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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.
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.
Washing step :
Excess antibody or unbound antibodies are removed by washing step and is followed by addition of
blocking solution.
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."
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.
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Overview of ELISA
Protein Biology Resource Library
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Introduction
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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.
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).
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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.
Advantages
Disadvantage
s
Quick because only one antibody and fewer steps are used.
Labeling primary antibodies for each specific ELISA system is time-consuming and
expensive.
Advantages
Versatile because many primary antibodies can be made in one species and the same
labeled secondary antibody can be used for detection.
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.
Disadvantage
s
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.
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.
and substrates.
Antibody Pair Kits contain only matched antibodies and standard (no plates or detection
reagents).
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Performing and Evaluating Spike and Recovery and Linearity of Dilution for ELISA
ELISA Protocols
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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.
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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.
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Primary Antibodies
Blocking Buffers
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Immunology
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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.
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:
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 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
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;
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.