Enzyme-Linked

ImmunoSorbantAssay (ELISA)
Dr Muhammad Zubair
• ELISA -an acronym for Enzyme-Linked
ImmunoSorbentAssay.
• The ELISA assay is a widely used biochemical
assay to detect in a sample the presence of
and quantity of proteins, such as hormones
and antibodies and bacteria or viruses.
• The ELISA assay uses the coupling of antigens and
antibodies and relies on the specificity and affinity
of antibodies for antigens. Specificity is the ability
to discriminate among diverse proteins.
• Affinity is the ability to tightly bind to molecules.
• One can determine how much antibody is
present by starting with an antigen, or one can
determine how much antigen or hormone is
present by starting with an antibody.
 What Are Antigens?
• Antigens are any foreign substance in the
body.
• Antigens include “not-self” molecules and
cells, such as:
• foreign proteins, viruses, environmental
pollutants and other foreign substances like
asbestos, tattoo ink, and cigarette smoked.
bacteria and parasites
 History of ELISA

• In 1971, Peter Permannand Eva Engvallin

Stockholm published the first paper on
Enzyme-Linked ImmunoSorbentAssay (ELISA)
showing they could quantify the amount of
IgG in rabbit serum using alkaline phosphatase
(an enzyme) as the reporter label.
• The same year, Anton Schuursand BaukeVan
Weemenin the Netherlands published a paper
showing that with an Enzyme
ImmunoAssay(EIA), they could quantify the
amount of human chorionic gonadotropin in
urine with horseradish peroxidase (an
enzyme) coupled with glutaraldehyde as the
reporter label.
ELISA Plate
ELISA Plate Reader
 Types of ELISA Methods

• The ELISA method has been used to detect

hepatitis B, rabies, and HIV through antibodies
in the blood serum, just to name a few
diseases, or to measure the amount of various
other proteins in the blood serum, such as
hormones, toxins, and allergens.
• There are five types of ELISA methods which include:

• Indirect ELISA

• Sandwich ELISA

• Direct ELISA
• Competitive ELISA

• Multiplex ELISA
 Types of ELISA Methods

• The indirect (to detect antibodies in the

sample) and the sandwich (to detect antigens
in the sample) ELISA methods are the two
most common types used
Types of ELISA Methods
Types of ELISA Methods
• ELISA results are reported as a number using a
spectrophotometer, spectrofluorometer, or
other optical device. This test is determining
the "cut-off" point between a positive and
negative result.
• Unknowns that generate a signal that is
stronger than the known sample are called
"positive"; those that generate weaker signal
are called "negative
 Advantages of ELISA

 Less costly and safest.

 Easy visualization of results with high level of accuracy.

 Specific and highly sensitive assay that can detect

protein at the picomolar to nanomolar range.

 Easily automated for performance of large numbers of

tests.
 Require minimal reagents.
 Qualitative detection or Quantitative
measurement of either antigen or antibody.
 Wells can be coated with antigens or antibodies.

 Can be done by personnel with only minimal

training.
• Limitations
– Expensive initial investment
– Variable sensitivity / specificity of variable tests

– Cross contamination
 Applications of ELISA

 Analysis of hormones, vitamins, metabolites,

and diagnostic markers.
 Therapeutic drug monitoring.

 Diagnostic procedures for detecting infection.

 Requirements for ELISA test

 Purified antigen (if you want to detect or quantify

antibody).
 Purified antibody (if you want to detect or quantify
antigen).
 Standard solutions (positive and negative controls).

 Sample to be tested.
 Micro-titer plates: plastic trays with small wells
in which the assay is done.
 Wash fluid (buffer).

 Enzyme-labeled antibody and enzyme substrate.

• ELISA reader (spectrophotometer) for
quantitative measurements.
 Enzyme labels

• Enzyme labels are used to detect the binding

of antigen-antibody complex.
• It should have high specific reactivity.

• It should be easily coupled to antigen-antibody

complex and must stable.
• Enzymes used in labelling should not be
normally present in the patient samples.
• Examples of enzyme labels are Horse radish
peroxidase, Alkaline phosphatase, and Glucose
oxidase.
 Stages in ELISA

1.The adsorption of either antigen or antibody to

the micro-titer plate.
2.The addition of the test sample and subsequent reagents.
3.The incubation of reactants (formation of antigen-
antibody complex).
4.The separation of bound and free reactants by washing.
5. The binding of enzyme to the target antigen or
antibody.

6. The addition of substrate (production of a

visible signal).

7. The visual or spectrophotometric reading of the

assay.
 ELISA-STEPS
• Addition of diluent and sample to plate and
QC sample
• Incubate at 37C for 30mins
• Wash with wash buffer(detergent +buffer) 5-6
washings
• Addition of conjugte (Ab conjugate e label)
• Incubate at 37C for 30mins
• Wash with buffer,5-6 washings
• Addition of substrate
• Incubate at dark at 18-30C for 30mins
• Stop with stop sol H2SO4 for 2mins
• Read @492nm via ELISA Reader
• Zero correction for substrate blank
• Calculate cutoff from mean
• ELFIA: (Enzyme linked fluorescent
immunoassays): Enzyme converts a substrate
to a reaction product that fluoresces when
excited by light of a particular wavelength
• Luminescent immunoassays: Enzyme converts
a substrate to a reaction product that emits
photons of light
• Polymerase chain reaction-enzyme linked
immunosorbent assay (PCR-ELISA): It is an
immunodetection method that can quantify PCR
product directly after immobilization of biotinylated
DNA on a microplate. This method, which detects
nucleic acid instead of protein, is a much more
sensitive method
• Peptide ELISA: It enables analysis and
screening on amino acid sequence level, e.g.
mapping of epitopes or definition of protein
interaction sites
• Nucleosome ELISA (NU-ELISA): It is used to
quantitatively determine global post-
translational modifications (PTM) signatures of
nucleosomes extracted from cells
Performance comparison of various ELISAs for
antibody detection
 ELISA data interpretation -3 steps

• Quantitative: ELISA data can be interpreted in

comparison to a standard curve (a serial
dilution of a known, purified antigen) 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.
• Semi-Quantitative: ELISAs can be used to
compare the relative levels of antigen in assay
samples, since the intensity of signal will vary
directly with antigen concentration
Thanks