The document discusses Enzyme-Linked Immunosorbent Assay (ELISA), a common immunoassay technique used to detect antigens in biological samples. It describes the basic ELISA principles including immobilizing the antigen and using a detection antibody conjugated to an enzyme. The document outlines the advantages of ELISA including high sensitivity and specificity, high throughput, and ability to test various sample types. It also discusses the different types of ELISA - direct, indirect, sandwich, and competitive/inhibition - and compares their features in a table. The key information provided is an overview of the ELISA technique and a comparison of the different types of ELISA assays.

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Enzyme-Linked Immunosorbent Assay (ELISA)
Enzyme-Linked Immunosorbent Assay (ELISA) is a common immunoassay, in which
antibodies, peptides, proteins, and small molecules can be detected and quantified using
a multi-well plate. It is a technique to detect the presence of antigens in biological
samples. An ELISA, like other types of immunoassays, relies on antibodies to detect a
target antigen using highly specific antibody-antigen interactions.
Basic ELISA principle:
In an ELISA assay, the antigen is immobilized to a solid surface. This is done either directly or
via the use of a capture antibody itself immobilized on the surface. The antigen is then
complexed to a detection antibody conjugated with a molecule amenable for detection such
as an enzyme or a fluorophore.
An ELISA assay is typically performed in a multi-well plate (96- or 384-wells), which provides
the solid surface to immobilize the antigen. Immobilization of the analytes facilitates the
separation of the antigen from the rest of the components in the sample. This characteristic
makes ELISA one of the easiest assays to perform on multiple samples simultaneously.
Advantages
➢ High sensitivity and specificity: it is common for ELISAs to detect antigens at the
picogram level in a very specific manner due to the use of antibodies.
➢ High throughput: commercial ELISA kits are normally available in a 96-well plate
format. But the assay can be easily adapted to 384-well plates.

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➢ Easy to perform: protocols are easy to follow and involve little hands-on time.
➢ Quantitative: it can determine the concentration of antigen in a sample.
➢ Possibility to test various sample types: serum, plasma, cellular and tissue extracts,
urine, and saliva among others.
Disadvantages
➢ Temporary readouts: detection is based on enzyme/substrate reactions and therefore
readout must be obtained in a short time span.
➢ Limited antigen information: information limited to the amount or presence of the
antigen in the sample.
TYPES OF ELISA
1.Direct ELISA:It is the simplest configuration in which the antigen is bound by passive
adsorption to the solid phase, washed to remove any unbound molecules and then directly
incubated with a conjugated antibody. Following the incubation period and additional washing,
substrate is added to produce signal that is allowed to develop. After certain time, the substrate

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reaction is stopped and the resulting signal quantified. It is commonly used for tittering
conjugated secondary antibodies and very useful to estimate antigen cross-reactivity.
2. Indirect ELISA :In this system, initial antigen binding and washing steps are the same as
the direct method. The main difference in this case is the use of unconjugated antibody to bind
the immobilized antigen upon incubation. Following a washing step to remove unbound
antibodies, the remaining antigen-bound antibodies are targeted by a conjugated secondary
antibody that will generate the readout signal as described for direct ELISA. This system allows
multiple sample evaluations using different primary antibodies to be screened with a single
conjugated secondary antibody.

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3.Sandwich ELISA:This assay requires a compatible antibody pair that recognize different
antigenic targets (epitopes) on the same antigen. The first antibody, called the capture antibody,
is coated on the plate and used to immobilize the antigen upon binding during incubation with
the sample. Free antigen is removed by a washing step and then a detection antibody is added
to bind the captured antigen and enable subsequent detection. Sandwich ELISA is divided into
two main systems:
I. Direct Sandwich :It uses a detection antibody conjugated to an enzyme or
fluorescence tag. Following incubation with the antibody-antigen complex
immobilized on the plate well, signal detection is performed upon successive
addition of substrate and stopping solution or appropriate excitation/emission of the
fluorescent tag.
II. Indirect Sandwich : uses an unconjugated antibody bound to the antibody-antigen
complex on the well. Following a washing step to remove unbound antibodies, the
remaining antigen-bound antibodies are targeted by a conjugated secondary
detection antibody. Signal detection is performed upon successive addition of
substrate and stopping solution or appropriate excitation/emission of the fluorescent
tag.

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4. Competitive and Inhibition ELISA:Each of the core systems described above can
be further modified to measure molecules based on their ability to interfere with a well-
known pre-titrated assay. Such assays can be used to study either antigens or antibodies and
they can be competitive or inhibitory based on the specific conditions of each assay. In both
types of assays, a pre-titrated system is challenged with the presence of the testing sample
whose binding activity is then determined from the degree of the resulting interference in the
established system.

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ELISA COMPARSIONS TABULATED FORMAT
Direct Indirect
Sandwich
Direct
Sandwich
Indirect
Coating (adsorption to solid
phase)
Antigen Antigen
Capture
antibody
Capture
antibody
Blocking Addition of blocking agent to prevent non-specific binding
Wash
Separate bound/unbound analytes
Analyte (addition of testing
sample)
Enzyme- or
fluorescence-
conjugated
antibody
Unconjugated
antibody
Antigen
sample
Antigen
sample
Wash
Separate bound/unbound analytes
Secondary reagent N/A
Enzyme- or
fluorescence-
conjugated
antibody
Enzyme- or
fluorescence-
conjugated
detection
antibody
Biotin-
conjugated or
unconjugated
detection
antibody
Wash Separate bound/unbound analytes
Additional reagent N/A N/A N/A
Enzyme- or
fluorescence-
conjugated
streptavidin
or secondary
antibody
Wash
Separate bound/unbound analytes
Signal development
Addition of substrate for enzyme-conjugated antibodies
Stop signal development
For end-point reading of enzyme-based detection systems
Signal Detection
Colorimetric, fluorescent, or chemiluminescent detection

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Which type of ELISA should I use?
Advantages Disadvantages
Direct ELISA Short protocol: saves time and
reagents.
No cross-reactivity from secondary
antibody.
Potential high background: all
proteins in the sample bind to the
surface.
No signal amplification.
Low flexibility: the primary
antibody must be conjugated.
Indirect ELISA Signal amplification: several
secondary antibodies will bind to
the primary antibody.
High flexibility: the same
secondary antibody may be used
for several primary antibodies.
Long protocol if compared to direct
ELISA.
Potential cross-reactivity from
secondary antibody.
Sandwich ELISA High specificity: involves two
antibodies detecting different
epitopes on the same antigen.
Suitable for complex samples.
High flexibility and sensitivity:
both direct and indirect methods
can be used.
Demanding design: finding two
antibodies against the same target
that recognize different epitopes
and work well together can be
challenging at times.
Competitive ELISA Depends on base ELISA selected.
Suitable for small antigens.
Depends on base ELISA selected
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