3. What is ELISA?
Enzyme-Linked Immunosorbent Assay (ELISA) is
biochemical assay technique used mainly in
immunology.
It is a plate-based assays designed for detecting
and quantifying substances such as peptides,
proteins, antibodies and hormones.
First and most basic test to determine if an individual
is positive for a selected pathogen, such as HIV.
Dimension of ELISA plant:
8 cm x 12 cm plastic plate which contains an 8 x 12 matrix
of 96 wells, each of which are about 1 cm high and 0.7 cm
in diameter.
4. Principle of ELISA
• A sensitive immunoassay that uses an enzyme linked to
an antibody or antigen as a marker for the detection of
a specific protein, especially an antigen or antibody.
• ELISA involves detection of “analyte” in a liquid sample
using liquid reagent (wet lab) or dry strips (dry lab).
• In dry analysis, strip can be read in reflectometry. The
quantitative reading usually based on detection of
intensity of transmitted light by spectrophotometry at
specific wavelength.
5. Principle of ELISA
The sensitivity of detection depends on amplification of
signal during the analytic reaction. In some enzymatic
reaction, the signal generated by enzyme which are
linked to the detection reagents in fixed proportions to
allow accurate quantification (enzyme linked).
There are two main variations on ELISA method is
ELISA can be used to detect the presence of
antigens that are recognized by an antibody or
it can be used to test for antibodies that recognize an
antigen.
6. Types of ELISA
Qualitative ELISA
Postive or Negative results
Quantitative ELISA
optical density or fluorescent units of the sample is
interpolated into a standard curve, which is
typically a serial dilution of the target.
8. Steps in ELISA
A general ELISA is a five-step procedure
coat the microtiter plate wells with antigen
block all unbound sites to prevent false positive results
add primary antibody (e.g. rabbit monoclonal antibody) to the
wells
add secondary antibody conjugated to an enzyme (e.g. anti-
mouse IgG)
reaction of a substrate with the enzyme to produce a colored
product
9. General ELISA protocol
Plate Preparation Working concentration of antibody/antigen coated
in 96-well microplate & incubate overnight at 4℃
(100 L/ well).μ Aspirate WITH 300 l washμ
buffer(3 times)
Aspirate with 300 l wash buffer(3 times) –μ
Step-2
Blockplates by adding 300 L of blocking bufferμ
incubate foran hour Aspiration/wash as in
step 2
Assay Procedure 100 L of sample/standards in dilution bufferμ
incubate for2 hours at roomtemp.
Aspiration/wash as in step 2
Add 100 L of the detection antibodyμ incubate
foran hour Repeat wash Add 200 L ofμ
substrate solution (each well) incubate for20 min
at roomtemperature 50 L of stop solutionμ
Avoid
placing the
plate in
direct light
10. General ELISA protocol
Calculation of Results
Calculate the mean absorbance
for each set of STD, control and
sample and subtract the mean
zero STD from each.
Calibration curve plotted using
absorbance (y-axis) against the
concentration (x-axis).
Unknown concentration
determined by interpolation
method.
11. Methods/ELISA Methods
Direct ELISA protocol
Direct ELISA is suitable for the detection of
proteinaceous antigens and may require pre-
purification of sample. Direct ELISA can be performed
when desired antibody is available in a pre-
conjugated sate (fluorometric, colorimetric, enzymatic)
Advantages:
Fast
Eliminates possible non-specific binding of secondary
antibody
Disadvantages
Reactivity of primary antibody may be reduced conjugation.
Little signal amplification.
12. Methods/ELISA Methods
In-direct ELISA protocol
If the primary antibody is not conjugated, then indirect
ELISA is required in which a conjugated secondary
antibody is targeted to the isotype (e.g. e.g., mouse IgG1,
goat IgM, rabbit IgG1,k, chicken IgY) of the primary
antibody.
Advantages:
Wide variety of secondary conjugates are available for detection
Immunoreactivity of the primary antibody is not compromised.
Multiple binding of the secondary affords some signal amplification
Disadvantages
Extra step in the protocol.
Some non-specific binding of the secondary may cause high
13. Apply a sample of known antigen
to a surface
Apply a sample of known antigen
to a surface
Direct ELISADirect ELISA
coated with blocking buffercoated with blocking buffer
Detecting antibody added &
incubated
Detecting antibody added &
incubated
Wash to remove unbound
antibody
Wash to remove unbound
antibody
Apply a substrate which is
converted by the enzyme to elicit
a chromogenic/ fluorescent signal
Apply a substrate which is
converted by the enzyme to elicit
a chromogenic/ fluorescent signal
View/quantify the result using a
spectrophotometer or other
optical device
View/quantify the result using a
spectrophotometer or other
optical device
Apply a sample of known antigen
to a surface
Apply a sample of known antigen
to a surface
coated with blocking buffercoated with blocking buffer
Detecting antibody added &
incubated
Detecting antibody added &
incubated
Wash, second antibodies (to form
antigen-antibody complexes),
wash
Wash, second antibodies (to form
antigen-antibody complexes),
wash
Apply a substrate which is
converted by the enzyme to elicit
a chromogenic/ fluorescent signal
Apply a substrate which is
converted by the enzyme to elicit
a chromogenic/ fluorescent signal
View/quantify the result using a
spectrophotometer or other
optical device
View/quantify the result using a
spectrophotometer or other
optical device
In-direct ELISAIn-direct ELISA
14. Sandwich ELISA protocol
The sandwich ELISA measures the amount of
antigen between two layers of antibodies (i.e.
capture and detection antibody).
The antigen to be measured must contain at least
two antigenic sites capable of binding to antibody,
since at least two antibodies act in the sandwich.
15. Sandwich
ELISA protocol
Steps in sandwich ELISA
Prepare a surface to which a known quantity of antibody is
bound.
Apply the antigen-containing sample to the plate.
Wash the plate, so that unbound antigen is removed.
Apply the enzyme-linked antibodies which are also specific
to the antigen.
Wash the plate, so that unbound enzyme-linked antibodies
are removed.
Apply a chemical which is converted by the enzyme into a
fluorescent signal.
View the result: if it fluoresces, then the sample contained
antigen.
