ELISA (enzyme-linked immunosorbent assay) is a biochemical technique used to detect the presence of antibodies and antigens in a liquid sample. It relies on an enzyme-linked antibody or antigen to detect the target protein. There are different types of ELISA including direct, indirect, sandwich, and competitive. The ELISA process involves coating a plate with an antigen or antibody, adding a sample and enzyme-linked antibody, washing unbound material, and detecting the enzyme's product to quantify the target. ELISAs are widely used in medical testing, food safety, and disease detection.

1. PRINCIPLES ANDPRINCIPLES AND
APPLICATION OFAPPLICATION OF
ELISAELISA
Dr. S. Parasuraman
Faculty of Pharmacy, AIMST University, Malaysia

2. Enzyme-Linked Immunosorbent Assay
(ELISA)
 ELISA
 Principles
 Types
 Applications
96-well polystyrene plate

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.

7. Methods/ELISA Methods
 Direct ELISA protocol
 Indirect ELISA protocol
 Sandwich ELISA protocol
 Competitive ELISA protocol or ELISPOT protocol

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.

16. Methods/ELISA Methods

17. Applications of ELISA
 Serum Antibody Concentrations
 Detecting potential food allergens
 (milk, peanuts, walnuts, almonds and eggs)
 Disease outbreaks- tracking the spread of disease
 e.g. HIV, bird flu, common, colds, cholera, STD etc
 Detections of antigens
 e.g. pregnancy hormones, drug allergen, GMO, mad cow
disease
 Detection of antibodies in blood sample for past
exposure to disease
 e.g. Lyme Disease, trichinosis, HIV, bird flu

18. Furtherreading
 http://www.elisa-antibody.com/ELISA-Introduction/ELISA-
Principle
 http://www.sigmaaldrich.com/life-science/cell-
biology/antibodies/antibodies-
application/protocols/elisa.html
 http://www.nj.gov/drbc/library/documents/PMP_Resources/
ELISA/definition.pdf