Introduction to Immunoassays

ELISA Technique

Dr. Maha Daghestani

Introduction to Immunoassays
ELISA Technique

Dr. Maha Daghestani

After completion of this lecture, you
will be able to:

• define immunoassay
• describe the structure and preparation of
antibodies
• define four categories of immunoassay
methodology
• (competitive and noncompetitive, and
homogeneous and heterogeneous)
Dr. Maha Daghestani
 Introduction

• An immunoassay is a test that uses antibody and

antigen complexes as a means of generating a
measurable result.

• An antibody:antigen complex is also known as an

immuno-complex.

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 “Immuno”& “assay”
• “Immuno” refers to an immune response that
causes the body to generate antibodies,
and
• “assay” refers to a test. Thus, an immunoassay
is a test that utilizes immunocomplexing when
antibodies and antigens are brought together

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• Immunoassays are different from other types
of laboratory tests, such as colorimetric tests,
because they use antibody:antigen
complexes to generate a signal that can be
measured.

• In contrast, most routine clinical chemistry

tests utilize chemical reactions between the
reagent (a solution of chemicals or other
agents) and patient sample to generate a test
result
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 Immunoassay: Antibodies, Antigens
and Analytes Defined

• An antibody is a protein that is produced by the

body in response to an “invading” (foreign)
substance.

• Antibodies are produced as part of the body’s

immune response to protect itself. For instance,
some immunoassays test for the presence of
antibodies to cancer molecules. Thus, if the
antibodies are present, it means invading cancer
cells are, too.
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 An antigen
• An antigen is the substance that the body
is trying to “fight off” (eliminate or reduce)
by mounting an immune response. Some
immunoassays test for antigens directly,
rather than looking for the antibodies. In a
test to measure the concentration of a
therapeutic drug, for example, the drug is
the antigen that binds to the antibody.

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 An analyte
• An analyte is anything measured by a
laboratory test. In immunoassay testing,
the analyte may be either an antibody, or
an antigen.

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 Immunoassays
• Immunoassays utilize one or more select
antibodies to detect analytes of interest. The
analytes being measured may be those
that are naturally present in the body (such as
a thyroid hormone), those that the body
produces but are not typically present (such as
a cancer antigen), or those that
do not naturally occur in the body (such as an
abused drug).
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 Antibodies

• Antibodies possess high

a) specificity
and
b) affinity
for a specific antigen. It is the specific binding of
an antibody to an antigen that allows the detection
of analytes by a variety of immunoassay methods.

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 Structure of Antibodies

• Antibodies (Ab) are a type of protein called

immunoglobulins.
• The most common one is immunoglobulin
G (IgG).
• IgG is a protein composed of two main
structural and functional regions

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Dr. Maha Daghestani
 Preparation of Polyclonal and
Monoclonal Antibodies
• Antibody reagents are developed from either
polyclonal or monoclonal antibodies.

• Polyclonal antiserum (serum from blood containing

the desired antibodies) is generated in animals,
most commonly sheep, rabbits, or goats.

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 Cont’ed
• The animals produce the antiserum - just as a
human would - as a defense mechanism when
exposed to an antigen.
• Antiserum contains a mixture of antibodies,
each of which may bind to different antigen
binding sites, or epitopes.

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 Producing antiserum
• The process of making an antiserum begins by injecting
a solution that contains the antigen of interest into an
animal. This antigen of interest is sometimes called an
immunogen, because it can stimulate an immune
response.
Over time, and in some cases with multiple injections,
the immune system of the animal produces antibodies to
the antigen that was injected.
Blood is collected from the animal, and serum is isolated
from the blood. This serum is usually rich in antibodies
that recognize the antigen, and is called
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the antiserum.
Dr. Maha Daghestani
 polyclonal antibodies
• Antiserum usually contains a mixture of
antibodies that recognize and bind to the same
antigen, but they may attach to different epitopes
(see Figure 1-2). An antigen that has multiple
sites for antibodies to bind is called a multivalent
antigen. These types of antibodies, present as a
diverse mixture, are called polyclonal antibodies.

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 Monoclonal antibodies
• Monoclonal antibodies differ from polyclonal
antibodies in that they are highly specific for a
single epitope on a multivalent antigen (see Figure
1-3). They are produced from a single cell line
using hybridoma technology and mouse myeloma
cell lines.
• Hybridomas are antibody-producing tumor cells
that produce many copies of the same antibody and
grow easily in laboratory cell culture.
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 An advantage
• An advantage of monoclonal antibodies is that the
hybridoma cell line that produces them is
potentially “immortal” and can produce the same
antibodies consistently and indefinitely.
• A polyclonal antisera produced by immunization
of animals can vary from animal to animal, and a
useful antiserum may no longer be available if the
single animal that produces it dies.

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Dr. Maha Daghestani
 procedure
Hybridomas are produced in a multi-step procedure (see
Figure 1-4):
• • injecting a specific antigen into a host animal (typically
a mouse);
• • isolating antibody-producing cells (B cells) from the
spleen of the mouse;
• • fusing these B cells with a specific type of tumor cell
that grows easily in culture and produces antibodies;
• • isolating successful hybridomas (fused cells) that
produce antibodies specific for the antigen of interest.
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Dr. Maha Daghestani
 In immunoassays
• In immunoassays, both monoclonal and polyclonal
antibodies are used for detecting antigens, each with
specific strengths for particular applications.
• Immunoassays that detect antibodies in patient sera
are likely to involve detection of polyclonal
antibodies generated by the patient’s immune
system.

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 Categories of Immunoassay
Methodologies

• The immunoassay methodologies are:

• noncompetitive and competitive immunoassays,
and
• homogeneous and heterogeneous immunoassays

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 labeled material

• All immunoassays require the use of labeled

material in order to measure the amount of antigen
or antibody present.
• A label is a molecule that will react as part
of the assay, so a change in signal can be measured
in the blood:reagent solution.

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 Examples of a label
• Examples of a label include a radioactive
compound, an enzyme that causes a change of
color in a solution, or a substance that produces
light.
• The label can be applied during the manufacture
of the reagent to either the antibody (Ab*, see
Figure 1-5) or antigen (Ag*, see Figure 1-6).
• Immunoassay technologies utilize different
formats to distinguish the bound antigen-antibody
complex from the free unbound
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Dr. Maha Daghestani
 Competitive and Noncompetitive
Immunoassays

• The measurement of analyte in an immunoassay is

achieved by using either

a competitive
or
a noncompetitive format.

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 Competitive Format

• In competitive formats,
• unlabelled analyte (usually antigen) in the test
sample is measured by its ability to compete
with labeled antigen in the immunoassay.
• The unlabeled antigen blocks the ability of the
labeled antigen to bind because that binding site
on the antibody is already occupied.

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 Con’ted
• Thus, in a competitive immunoassay, less label
measured in the assay means more of the unlabeled
(test sample) antigen is present. The amount of
antigen in the test sample is inversely related to the
amount of label measured in the
• competitive format (Figure 1-7).

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 One step competitive format
• In the one step competitive format (see Figure 1-8),
both the labeled antigen reagent (Ag*) and the
unlabeled specimen (or test sample analyte) compete
for a limited amount of antibody.

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 Two step competitive format

• In the two step competitive format, the antibody

concentration of the reaction solution is present in
excess in comparison to the concentration of
antigen. Antibody reagent is first incubated with
specimen containing antigens of interest; then in
the second step, labeled antigen is added.
Remember that in the competitive format, less
bound labeled antigen indicates more antigen
present in the test sample. Two step competitive
assay formats provide several fold improved assay
sensitivity compared to Daghestani
Dr. Maha one step assay formats.
Dr. Maha Daghestani
 Noncompetitive (Sandwich) Method

• Noncompetitive assay formats generally provide the

highest level of assay sensitivity and specificity and
are applied to the measurement of critical analytes
such as cardiac and hepatitis markers. This format is
referred to as a “sandwich” assay because analyte is
bound (sandwiched) between two highly specific
antibody reagents (Figure 1-10).

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Dr. Maha Daghestani
 Noncompetitive assay
• Noncompetitive assay formats can also utilize either
one step or two step methods, as with the
competitive assay.
• The two step assay format employs wash steps in
which the sandwich binding complex is isolated and
washed to remove excess unbound labeled reagent
and any other interfering substances.
• The two step noncompetitive format usually offers
the highest specificity and sensitivity of all the
• assay formats discussed here.
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Dr. Maha Daghestani
 Homogeneous and Heterogeneous
Immunoassay Methods
• Immunoassay methods that require separation of
bound Ab-Ag* complex are referred to as
heterogeneous immunoassays. Those that do not
require separation are referred to as homogeneous
immunoassays.
• Homogeneous methods have been generally applied
to the measurement of small analytes such as abused
and therapeutic drugs. Since homogeneous methods
do not require the separation of the bound Ab-Ag*
from the free Ag*, they are generally much easier
and faster to perform.
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Dr. Maha Daghestani
 Summary
• Immunoassays are tests that use antibody and
antigen complexes (also called
immunocomplexes) to measure the presence of a
specific analyte in a sample.
• Antibodies are proteins that are normally
produced by the immune system in response to a
foreign substance.
• Antigens are the molecules that antibodies bind
to, which in the body could be an invading
pathogen, or the foreign molecules injected into
an animal to trigger the immune response.
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 Summary
• Antibodies are comprised of two major regions,
the Fab region (antigen specific) and the Fc
region.
• Antibody preparations are either polyclonal
antisera, which recognize multiple sites on
antigens, or monoclonal antibodies, which
recognize single sites on antigens.

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 Summary

• In immunoassays, the antibody or antigen is

labeled in order to have a measurable signal that
corresponds to the concentration of the analyte.
• Immunoassays can be either competitive or
noncompetitive. In competitive immunoassays,
the amount of antigen is indirectly proportional
to the amount of signal. In noncompetitive
immunoassays, the amount of antigen is directly
proportional to the amount of signal.

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 Summary
• Homogeneous immunoassays do not require
separation of unbound complexes from the bound
complexes, and thus are faster and easier to
perform then heterogeneous immunoassays.

• Heterogeneous immunoassays require the

separation of unbound complexes, often utilizing a
solid phase reagent such as a magnetic particle or
plastic bead.
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Questions?

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