Enzyme-Linked

Immunosorbent Assay
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Introduction
ELISA
Types
Applications
Principles
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What is ELISA?
Biochemical technique
used mainly in
immunology.
first and most basic test to
determine if an individual
is positive for a selected
pathogen, such as HIV.
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.

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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.
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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
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Basic principles of ELISA
the antibodies fixed to a
solid surface, such as the
inner surface of a test
tube;
a preparation of the same
antibodies coupled to an
enzyme. This is one (e.g.,
-galactosidase) that
produces a colored
product from a colorless
substrate.
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Objectives
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To be able to determine if a serum sample
is positive or negative
which may help in the diagnosis and
treatment of a particular disease
like HIV and leptospirosis and alike.

To be able to apply the proper methods in
achieving an accurate
results.

To be able to know the different reagents,
their properties and
functions.

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Methodology
ELISA for Tracking Disease
Outbreaks
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Methods
A yellow tube and plastic transfer was
labeled.
The bodily fluid was then transferred
into the tube of another group. The
samples were then mixed and after which,
the half of the shared sample (750l) was
placed in the groups tube.

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Methods
The sharing protocol was repeated twice.
The 12-well strip was then labeled. On the
first 3 wells, it was labeled as + while
for the next 3 it was labeled as -.
The remaining wells were labeled with two
of the members initials.
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Methods
A fresh pipette tip was used to transfer
50l of the positive control into the +
wells (the same was done for the -
wells).
50l of the groups sample was
transferred into the the appropriately
initialed three wells.

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Methods
There was a 5 minute waiting period so as
to allow the proteins in the samples to
bind to the plastic wells.
The microplate strip was then tapped onto
the paper towels provided.
The well was then filled with wash buffer.
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Methods
A 50l of primary antibody was then
transferred into the 12 wells of the
microplate strip.
The fluids was then allowed to stand for
another five minutes so as to allow the
antibody to bind.
Washing of the wells was again performed.
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Methods
A 50l of secondary antibody was then
transferred into the 12 wells of the
microplate strip.
A 5 minute waiting period and washing of
the microplate wells was again performed
(2x).
A 50l of enzyme substrate was then
transferred into the 12 wells of the
microplate strip.

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Methods
The enzyme substrate was let to stand for
five minutes.
The results were then observed and
recorded.
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Results and discussion
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