ELISA (Enzyme-linked Immunosorbent Assay)

INTRODUCTION

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

for detecting and quantifying peptides, proteins, antibodies and hormones. In an ELISA, an
antigen must be immobilized to a solid surface and then complexed with an antibody that is
linked to a n 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. 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 non-bound 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.

General ELISA Procedure

Unless you are using a kit with a plate that is pre-coated with antibody, an ELISA begins
with a coating step, in which the first layer, consisting of a target antigen or antibody, is adsorbed
onto a 96-well polystyrene plate. This is followed by a blocking step in which all unbound sites
are coated with a blocking agent. Following a series of washes, the plate is incubated with
enzyme-conjugated antibody. Another series of washes removes all unbound antibody. A
substrate is then added, producing a calorimetric signal. Finally, the plate is read.

Because the assay uses surface binding for separation, several washes are repeated in each
ELISA step to remove unbound material. During this process, it is essential that excess liquid is
removed in order to prevent the dilution of the solutions added in the next assay step. To ensure
uniformity, specialized plate washers are often used.

ELISAs can be quite complex and include multiple intervening steps, especially when measuring
protein concentration 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.
ELISA TYPES

ELISAs can be performed with a number of modifications to the basic procedure: direct,
indirect, sandwich or competitive. 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 (enzyme-labeled primary
antibody) or indirectly (enzyme-labeled secondary antibody). The detection antibodies are
usually labeled with alkaline phosphatase (AP) or horseradish peroxidase (HRP). 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).

Among the standard assay formats discussed and illustrated below, 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.
1. Direct ELISA
For direct detection, an antigen coated to a multi-well plate is detected by an antibody
that has been directly conjugated to an enzyme. This detection method is a good option if
there are no commercially available ELISA kits for your target protein.

Advantages

• Quick because only one antibody and fewer steps are used.

• Cross-reactivity of secondary antibody is eliminated.

Disadvantages

• Immunoreactivity of the primary antibody might be adversely affected by labeling with

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.

2. Indirect ELISA
For indirect detection, the antigen coated to a multi-well plate is detected in two stages or
layers. First an unlabeled primary antibody, which is specific for the antigen, is applied.
Next, an enzyme-labeled secondary antibody is bound to the first antibody. The
secondary antibody is usually an anti-species antibody and is often polyclonal. The
indirect assay, the most popular format for ELISA, has the advantages and disadvantages:

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.

Disadvantages

• Cross-reactivity might occur with the secondary antibody, resulting in nonspecific signal. • An
extra incubation step is required in the procedure.

3. Sandwich ELISA

Sandwich ELISAs typically require the use of matched antibody pairs, where each antibody
is specific for a different, non-overlapping part (epitope) of the antigen molecule. A first
antibody (known as capture antibody) is coated to the wells. The sample solution is then
added to the well. A second antibody (known as detection antibody) follows this step in order
to measure the concentration of the sample.
This type of ELISA has the following advantages:

• High specificity: the antigen/analyte is specifically captured and detected

• Suitable for complex (or crude/impure) samples: the antigen does not require purification
prior to measurement

• Flexibility and sensitivity: both direct or indirect detection methods can be used.

4. Competitive ELISA
The key event of competitive ELISA (also known as inhibition ELISA) is the process of
competitive reaction between the sample antigen and antigen bound to the wells of a
microtiter plate with the primary antibody. First, the primary antibody is incubated with
the sample antigen and the resulting antibody– antigen complexes are added to wells that
have been coated with the same antigen. After an incubation period, any unbound
antibody is washed off.
The more antigen in the sample, the more primary antibody will be bound to the sample
antigen. Therefore, there will be a smaller amount of primary antibody available to bind
to the antigen coated on the well, resulting in a signal reduction. The main advantage of
this type of ELISA arises from its high sensitivity to compositional differences in
complex antigen mixtures, even when the specific detecting antibody is present in
relatively small amounts.