2. • ELISA is short for enzyme-linked immunosorbent assay (also
referred to as EIA: Enzyme Immunoassay)
• It is a very mature method for the detection of various targets
(antibodies, hormones, peptides and proteins in the blood) involving
the use of enzymes and the specific binding of antibody and
antigen.
• ELISA is a distinguished analysis compared to other antibody-assays
as it yields quantitative results and separation of non-specific and
specific interactions that take place through serial binding to solid
surfaces, which is normally a polystyrene multiwell plate.
• The four main types of ELISAs are direct, indirect, sandwich, and
competitive.
3. • Direct ELISA is used when assessing antibody affinity and
specificity or when investigating blocking/inhibitory
interactions.
• An antigen or sample is immobilized directly on the plate and a
conjugated detection antibody binds to the target protein.
• Substrate is then added, producing a signal that is proportional
to the amount of analyte in the sample.
• Since only one antibody is used in a direct ELISA, they are less
specific than a sandwich ELISA.
Direct ELISA
4.
5. • Used when measuring endogenous antibodies.
• An indirect ELISA is similar to a direct ELISA in that an
antigen is immobilized on a plate, but it includes an additional
amplification detection step.
• First, an unconjugated primary detection antibody is added
and binds to the specific antigen.
• A conjugated secondary antibody directed against the host
species of the primary antibody is then added.
• Substrate then produces a signal proportional to the amount
of antigen bound in the well.
Indirect ELISA
6.
7. • Used when determining analyte concentration in a biological sample.
• Sandwich ELISAs are the most common type of ELISA. Two
specific antibodies are used to sandwich the antigen, commonly
referred to as matched antibody pairs.
• Capture antibody is coated on a microplate, sample is added, and the
protein of interest binds and is immobilized on the plate.
• A conjugated-detection antibody is then added and binds to an
additional epitope on the target protein.
• Substrate is added and produces a signal that is proportional to the
amount of analyte present in the sample. Sandwich ELISAs are
highly specific, since two antibodies are required to bind to the
protein of interest.
Sandwich ELISA
8.
9. • Used when determining concentrations of a small molecules
and hormones.
• Commonly used for small molecules, when the protein of
interest is too small to efficiently sandwich with two
antibodies. Similar to a sandwich ELISA, a capture antibody is
coated on a microplate.
• Instead of using a conjugated detection antibody, a
conjugated antigen is used to complete for binding with the
antigen present in the sample. The more antigen present in the
sample, the less conjugated antigen will bind to the capture
antibody.
• Substrate is added and the signal produced is inversely
proportional to the amount of protein present in the sample.
Competitive ELISA
10.
11. Advantages Disadvantages
Direct ELISA 1. Simple protocol, time-saving,
and reagents-saving.
2. No cross-reactivity from
secondary antibody.
1. High background.
2. No signal amplification, since
only a primary antibody is used
and a secondary antibody is not
needed.
3. Low flexibility, since the
primary antibody must be
labeled.
Indirect ELISA 1. Signal amplification, since one
or more secondary antibodies
can be used to bind to the
primary antibody.
2. High flexibility, since the same
secondary antibody can be used
for various primary antibodies.
1. Complex protocol compared
with direct ELISA.
2. Cross-reactivity from secondary
antibody.
12. Advantages Disadvantages
Sandwich ELISA 1. High flexibility.
2. High sensitivity.
3. High specificity, since different
antibodies bind to the same
antigen for detection.
1. The antigen of interest must
be large enough so that two
different antibodies can bind
to it at different epitopes.
2. It's sometimes difficult to find
two different antibodies that
recognize different epitopes
on the antigen of interest and
cooperate well in a sandwich
format.
Competitive ELISA 1. High flexibility.
2. High sensitivity.
3. Best for the detection of small
antigens, even when they are
present in low concentrations.
1. Relatively complex protocol.
2. Needs the use of inhibitor
antigen.
13. • ELISAs are preferred in many cases due to their sensitivity,
specificity, accuracy, and ability to tolerate harsh buffers or
pretreatments.
• Comparing an ELISA to a Western blot, sandwich ELISAs use
2 specific antibodies rather than one and allow for completely
quantitative results, while a Western blot can see non-specific
bands and are semi-quantitative at best.
• An advantage of ELISAs over different multiplexing platforms
is the ability to customize the assay for that antigen and not
having to worry about many other antibodies and proteins
working together.
• The potential of observing cross-reactivity or interference is
minimized and you can push the sensitivity limits.
Why use an ELISA over other
techniques?
14. Group 9 Members
Tatenda Makwavarara R207648Q
Tertia Nene R195641H
Dorcas Chibondo R207211H
Nyasha A Bepe R204773X
Rejoice T Gwenzi R171OO29
Shelton M Chuma R207309B
Steven Gweshelo R206718M
Tadiwanashe F Gwara R207619N
Nothando Makosa R206710B
Tadiwa F Matanga R205500T
