The document describes the enzyme-linked immunosorbent assay (ELISA), a commonly used laboratory technique for detecting and quantifying antigens and antibodies. It involves immobilizing an antigen or antibody to a plate and detecting it using an antibody or antigen linked to an enzyme. There are four basic ELISA formats - direct, indirect, sandwich, and competitive - that allow flexibility based on the antibodies and results required. The general ELISA procedure involves coating a plate with a capture antibody or antigen, adding the sample, washing unbound material, adding a detection antibody linked to an enzyme, washing again, and detecting the enzyme's activity using a substrate to produce a measurable signal correlated to analyte concentration.

1. Enzyme-linked
immunosorbent assay
Dr. Shaymaa Z. Nada
Asst. Prof. In Clinical Biochemistry

2. Enzyme-linked immunosorbent
assay (ELISA)
ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA)
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The enzyme-linked immunosorbent assay (ELISA) is an immunological assay
commonly used to measure antibodies, antigens, proteins and glycoproteins in
biological samples. Some examples include: diagnosis of HIV infection, pregnancy
tests, and measurement of cytokines or soluble receptors in cell supernatant or
serum. ELISA assays are generally carried out in 96 well plates, allowing multiple
samples to be measured in a single experiment. These plates need to be special
absorbant plates (e.g. NUNC Immuno plates) to ensure the antibody or antigen sticks
to the surface. Each ELISA measures a specific antigen, and kits for a variety of
antigens are widely available.
The ELISA pictured in Figure 1 is what is known as a sandwich ELISA, here two sets
of antibodies are used to detect secreted products, e.g. cytokines. The method is
stepwise in the order shown. The 1st step is to coat the ELISA plate with capture
antibody, any excess, unbound antibody is then washed from the plate. The capture
antibody is an antibody raised against the antigen of interest.
Next the sample (e.g. urine, serum, or cell supernatant) is added. Any antigen found
in the sample will bind to the capture antibody already coating the plate. Samples are
usually added in duplicate or triplicate (to allow for statistical analysis), and in varying
concentrations to guarantee it falls within the levels of detection of the assay. Again
any excess sample is washed from the plate.
In step 3, detection antibody is added. This antibody is labelled with an enzyme,
usually horse radish peroxidase or alkaline phosphatase. Detection antibody binds to
any target antigen already bound to the plate. Finally, a substrate is added to the
plate. ELISA assays are usually chromogenic using a reaction that converts the
substrate (e.g. TMB or ABTS) into a coloured product which can be measured using
a plate reader.
Figure 2. A typical standard curve. Shown is a standard
curve for an IFN-γ ELISA. To work out the concentration
of antigen in a sample, a standard curve using a solution
of known concentration needs to be prepared.
Figure 1. ELISA method. Described above is a sandwich
ELISA, showing the steps in the assay, numbered in
order 1-4.
Determination of antigen concentration in a sample requires production of a standard curve using antigens of a known concentration (shown
in Figure 2). The concentration of antigen in a sample can then be calculated using the optical density (OD).

3. The Enzyme Linked Immunosorbent Assay (ELISA) is
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. It 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 an enzyme. Detection is accomplished by
assessing the conjugated enzyme activity via incubation
with a substrate to produce a measurable product. The
most crucial element of the detection strategy is a highly
specific antibody-antigen interaction.

4. • ELISA stands for enzyme-linked immunoassay. It
is a commonly used laboratory test to detect
antibodies in the blood. An antibody is a protein
produced by the body’s immune system when it
detects harmful substances, called antigens. ELISA
is an effective and widely used technique in
microbiology and virology—in particular, for
investigating infectious pathogens.

5. ELISA Principle
What is the principle of enzyme linked immunosorbent assay?
The Enzyme Linked Immunosorbent Assay (ELISA) is a common
laboratory technique which is used to measure the concentration of the
analyte (usually antibodies or antigens) in solutions.
The steps of the ELISA result in a colored end product which correlates to
the amount of the analytes present in the original sample.
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.
The ELISA principle depends on having the reactants of the ELISA
immobilized to the microplate surface makes it easy to separate bound
from unbound 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.

6. General ELISA Procedure
How does the ELISA test work?
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 (Blocking ELISA principle: 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 signals.

8. There are four basic ELISA formats, allowing for a certain amount
of flexibility which can be adjusted based on the antibodies
available, the results required, or the complexity of the samples:
1.Direct ELISA
2.Indirect ELISA
3.Sandwich ELISA
4.Competition or Inhibition ELISA

9. Direct ELISA
Direct ELISA principle
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.

11. 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.
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

12. 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.
What is the principle of competitive ELISA technique?
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 antigens in the sample, the more primary
antibodies 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.

13. General ELISA Test Steps

14. 1- Prepare a surface to which a known quantity of capture antibody is
bound.
2- Block any nonspecific binding sites on the surface.
3- Add an antigen-containing sample to the plate.
4- Wash the plate, so that unbound antigen is removed.
5- A specific antibody is added, and binds to antigen (hence the
‘sandwich, the Ag is stuck between two antibodies)
6- Add enzyme-linked secondary antibodies as detection antibodies
that also bind specifically to the antibody’s Fc region (non-specific).
7- Wash the plate, so that the unbound antibody-enzyme conjugates
are removed.
8- Add a substrate that is converted by the enzyme into a color or
fluorescent or electrochemical signal.
9- Measure the absorbance or fluorescence or electrochemical signal
(e.g., current) of the plate wells to determine the presence and quantity
of antigen.

15. https://youtu.be/FN65qr4G0TA