ELISA, Principles of ELISA, Types of ELISA- Direct ELISA Indirect ELISA, Sandwich ELISA, Competitive ELISA, and other Types i.e. ELISPOT (enzyme-linked immunospot assay) and In-cell ELISA, Advantages and disadvantages of ELISA detection methods, Different types of microplates for ELISA, Detection strategies for ELISA

1. ELISA (Enzyme-linked immunosorbent
assay)
By
Dr. Shilpy Singh
NIU, Gr. Noida

2. What is an ELISA?
 ELISA (which stands for enzyme-linked immunosorbent
assay) 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.
 Originally described by Engvall and Perlmann (1971), the
method enables analysis of protein samples immobilized
in microplate wells using specific antibodies.

3. Principle of ELISA
 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.
Fig. The basic setup of an ELISA assay. A capture
antibody on a multi-well plate will immobilize
the antigen of interest. This antigen will be
recognized and bound by a detection antibody
conjugated to biotin and streptavidin-HRP.

4. ELISA depend on the same basic elements:
Coating/capture–direct or indirect immobilization of antigens to the surface of polystyrene
microplate wells.
Plate blocking–addition of irrelevant protein or other molecule to cover all unsaturated
surface-binding sites of the microplate wells.
Probing/detection–incubation with antigen-specific antibodies that affinity-bind to the
antigens.
Signal measurement–detection of the signal generated via the direct or secondary tag on the
specific antibody.
 The most commonly used enzyme labels are horseradish peroxidase (HRP) and alkaline
phosphatase (AP). Other enzymes have been used as well; these include β-galactosidase,
acetylcholinesterase, and catalase. A large selection of substrates is available commercially
for performing 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).

5. Types of ELISA
There are four main types of ELISA: direct ELISA, indirect
ELISA, sandwich ELISA and competitive ELISA.
Direct ELISA
 In a direct ELISA, the antigen is
immobilized to the surface of the
multi-well plate and detected with
an antibody specific for the antigen
 The antibody is directly conjugated
to HRP or other detection
molecules.

6. Indirect ELISA
 Indirect ELISA is a technique that uses a two-
step process for detection, whereby a primary
antibody specific for the antigen binds to the
target, and a labeled secondary antibody
against the host species of the primary
antibody binds to the primary antibody for
detection. As for direct ELISA assays, the
antigen is immobilized to the surface of the
multi-well plate.
 The method can also be used to detect
specific antibodies in a serum sample by
substituting the serum for the primary
antibody.

7. Sandwich ELISA (or sandwich
immunoassay)
 The most commonly used type of
ELISA. This requires two antibodies
specific for different epitopes of the
antigen. These two antibodies are
normally referred to as matched
antibody pairs.
 One of the antibodies is coated on the
surface of the multi-well plate and used
as a capture antibody to facilitate the
immobilization of the antigen. The
other antibody is conjugated and
facilitates the detection of the antigen.

8. Advantages of ELISA detection methods
Direct Indirect Sandwich
• Quick because only
one antibody and
fewer steps are
used.
• Cross-reactivity of
secondary antibody
is eliminated.
• 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.
• Different detection methods can be used
with the same primary antibody
(colorimetric, chemiluminescent, etc.).
• Highly sensitive and
highly specific for
target antigen as two
antibodies are used
for capture and
detection.
• Different detection
methods can be used
with the same
capture antibody.

9. Disadvantages of ELISA detection methods
Direct Indirect Sandwich
• Immunoreactivity of the
primary antibody might be
adversely affected by labeling
with reporter enzymes or tags.
• Labeling primary antibodies for
each specific ELISA system is
time-consuming and expensive.
• Limited number of conjugated
primary antibodies available
commercially.
• No flexibility in choice of
primary antibody label from
one experiment to another.
• Minimal signal amplification.
• Cross-reactivity might
occur with the
secondary antibody,
resulting in nonspecific
signal.
• An extra incubation step
is required in the
procedure.
• Requires more
optimization to
identify antibody pairs
and to ensure there is
limited cross-reactivity
between the capture
and detection
antibodies.

10. Competitive ELISA and other Types
Besides the standard direct and sandwich formats
described above, several other styles of ELISA exist:
Competitive ELISA
 Competitive ELISA is a strategy that is commonly used
when the antigen is small and has only one epitope or
antibody binding site. One variation of this method
consists of labeling purified antigen instead of the
antibody. Unlabeled antigen from samples and the
labeled antigen compete for binding to the capture
antibody. A decrease in signal from the purified antigen
indicates the presence of the antigen in samples when
compared to assay wells with labeled antigen alone.
 In competitive ELISA, also referred to as inhibition
ELISA, the concentration of the target antigen is
determined by detection of signal interference. The
target antigen in the sample competes with a labeled
reference or standard for binding to a limited amount of
antibodies immobilized on the plate.

11. ELISPOT (enzyme-linked immunospot assay)
 ELISPOT refers to ELISA-like capture and measurement of proteins secreted by cells
that are plated in PVDF-membrane-backed microplate wells. It is a "sandwich" assay in
which the proteins are captured locally as they are secreted by the plated cells, and
detection is with a precipitating substrate. ELISPOT is like a western blot in that the
result is spots on a membrane surface.
In-cell ELISA
 is performed with cells that are plated and cultured overnight in standard microplates.
After the cultured cells are fixed, permeabilized, and blocked, target proteins are
detected with antibodies. This is an indirect assay, not a sandwich assay.
 The secondary antibodies are either fluorescent (for direct measurement by a
fluorescent plate reader or microscope) or enzyme-conjugated (for detection with a
soluble substrate using a plate reader).

12. Different types of microplates for ELISA
ELISA plate Coating Applications
Modified polymer surfaces Various modifications to the
plate surface to increase
hydrophobicity or hydrophilicity
Enhance passive binding of
biomolecules based on their
physiochemical characteristics
Antibody-binding plates Protein A, G, L, or A/G Binds to the FC region (VL for
protein L) of capture
antibodies to properly orient
while leaving antigen binding
capability
Fusion-tag binding plates Glutathione (GST tag binding) or
nickel or copper coated (His tag
binding)
Study of genetically engineered
fusion proteins or protein-
protein interactions
Biotin-binding plates Streptavidin or neutravidin Binds small biotinylated
peptides and other small
molecules that are difficult to
bind by passive adsorption

13. Detection strategies for ELISA
Chromogenic
(colorimetric) Fluorescence Chemiluminescence
Sensitivity Low Moderate High
Equipment required Standard absorbance
plate reader
Fluorometer Luminometer plate
reader
Enzyme HRP or AP Fluorescent tag or HRP
(with chemifluorescent
substrates)
HRP or AP
Advantages Direct visualization,
high reproducibility
between plates
High reproducibility
between plates, wide
dynamic range
Most sensitive
detection strategy,
wide dynamic range
Considerations Requires black
microplates
Requires opaque or
black microplates