Exercise Your Options – Appendix
Appendix
“Exercise Your Options”
Immunology Overview
 Antigens
Antigen is short for antibody-generating. Antibodies are protective proteins that are
produced by the immune system when an animal is exposed to an antigen. Antigens
are also referred to as foreign entities because the immune system is designed to fight
off foreign invaders. Antigens are usually proteins, but may also be carbohydrates,
lipids, and nucleic acids. Some macromolecules are better at generating an immune
response than others and proteins are the “best” antigens. Therefore, we will restrict
our discussion of antigens to protein antigens.
When thinking of foreign entities that trigger an immune response, many people think
in terms of whole organisms (such as bacteria, viruses, and fungi). While it is true
that these are antigens, so are bee venom, pollen, and vaccines. Remember, an
antigen is something that results in an immune response and can therefore be just
about any protein. In fact, one protein can actually be more than one antigen! This is
because the part of the immune system that responds to an antigen only recognizes
and responds to a small part of that antigen, called an epitope. Epitopes are usually
composed of 7-25 amino acids, so, one antigen (such as a whole E. coli) will consist
of many different proteins and each protein will contain several different epitopes.
Each epitope results in a different antibody being formed, with each antibody being
specific for one, and only one, epitope. Although epitopes will become important
when discussing the types of reagents needed for an ELISA, the word antigen will be
used here to refer to epitopes. Figure 1 illustrates the principle of an epitope.
Figure 1: Epitopes (antigenic determinants)
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 Antibodies
Antibodies are proteins produced by the body in response to an antigen. Several
features of antibodies are important to understand in order to appreciate and design
ELISAs. First, antibodies are specific for their corresponding antigen. An E. coli, for
example, that induces an immune response will result in antibodies to E. coli and not
to HIV or any other antigen. Second, antibodies bind to their corresponding antigen.
Again, antibodies to E. coli will bind only to E. coli and not to HIV or any other
antigen. The specificity of antibody-antigen binding is due to the types of amino
acids present in these two proteins that are able to form hydrogen bonds with each
other. Antibody-antigen interactions, then, are specific, strong, and mediated by
hydrogen bonds. The specificity of antibody-antigen binding is what makes an
ELISA so effective. As with all matters biological, this specificity has its exceptions.
When an antibody against one antigen recognizes and binds to another antigen, we
say that antibody is cross-reactive. This is most common when the antigens are very
closely related (such as the same protein from different animals) and needs to be
considered when choosing reagents for an ELISA.
The structure of an antibody molecule is like the letter “Y.” The tips of the “Y” are
the antigen-binding sites and the amino acid sequences in this region of the antibody
molecule show tremendous variation. The stalk of the “Y” is what gives the antibody
its ability to eliminate the antigen, often by increasing the effectiveness of the body’s
phagocytic white blood cells. Unlike the variable, antigen-binding region, there are
only a few possible amino acid sequences that make up the stalk of the “Y.”
Therefore, this region is called the constant region, or isotype. There are five
different isotypes and they are identified by letters. The five isotypes are IgD, IgM,
IgG, IgA, and IgE. Ig stands for immunoglobulin which is another name for
antibody. The most important isotypes used in an ELISA are IgG and IgA,
occasionally IgM. IgG is the isotype found in serum, IgA is the isotype found in
mucous secretions, and IgM is the first isotype found upon antigen exposure. Figure
2 illustrates antibody structure.
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Figure 2: Antibody Structure
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Because antibodies are proteins, they can function as antigens when injected into
other animals. For example, human IgG can be injected into sheep and the sheep will
make antibodies to this. This product will be called “sheep anti-human IgG.” This
antibody is available as an ELISA reagent, and the translation means “made in sheep
against human immunoglobulin isotype G.”
This brings us to a discussion of polyclonal vs. monoclonal antibodies.
Polyclonal means “from many clones” and refers to the fact that the antibodies
present in a polyclonal sample are from different B-cell clones. B-cells are the white
blood cells of the immune system that produce antibodies. Remember that antigens
have different epitopes, so each B-cell recognizes, responds, and produces antibodies
to a different epitope of the same antigen. Polyclonal antibodies are all directed
against the same antigen (E. coli, for example), but recognize many different epitopes
of that antigen. Polyclonal antibodies are easy to produce. They are made by
injecting an animal (goat, sheep, rabbit and donkey are the most common) with the
desired antigen and collecting blood from that animal. The antibodies against the
desired antigen are then purified from the blood. So, using our example above,
“sheep anti-human IgG” is a polyclonal antibody. How do you know? Because it is
made in a sheep. Any large animal will be used to make polyclonal antibodies only.
Monoclonal means “from one clone.” Monoclonal antibodies can be thought of as
purified polyclonals and are produced by one B-cell clone against one epitope of one
antigen. The method by which monoclonal antibodies are produced is very different
from the production of polyclonal antibodies. Monoclonal antibodies are made in
mice. First, mice are injected with the desired antigen. Then, the spleen is removed
from the mice and the spleen cells are mixed with another type of cell in a process
called a fusion. These two cell types result in a hybridoma that produces (one hopes)
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antibodies to the desired antigen. The entire process of producing monoclonal
antibodies may take years, so they are much more expensive than polyclonal
antibodies. If our example antibody was called “mouse anti-human IgG” then you
would know it was a monoclonal (can you imagine collecting enough blood from a
mouse to purify polyclonal antibodies?!).
Either polyclonal or monoclonal antibodies may be used in an ELISA—it really
depends on the application and the laboratory. Most labs develop protocols using
polyclonal antibodies, if possible, due to their decreased cost. However, sometimes
the polyclonals available don’t work, and monoclonals may be used instead. There is
no hard and fast rule about when to use monoclonals over polyclonals.
One last type of antibody worth mentioning is the conjugated antibody. This is the
last step to an ELISA (discussed below). A conjugated antibody has an enzyme, or
other indicator molecule, chemically attached to the isotype region and is purchased
in this form. There are many types of molecules that can be conjugated to antibodies,
but in an ELISA the conjugated molecule is always an enzyme.
All of these antibodies (polyclonal, monoclonal, conjugated) can be ordered from
companies that specialize in this type of product.
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ELISA
ELISA stands for enzyme-linked immunosorbent assay. It is an assay, or test, that uses
the principles of antigen-antibody specificity to detect the presence of one specific protein in a
complex mixture of proteins. Typical uses for the ELISA include diagnostic (HIV infection,
pregnancy) and research (monoclonal antibodies, recombinant proteins) applications. There are
three main types of ELISAs: direct, indirect, and sandwich. The type of ELISA that is chosen
for a particular application depends on many factors, including type and amount of protein
product, form of the material being tested, and availability of appropriate reagents. All ELISAs,
however, use an enzyme-conjugated antibody to detect the desired protein (hence, “enzymelinked”). Descriptions and diagrams of each type of ELISA are to follow.
All ELISAs are done in specially designed 96-well microtiter plates, using typical
volumes ranging from 50l – 100l per well. The first step to an ELISA is “coating the plate.”
In this step, some type of protein (which varies with the type of ELISA) is mixed with a buffer
that enables the protein to stick to the wells of the microtiter plate. Repeated washings and
additions of other reagents will not remove the proteins from the plate once the plate has been
coated. After coating the plate, there is typically a “blocking” step, where another protein (such
as gelatin or bovine serum albumin) is added to the plate to prevent non-specific binding to the
proteins coated on the plate. After blocking, there may be several other steps (again, varies with
the type of ELISA) of protein addition. The last steps of all ELISAs are the addition of the
“conjugate” (the antibody with the enzyme linked to it) and then the addition of the proper
substrate of the enzyme. This substrate is designed to change color if the enzyme is present. So
a positive result in an ELISA is a color change. Many different enzyme-substrate combinations
can be used, but typically the enzyme is either alkaline-phosphatase (AP) or horseradishperoxidase (HRP). Substrates can be chosen that turn blue, green, orange, or yellow and the
absorbance is read on a special spectrophotometer (called a plate-reader) that is designed for 96well plates. The higher the absorbance number, the more specific protein present in the mixture.
Therefore, ELISAs are not just qualitative, but also quantitative, assays.
Think of ELISAs, then, as stacks of proteins. In each type of ELISA, the proteins stick to
each other in a specific manner (via hydrogen bonds) and the main difference between the types
of ELISAs is the order in which proteins are stacked.
 Direct
A direct ELISA is the simplest, and least used, type of ELISA. It is used to detect a
specific antigen present in a complex mixture of proteins. Because of sensitivity and
cross-reactivity problems, it has limited application in research. In this ELISA, the
first step is to coat the plate with a series of dilutions of the complex mixture of
proteins*. All proteins, therefore, will bind to the microtiter plate. A blocking step
follows, and then the conjugate is added. The conjugate will bind to its corresponding
antigen on the plate. The last step is substrate addition. Because conjugates are
almost always anti-immunoglobulins, the direct ELISA is mainly used to detect
immunoglobulins in serum or supernatant.
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Figure 3: Direct ELISA
Step 1: A complex mixture of proteins, containing the desired antigen (protein of interest) are
added to the wells.
Step 2: An enzyme-labeled antibody to the protein of interest is added to the wells.
e
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Step 3: A chromogenic substrate is added that in the presence of the enzyme, changes color. The
amount of color that develops is inversely proportional to the amount of the protein of interest in
the protein mixture.
color color
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*When coating plates with a lot of different proteins, the antigen of interest may be
hidden and hard to find. A larger concentration of total protein, therefore, may
actually give a lower absorbance (which is the opposite of what is expected). This
phenomenon is known as antigen excess and is a problem with direct ELISAs. Think
of it in terms of the well being too crowded, so the conjugated antibody cannot find its
antigen. Therefore, when doing a direct ELISA, the plates are coated with a series of
dilutions of the protein mixture. This will ensure that the protein of interest can be
found by the conjugated antibody. Antigen excess is not a problem with the indirect
or sandwich ELISAs because in these assays purified proteins are used to coat the
plate.
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 Indirect
An indirect ELISA is probably the most commonly used type of ELISA in research
applications. It is used to detect a specific antibody present in a complex mixture of
proteins. In this ELISA, the first step is to coat the plate with purified antigen. A
blocking step follows, and then the complex mixture of proteins is added. Any
antibodies present in the mixture that are specific for the antigen on the plate will bind
to the antigen. All other antibodies and proteins will be washed off. The next step is
the conjugate step. The conjugate is an anti-immunoglobulin that recognizes the first
antibody bound to the antigen. Remember that antibodies are proteins and can be
antigens in other animal species! The enzyme-conjugated antibody binds the antibody
that is bound to the antigen that is stuck to the plate. This is visualized using the
proper substrate. Indirect ELISAs are used to detect the presence of anti-HIV
antibodies in human serum (diagnostic application) and to detect the presence of
monoclonal antibodies in tissue culture supernatant (research application). See
Figure 4(a) for a diagram of an indirect ELISA.
 Sandwich
A sandwich ELISA is another commonly used ELISA in research applications. It can
be thought of as a combined direct and indirect, and it is used to detect a specific
antigen in a complex mixture of proteins. It gives more flexibility and sensitivity to
the direct ELISA that is also used to detect antigens. The first step to a sandwich
ELISA is to coat the plate with an antibody that is specific for the desired protein.
After a blocking step, the complex mixture of proteins is added. If the desired protein
is present in this mixture, it will bind to the antibody on the plate. Because this
antibody removes the antigen from the other proteins in the mixture, it is often
referred to as the capturing antibody. To detect the desired protein it is usually
necessary to use two more antibodies. The only exception to this is if the desired
protein is actually an antibody or if there is a conjugate available to the desired
protein (highly unlikely). We will assume two more antibodies are needed. The
second antibody that is used will differ slightly from the capturing antibody. It will
bind to other epitope sites on the desired protein. The conjugate is added last and it
will bind to this second antibody. The addition of substrate follows and a color
change will result. Sandwich ELISAs can be used to detect recombinant proteins
produced in cell culture (research application). Two types of sandwich ELISAs are
depicted in Figure 4(b).
Figure 4(c) depicts a competitive ELISA. This type of ELISA is not discussed as a
part of this case study and laboratory exercise.
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Figure 4: More ELISA Types
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Some Common Abbreviations/Symbols Associated with ELISAs
ELISA: enzyme-linked immunosorbent assay
:
anti (refers to an antibody, as in -necrotin)
animal designations:
dk:
donkey
gt:
goat
hu:
human
mo: mouse
rb:
rabbit
sh:
sheep
POI:
protein of interest (refers to what protein you are looking for)
enzymes:
AP: alkaline phosphatase
HRP: horseradish peroxidase
sn:
supernatant
PBS: phosphate-buffered saline
Y:
Ig:
IgG:
IgA:
IgE:
IgM:
IgD:
antibody
immunoglobulin
immunoglobulin type G
immunoglobulin type A
immunoglobulin type E
immunoglobulin type M
immunoglobulin type D
Ab:
Ag:
antibody
antigen
moAb: monoclonal antibody
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