ELISA

(ENZYME LINKED IMMUNOSORBENT ASSAY)

BY
Dr Anwar Ullah
 HISTORY

•Prior to the development of the EIA/ELISA, the only option for

conducting an immunoassay was radioimmunoassay, a
technique using radioactively-labeled antigens or antibodies.

•Avrameas (1966, 1969) and Pierce (1967) developed methods

to chemically link antibodies to biological enzymes whose
activities produce a measurable signal with solutions
containing appropriate substrates.

•This signal has to be associated with the presence of antibody

or antigen .
 COMPONENTS OF ELISA

Antibody: proteins produced by the immune system which

help defend against antigens

Enzyme: Horse Radish Peroxidase (HRP) MW 44, 000,

glycoprotein with 4 lysine residues.

Substrate: TMB (3,3',5,5', tetramethylbenzidine) The enzyme

acts as a catalyst to oxidize substrate in the presence of
Hydrogen peroxide to produce a blue color.
 INTRODUCTION TO ELISA

A test that uses antibodies and color

change to identify a substance.

ELISA involves at least one

antibody with specificity for a
particular antigen.

A 96 - well microtiter plate being used

for ELISA.
 PRINCIPLE

• The basic principle of an ELISA is to use an enzyme to detect

the Ag-Ab binding (antigen- antibody binding). The enzyme
converts a colorless substrate (chromogen) to a colored
product, indicating the presence of Ag:Ab binding.
BASIC STEPS OF ELISA
 Secondary antibody

substrate

Colored

product
Primary
antibody

Different antigen in sample

 TYPES OF ELISA

 INDIRECT ELISA
 DIRECT ELISA NON -COMPETETIVE ELISA
 SANDWICH ELISA

 COMPETETIVE ELISA
 INDIRECT ELISA

 Antigen is added to plate.

 Added buffer.

 Suitable primary antibody is added.

 Secondary antibody- HRPO is then

added which recognizes and binds
to primary antibody.

 TMB substrate is added, is

converted to detectable form.
 ADVANTAGES OF INDIRECT DETECTION

Wide variety of labeled secondary antibodies are available

commercially.
 Versatile, since many primary antibodies can be made in one
species and the same labeled secondary antibody can be used for
detection.
Immunoreactivity of the primary antibody is not affected by
labeling.
 Sensitivity is increased because each primary antibody contains
several epitopes that can be bound by the labeled secondary
antibody, allowing for signal amplification.
DISADVANTAGES OF INDIRECT DETECTION

Cross-reactivity may occur with the secondary antibody,

resulting in nonspecific signal.

An extra incubation step is required in the procedure.

 DIRECT ELISA

 Apply a sample of known antigen to a

surface.

 Enzyme linked primary antibody is

applied to the plate.

 Washed, After this wash, only the

antibody-antigen complexes remain
attached.

 Apply a substrate which is converted by

the enzyme to elicit a chromogenic
signal.
 ADVANTAGES OF DIRECT DETECTION

 Quick methodology since only one antibody is used.

 Cross-reactivity of secondary antibody is eliminated.

 DISADVANTAGES OF DIRECT DETECTION

 Immunoreactivity of the primary antibody may be reduced as

a result of labeling.

 Labeling of every primary antibody is time-consuming and

expensive.

 No flexibility in choice of primary antibody label from one

experiment to another.
 SANDWICH ELISA
1. a. Plate is coated with suitable antibody.
b. Buffer is added.
2. Sample is added to plate so antigen is bounded by capture antibody.
3. A suitable biotin labeled detection antibody is added to plate.
4. Enzyme HRPO is added and binds the biotin labeled detection
antibody.
5. TMB substrate is added and converted by HRPO to colored product.
COMPETETIVE ELISA
Solid phase coated with
antibody
Add unknown amount of
unlabeled antigen and known
amount of labeled antigen

Free and labeled antigen are

captured

Color formation by oxidation of substrate

into a colored compound
ADVANTAGES
 Suitable for complex (crude or impure) samples, since the
antigen does not require purification prior to measurement.

DISADVANTAGES

 Each antigen may require a different method to couple it

to the enzyme.
COMPARISON BETWEEN INDIRECT SANDWICH & COMPETITIVE ELISA
 ELISA RESULTS

The ELISA assay yields three different types of data output:

 Quantitative: ELISA data can be interpreted in comparison to a

standard curve in order to precisely calculate the concentrations of
antigen in various samples.

 Qualitative: ELISAs can also be used to achieve a yes or no

answer indicating whether a particular antigen is present in a
sample, as compared to a blank well containing no antigen or an
unrelated control antigen.
 ELISA data is typically graphed with Optical density Vs
Log concentration to produce a sigmoidal curve.
 Known concentrations of antigen are used to produce a standard
curve and then this data is used to measure the concentration of
unknown samples by comparison to the linear portion of the
standard curve.
 APPLICATIONS

 Screening donated blood for evidence of viral contamination by

 HIV-1 and HIV-2 (presence of anti-HIV antibodies)
 Hepatitis C (presence of antibodies)
 Hepatitis B (testing for both antibodies and a viral antigen)
 Measuring hormone levels
 HCG (as a test for pregnancy)
 LH (determining the time of ovulation)
 TSH, T3 and T4 (for thyroid function)
THANK YOU