Enzyme-Linked

ImmunoSorbant Assay
(ELISA)
U S I N G ELISA T O M E A SURE C O N C EN T R A
TIONS OF SEX HORMONES OVER
M E N S T R UA L C Y C L E I N F E M A LES A N D O
VER LI F E S PA N I N M A LES

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INTERNATIONAL LICENSE.
 ELISA

 ELISA - an acronym for Enzyme-Linked ImmunoSorbent

Assay.

 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:
a. foreign proteins
b. viruses
c. environmental pollutants and
other foreign substances like
asbestos, tattoo ink, and cigarette
smoke
d. bacteria and parasites (Protista,
Fungi, Plantae, and
Animalia cells)
e.foreign transplanted tissue
What Are Antibodies and How Are They
Produced?
Antibodies are large glycoprotein
molecules produced by B-lymphocytes
during the humoral immune response to
antigens introduced into the body.
Lymphocytes include B-lymphocytes (B-cells)
and T- lymphocytes (T-cells) which are white
blood cells form from the hematopoietic
(blood) stem cells in the bone marrow.
The immune system is made of two parts –
humoral (antibody-mediated) and cellular
(cell-mediated).
 Formation of B Lymphocytes and T
Lymphocytes

http://cnx.org/contents/[email protected]:139/Anatomy-&-
Physiology
 B Lymphocytes Mature in the Bone Marrow and T
Lymphocytes Mature in the Thymus Gland

http://cnx.org/contents/[email protected]:139/Anatomy-&-
Physiology http://creativecommons.org/licenses/by/4.0/ No changes made.
Humoral (Antibody-Mediated) Immune
System
B-lymphocytes produce large glycoproteins
called antibodies in response to antigens
(any foreign substance) and then mark
those antigens-antibody complex to be
destroyed by the T-lymphocytes.

Each B-cell makes its own distinct antibody in

response to a specific antigen which comes in
contact with it. Each antibody is designed to
bind to a specific surface binding site or epitope
on the antigen.

There are millions of different types of

antibodies circulating in an individual’s
bloodstream and they are based on exposure to
antigens in his/her environment.
 Structure of An Antibody

Over 80% of human glycoprotein antibodies are

in the immunoglobulin class IgG. They are
shaped like a Y and are found in the blood,
lymph, and intestine.

IgG molecules have a molecular weight of

150,000 Daltons and are made of 2 long (heavy)
chains coded from chromosome 14, and 2 short
(light) chains coded from either chromosome 2
or 22, and then all connected by disulphide
bonds.

Most of the molecule is composed of a constant

region that doesn’t change from one IgG
molecule to another. However, the ends of the Y
are variable, which accounts for each IgG
 Antibody Structure and Antigen-Antibody
Interactions

For images of Antibody Structure and of

Antigen- Antibody Interactions, go to
http://www.ncbi.nlm.nih.gov/books/NBK22420/figure/A508/?report=
objectonly

and scroll down to Figures 4.30 and

4.31.

Also, go to http://www.ncbi.nlm.nih.gov/books/NBK27144/ and

scroll down to Figures 3.1, 3.2, and 3.5.

For an image of different antigens

binding in a specific binding site, go to
Figure 3.8 at
 History of Antibody-Antigen
Interactions 1
In 1890, Emil von Behring from Germany and
Shibashuro Kitasato from Japan noticed an
“antitoxin” formed in the blood of animals
infected with diphtheria bacillus. When
antitoxin serum was transferred to an animal
given a lethal dose of toxin, the animal
survived.

They proposed the humoral theory of immunity

based on their toxin-antitoxin theory.

In 1901, Emil Von Behring was awarded the

first Nobel Prize in Physiology or Medicine “for
his work on serum therapy, especially its
History of Antibody-Antigen
Interactions - 2
In 1891, Paul Erlich was the first to use the
term “antikorper”, the German word for
antibody, in an article he wrote.

In 1897, he proposed the idea that the “side

chain” receptors on the surface of cells could
bind to specific toxins in a “lock-and-key”
interaction.

Paul Erlich shared the Nobel Prize in

Physiology or Medicine in 1908 with Ilya
Mechnikov, a Russian scientist, “in recognition
for their work on immunity”.
History of Antibody-Antigen
Interactions - 3
In 1940, Linus Pauling at the California Institute
of Technology confirmed the lock-and-key theory
proposed by Erlich and was awarded the Nobel
Prize in Chemistry in 1954 “for his research into
the nature of the chemical bond and its
application to the elucidation of the structure of
complex substances” including antibodies and
the nature of serological reactions.

In 1948, Astrid Fagreaus at the Karolinska

Institutet in Stockholm, Sweden presented
evidence that B- lymphocytes in the form of
plasma cells formed the antibodies circulating
in the bloodstream.
History of Antibody-Antigen
Interactions - 4
By the 1960s, Gerald Edelman at
Rockefeller University in New York and
Rodney Porter at the University of Oxford,
England worked out the structure and
complete amino acid sequence of the
antibody, IgG.

In 1972, Gerald Edelman and Rodney

Porter were shared the Nobel Prize in
Physiology or Medicine “for their
discoveries concerning the chemical
structure of antibodies”.
 RadioImmunoAssay (RIA)

 Based on their understanding of the specificity and

affinity of the antigen-antibody interaction,
Solomon Berson and Rosalyn Yalow at the
Veterans Administration Hospital in New York
developed a method called RadioimmunoAssay
(RIA) in 1960.

 RIA was used to measure the amount of

endogenous plasma insulin by tagging the insulin
with a radioactive label.

 Originally, they used iodine-131, half-life=8.1 days, a

beta and gamma emitter. Later, for safety reasons,
iodine-125, half- life=60.14 days, weak gamma
emitter, was used instead.

 RadioimmunoAssays opened the door for others to

 RadioImmunoAssay
(RIA)

For a diagram of RIA, go to

http://
157.93.252.5/bmia/flx0/bmia_ria_science_tab
and scroll to the bottom of the page.
 History of ELISA/EIA

 The development of the radioimmunoassay opened the

door for others to develop similar methods, like ELISA,
to test for the presence of proteins but without the use of
radioactive substances.

 In
1971, Peter Permann and Eva Engvall in Stockholm
published the first paper on Enzyme-Linked
ImmunoSorbent Assay (ELISA) showing they could
quantify the amount of IgG in rabbit serum using alkaline
phosphatase (an enzyme) as the reporter label.

 Thesame year, Anton Schuurs and Bauke Van Weemen in

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.

 The assays were highly sensitive and compared favorably

 Development of ELISA/EIA Test
Kits

http://commons.wikimedia.org/wiki/
File:ELISA.jpg
 ELISA Reader Spectrophotometer
A microplate reader with a 96-well plate in the sample
drawer

http
://en.wikipedia.org/wiki/Plate_reader#mediaviewer/File:Microplate_reader.
jpg
 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

 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.
 The Indirect ELISA Method – Part
1
a)Binding Known Antigen - The indirect ELISA method begins with
a sample of known antigen being bound to the wells of a microtiter plate.

b)Blocking - The other unoccupied sites in each well are then bound by a
concentrated solution of non-interacting protein, like casein or bovine serum
albumin, to block or prevent other proteins in the test sample from adhering.

c) Washing – Rinse to remove any unbound antigen and non-interacting

protein.

d)Adding Test Sample Primary Antibody - The test sample of serum

containing the primary antibodies is added to each well. Antibodies could be
HIV, rabies, or hepatitis B antibodies, for example.

e) Washing – Rinse to remove any antibodies that did not bind to the known
antigen.
 The Indirect ELISA Method – Part
2
f)Adding Enzyme-linked Secondary Antibody - An enzyme-
linked secondary antibody is added next to bind to the test sample
antibodies. The enzyme on the secondary antibodies are proteins, such
as horse radish peroxidase or alkaline phosphatase.

g)Washing – Rinse to remove any secondary antibodies that did

not bind to the primary antibody.

h)Adding Substrate - A substrate is then applied which is

converted by the enzyme to give a color or fluorescence or
electrochemical signal. In the presence of horse radish peroxidase,
ABTS turns green, OPD turns orange, and TMB turns blue. In the
presence of alkaline phosphatase, pNPP turns yellow.

i)Reading Results - By using a spectrophotometer,

spectrofluorometer, or electrochemical device, the results can be read
and recorded. The amount of color produced is proportional to the
amount of primary antibody bound to the antigen proteins on the
bottom of the wells.
 Indirect ELISA-
ELISA to Measure Specific Serum
Antibodies
For a 9 minute tutorial overview of Direct,
Indirect, and Sandwich ELISA, go to
https://www.youtube.com/watch?v=nNjlBCnpGZ4

For a diagram of Indirect ELISA, go to

http://www.ncbi.nlm.nih.gov/books/NBK22420/figure/A515/?report=objectonly

and scroll down the Figure 4.35. Also

included is the Sandwich ELISA method.

For a 2 minute animation of Indirect ELISA

method and test results presented by Dr.
Cary Engleberg, MD, Professor, University of
Michigan Medical School, go to
https://www.youtube.com/watch?v=RRbuz3VQ100
 Use of the Indirect ELISA Method to Test
for HIV
HIV Test Flow Chart

http://www.cdc.gov/mmwr/preview/mmwrhtml/mm6224a2
.htm
 Sandwich ELISA

As the substrate is added, the enzyme converts it into a colored

product. The rate of color formation is proportional to the amount of
antigen present.

For a diagram showing both a positive and negative result if testing for
an antigen using the sandwich ELISA method, see
https://cellularphysiology.wikispaces.com/Enzyme-
linked+immunosorbent+assay+(ELISA)
http://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm073
674.htm
 Sandwich ELISA –
Enzyme Immunoassay to Detect Antigens

For a 2 minute animation of the sandwich

ELISA method and test results presented
by Dr. Cary
Engleberg, MD, Professor, University of
Michigan Medical School, go to https://
www.youtube.com/watch?v=70TPrfL_8-M
 Other ELISA Methods

For an image of Direct ELISA method, go

to
http://en.wikipedia.org/wiki/ELISA and
scroll down to Types, Direct ELISA.

For a YouTube tutorial about Competitive

ELISA method can be found at https://
www.youtube.com/watch?v=Kb26nQVMH
ds

Multiplex ELISA method

 Immunohistochemical Staining

 While the ELISA tests are routinely used to test

antigen and antibody presence in patient blood
serum, the direct ELISA and indirect ELISA
methods have also been applied in
immunohistochemistry.
 The tissue being studied would be embedded in
paraffin and thinly sliced with a microtome onto a
glass microscope slide.
 In order to fluorescently tag a particular cell
component, the paraffin would be removed, the
antigens of the tissues retrieved, and a blocking
non-interacting protein would be added to bind all
unoccupied sites on the slide.
 Then the slide would be washed to remove any
unbound non- interacting protein.

Indirect Method-Immunohistochemical
Staining
After deparaffinization, antigen retrieval,
blocking, and washing, an antigen-specific
primary antibody is added and then washed to
remove any unbound primary antibody.
Then an enzyme-linked secondary antibody is
added and then washed to remove unbound
secondary antibodies.
As substrate is added, the interaction
between the substrate and enzyme occurs
and the cell component becomes visible.
While the direct immunohistochemical method
is much quicker, the indirect
immunohistochemical method is thought to be
more sensitive.
 Direct and Indirect Methods of
Immunohistochemistry

For a diagram of direct and indirect methods of

immunohistochemistry, go to
http://www.piercenet.com/method/immunodetection-
ihc and scroll down to Direct vs. Indirect Staining.
This site has
more examples of stained tissues.
 Immunohistochemistry Staining of the
http://en.wikipedia.org/wiki/Immunohistochemistry#mediaviewer/
Kidney File:Kidney_cd10_ihc.jpg
http://creativecommons.org/licenses/by-sa/3.0/ No changes have been made.

Neo-plastic kidney stained with CD10 which stains both the glomeruli and proximal
convoluted tubules.