or polystyrene tube, inside the well of a microdilution tray or outside of
spherical plastic or metal bead. Such systems are also called Solid Phase Immunosorbent Assay.
The enzyme system consists of;
1. An enzyme: horse radish peroxidase, alkaline phosphatase which is labelled or linked, to
a specific antibody.
2.

A specific substrate:

O-Phenyl-diamine-dihydrochloride for peroxidase

P Nitrophenyl Phosphate- for Alkaline Phosphatase

Which is added after the antigen-antibody reaction. The enzyme catalyses (usually hydrolyses)
the substrate to give a colour end point (yellow compound in case of Alkaline Phosphatase). The
intensity of the colour gives an indication of the amount of bound antibody or antigen.

Indirect ELISA, conventional but efficient

Indirect ELISA is a two-step ELISA which involves two binding process of primary antibody and labeled
secondary antibody. The primary antibody is incubated with the antigen followed by the incubation
with the secondary antibody. However, this may lead to nonspecific signals because of cross-reaction
that the secondary antibody may bring about.
1. Micro-well plates are incubated with antigens, washed up and blocked with BSA.
2. Samples with antibodies are added and washed.
3. Enzyme linked secondary antibody are added and washed.
4. A substrate is added, and enzymes on the antibody elicit a chromogenic or fluorescent signal.
Learn more about indirect ELISA protocol

Indirect ELISA advantages

:

High sensitivity: More than one labeled antibody is bound per antigen molecule;

Flexible: Different primary detection antibodies can be used with a single labeled secondary
antibody;

Cost-saving: Fewer labeled antibodies are required.

In the indirect ELISA test, the sample antibody is sandwiched between the antigen coated on the plate
and an enzyme-labeled, anti-species globulin conjugate. The addition of an enzyme substratechromogen reagent causes color to develop. This color is directly proportional to the amount of bound

sample antibody. The more antibody present in the sample, the stronger the color development in the
test wells. This format of indirect ELISA is suitable for determining total antibody level in samples
(Newcastle disease virus, B. abortus, etc.). Detailed information about indirect ELISA application in
thedetermination of antibody titer and procedures of antibody concentration
determination are discussed in the following section of ELISA applications.