The Immunologist's Toolbox

Immunization.
Natural adaptive immune responses are normally directed at antigens borne
by pathogenic microorganisms. The immune system can also be induced
to respond to simple nonliving antigens, and experimental immunologists
have focused on the responses to these simple antigens in developing our
understanding of the immune response. The deliberate induction of an
immune response is known as immunization. Experimental immunizations
are routinely carried out by injecting the test antigen into the animal or
human subject. The route, dose, and form in which antigen is administered
can profoundly affect whether a response occurs and the type of response
that is produced, and are considered in Sections A-1-A-4. The induction
of protective immune responses against common microbial pathogens in
humans is often called vaccination, although this term is correctly only applied
to the induction of immune responses against smallpox by immunizing with
the cross-reactive cowpox virus, vaccinia.
To determine whether an immune response has occurred and to follow its
course, the immunized individual is monitored for the appearance of immune
reactants directed at the specific antigen. Immune responses to most antigens
elicit the production of both specific antibodies and specific effector T cells.
Monitoring the antibody response usually involves the analysis of relatively
crude preparations of antiserum (plural: antisera). The serum is the fluid
phase of clotted blood, which, if taken from an immunized individual, is called
antiserum because it contains specific antibodies against the immunizing
antigen as well as other soluble serum proteins. To study immune responses
mediated by T cells, blood lymphocytes or cells from lymphoid organs such
as the spleen are tested; T-cell responses are more commonly studied in
experimental animals than in humans.
Any substance that can elicit an immune response is said to be immunogenic
and is called an immunogen. T here is a clear operational distinction between
an immunogen and an antigen. An antigen is defined as any substance that
can bind to a specific antibody. All antigens therefore have the potential to
elicit specific antibodies, but some need to be attached to an immunogen
in order to do so. This means that although all immunogens are antigens,
not all antigens are immunogenic. The antigens used most frequently in
experimental immunology are proteins, and antibodies against proteins are
of enormous utility in experimental biology and medicine. Purified proteins
are, however, not always highly immunogenic, and to provoke an immune
response they have to be administered with an adjuvant (see Section A-4).
Carbohydrates, nucleic acids, and other types of molecules are all potential
antigens, but will often only induce an immune response if attached to a
protein carrier. Thus, the immunogenicity of protein antigens determines the
outcome of virtually every immune response.

APPENDIX I

Appendix I: The Immunologist's Toolbox

Primary Immunization with different

doses of antigen

than the immunogen; most antibodies cross-react with closely related anti
the immunogen. These cross-reacting antibodies can create problems when

105

the antiserum is used to detect a specific antigen. They can be removed from

:e

different ways. Some of the antibodies in an antiserum are cross-reactive. A

gens but, on occasion, some bind antigens with no clear relationship to

106

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tain many different antibody molecules that bind to the immunogen in slightly

cross-reaction is defined as the binding of an antibody to an antigen other

Primary response

2
"

Antisera generated by immunization with even the simplest antigen will con

an antiserum by

104

absorption with the cross-reactive antigen, leaving behind

the antibodies that bind only to the immunogen.Absorption can be performed

103

by affinity chromatography using immobilized antigen, a technique that is

also used for the purification of antibodies or antigens (see SectionA-5). Most

102

problems of cross-reactivity can be avoided, however, by making monoclonal

101

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antibodies (see SectionA-12).

1 1 1 1 1 1 1 1
Antigen dose

Although almost any structure can be recognized by antibody as an antigen,

usually only proteins elicit fully developed adaptive immune responses.
This is because proteins have the ability to engage T cells, which contribute
to inducing most antibody responses and are required for immunological

Secondary Immunization with

single antigen dose of 1 oa units

memory. Proteins engage T cells because the T cells recognize antigens as

peptide fragments of proteins bound to major histocompatibility complex
(MHC) molecules. An adaptive immune response that includes immuno

Secondary response

logical memory can be induced by nonpeptide antigens only when they are
attached to a protein carrier that can engage the necessaryT cells (see Section
10-3 and Fig. 10.5).

106
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primary
immunization, which evokes the primary immune response. This is also
known as priming, because the animal or person is now 'primed' like a pump

Immunological memory is produced as a result of the initial or

to mount a more potent response to subsequent challenges with the same

antigen. The response to each immunization is increasingly intense, so that

secondary, tertiary, and subsequent responses are of increasing magnitude

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(Fig. A.1). Repetitive challenge with antigen to achieve a heightened state of

1
10
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immunity is known as

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hyperimmunization.

101 102 103 104 105 106 107 108

Certain properties of a protein that favor the priming of an adaptive immune

Antigen dose given in primary immunization

response have been defined by studying antibody responses to simple natural

proteins such as hen egg-white lysozyme and to synthetic polypeptide

Fig. A.1 The dose of antigen used in an
initial immunization affects the primary
and secondary antibody response.
The typical antigen dose-response curve

antigens (Fig. A.2). The larger and more complex a protein, and the more
distant its relationship to self proteins, the more likely it is to elicit a response.
This is because such responses depend on the proteins being degraded into
peptides that can bind to MHC molecules, and on the subsequent recognition

6). The larger

shown here illustrates the influence

of these peptide:MHC complexes by T cells (see Chapter

of dose on both a primary antibody

and more distinct the protein antigen, the more likely it is to contain such

response (amounts of antibody produced

peptides. Particulate or aggregated antigens are more immunogenic because

expressed in arbitrary units) and the

they are taken up more efficiently by the specialized antigen-presenting

effect of the dose used for priming on a

secondary antibody response elicited by
a dose of antigen of 1 03 arbitrary mass
units. Very low doses of antigen do not
cause an immune response at all. Slightly

cells responsible for initiating a response. Indeed, small soluble proteins are
unable to induce a response unless they are made to aggregate in some way.
Many vaccines, for example, use aggregated protein antigens to potentiate
the immune response.

higher doses seem to inhibit specific

antibody production, an effect known as
low-zone tolerance. Above these doses

A-1

Haptens.

there is a steady increase in the response

with antigen dose to reach a broad

Small organic molecules of simple structure, such as phenyl arsonates and

optimum. Very high doses of antigen

nitrophenyls, do not provoke antibodies when injected by themselves.

also inhibit immune responsiveness to a

subsequent challenge, a phenomenon
known as high-zone tolerance.

However, antibodies can be raised against them if the molecule is attached

covalently, by simple chemical reactions, to a protein carrier. Such small
molecules were termed

haptens (from the Greek haptein, to fasten) by the

immunologist Karl Landsteiner, who first studied them in the early 20th

Immunization
-------

Fig. A.2 Intrinsic properties and

Factors that influence the immunogenicity of proteins

extrinsic factors that affect the

immunogenicity of proteins.

Parameter
Size

Dose

Route

Composition

I
I
I

Increased immunogenicity

Decreased immunogenicity

Large

Small (MW<2500)

Intermediate

Subcutaneous

>

intraperitoneal

Complex

Particulate
Form
Denatured

Similarity to self protein

II
II
II

High or low

>

intravenous or intragastric

Soluble

Native

Few differences

Slow release

Rapid release

Bacteria

Effective

II
II

I
I
I

Simple

Multiple differences

Adjuvants

Interaction with host MHC

II

MW, molecular weight in daltons.

No bacteria

Ineffective

Immunize rabbit with

hapten-carrier conjugate

century. He found that animals immunized with a hapten-carrier conjugate

produced three distinct sets of antibodies (Fig. A.3). One set comprised
hapten-specific antibodies that reacted with the same hapten on any carrier,
as well as with free hapten. The second set of antibodies was specific for the
carrier protein, as shown by their ability to bind both the hapten-modified

and unmodified carrier protein. Finally, some antibodies reacted only

Antiserum

with the specific conjugate of hapten and carrier used for immunization.
Landsteiner studied mainly the antibody response to the hapten, as these
small molecules could be synthesized in many closely related forms. He
observed that antibodies raised against a particular hapten bind that hapten
but, in general, fail to bind even very closely related chemical structures. The
binding of haptens by anti-hapten antibodies has played an important part
in defining the precision of antigen binding by antibody molecules. Anti
hapten antibodies are also important medically because they mediate allergic
reactions to penicillin and other compounds that elicit antibody responses
when they attach to self proteins (see Section 14-10).

Binding
to free
carrier

~ .;..*_,

antibodies are produced. One set (blue) binds the carrier protein alone and is called

carrier-specific. One set (red) binds to the hapten on any carrier or to free hapten in
of hapten and carrier used for immunization, apparently binding to sites at which the
hapten joins the carrier, and is called conjugate-specific. The amount of antibody of
each type in this serum is shown schematically in the graphs at the bottom; note that
the original antigen binds more antibody than the sum of anti-hapten and anti-carrier
antibodies as a result of the additional binding of conjugate-specific antibody.

Binding to
hapten-carrier
conjugate

Fig. A.3 Antibodies can be elicited by small chemical groups called haptens
only when the hapten is linked to an immunogenic protein carrier. Three types of

solution and is called hapten-specific. One set (purple) only binds the specific conjugate

Binding
to hapten on
unrelated
carrier

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Antigen

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Antigen

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Antigen

Appendix I: The Immunologist's Toolbox

A-2

Routes of immunization.

The route by which antigen is administered affects both the magnitude and
the type of response obtained. The most common routes by which antigen
is introduced experimentally or as a vaccine into the body are injection
into tissue by subcutaneous (s.c.) injection into the fatty layer just below
the dermis, or by intradermal (i.d.) or intramuscular (i.m.) injection; by

intravenous (i.v.) injection or transfusion directly into the bloodstream; into

the gastrointestinal tract by oral administration; and into the respiratory tract
by intranasal (i.n.) administration or inhalation.
Antigens injected subcutaneously generally elicit the strongest responses,
most probably because the antigen is taken up by Langerhans cells in the
skin and efficiently presented in local lymph nodes, and so this is the method
most commonly used when the object of the experiment is to elicit specific
antibodies or T cells against a given antigen. Antigens injected or transfused
directly into the bloodstream tend to induce immune unresponsiveness or
tolerance unless they bind to host cells or are in the form of aggregates that
are readily taken up by antigen-presenting cells.
Antigen administration via the gastrointestinal tract is used mostly in the
study of allergy. It has distinctive effects, frequently eliciting a local antibody
response in the intestinal lamina propria, while producing a systemic state
of tolerance that manifests as a diminished response to the same antigen if
subsequently administered in immunogenic form elsewhere in the body
(see Chapter 12). This 'split tolerance' may be important in avoiding allergy
to antigens in food, because the local response prevents food antigens
from entering the body, while the inhibition of systemic immunity helps to
prevent the formation of IgE antibodies, which are the cause of such allergies
(see Chapter 14).
Introduction of antigen into the respiratory tract is also used mainly in the
study of allergy. Protein antigens that enter the body through the respiratory
epithelium tend to elicit allergic responses, for reasons that are not clear.

A-3

Effects of antigen dose.

The magnitude of the immune response depends on the dose of immunogen

administered. Below a certain threshold dose, most proteins do not elicit any
immune response. Above the threshold dose, there is a gradual increase in
the response as the dose of antigen is increased, until a broad plateau level
is reached, followed by a decline at very high antigen doses (see Fig. A.l).
Because most infectious agents enter the body in small numbers, immune
responses are generally elicited only by pathogens that multiply to a level
sufficient to exceed the antigen dose threshold. The broad response optimum
allows the system to respond to infectious agents across a wide range of
doses. At very high antigen doses, the immune response is inhibited, which
may be important in maintaining tolerance to abundant self proteins such as
plasma proteins. In general, secondary and subsequent immune responses
occur at lower antigen doses and achieve higher plateau values, which is a
sign of immunological memory. However, under some conditions, very low
or very high doses of antigen may induce specific unresponsive states, known
respectively as acquired low-zone or high-zone tolerance.

A-4

Adjuvants.

Most proteins are poorly immunogenic or nonimmunogenic when adminis

tered by themselves. Strong adaptive immune responses to protein antigens
almost always require that the antigen be injected in a mixture known as an

Immunization
-------

adjuvant. An adjuvant is any substance that enhances the immunogenicity of

substances mixed with it. Adjuvants differ from protein carriers in that they
do not form stable linkages with the immunogen. Furthermore, adjuvants are
needed primarily for initial immunizations, whereas carriers are required to
elicit not only primary but also subsequent responses to haptens. Commonly
used adjuvants are listed in Fig. A.4.
Adjuvants can enhance immunogenicity in two different ways. First, adju
vants convert soluble protein antigens into particulate material, which is
more readily ingested by antigen-presenting cells such as macrophages. For
example, the antigen can be adsorbed on particles of the adjuvant (such as
alum), made particulate by emulsification in mineral oils, or incorporated
into the colloidal particles of immune stimulatory complexes (ISCOMs). This
enhances immunogenicity somewhat, but such adjuvants are relatively weak
unless they also contain bacteria or bacterial products. Such microbial con
stituents are the second means by which adjuvants enhance immunogenic
ity, and although their exact contribution to enhancing immunogenicity is
unknown, they are clearly the more important component of an adjuvant.
Microbial products may signal macrophages or dendritic cells to become
more effective antigen-presenting cells (see Section 3-10). One of their effects
is to induce the production of inflammatory cytokines and potent local
inflammatory responses; this effect is probably intrinsic to their activity in
enhancing responses, but precludes their use in humans.
Nevertheless, some human vaccines contain microbial antigens that can also
act as effective adjuvants. For example, purified constituents of the bacterium

Bordetella pertussis, which is the causative agent of whooping cough, are

used as both antigen and adjuvant in the triplex DPT (diphtheria, pertussis,
tetanus) vaccine against these diseases.
B cells contribute to adaptive immunity by secreting antibodies, and the

response of B cells to an injected immunogen is usually measured by

Adjuvants that enhance immune responses

Fig. A.4 Common adjuvants and their

use. Adjuvants are mixed with the antigen
and usually render it particulate, which

Adjuvant name

Incomplete Freund's adjuvant

Complete Freund's adjuvant

Mechanism of action

Composition

Delayed release of antigen;

enhanced uptake by
macrophages

Oil-in-water emulsion

Delayed release of antigen;

enhanced uptake by
macrophages; induction of
co-stimulators in macrophages

Oilinwater emulsion
with dead mycobacteria

helps to retain the antigen in the body

and promotes uptake by macrophages.
Most adjuvants include bacteria or
bacterial components that stimulate
macrophages, aiding in the induction
of the immune response. ISCOMs are
small micelles of the detergent Qui I A;
when viral proteins are placed in these
micelles, they apparently fuse with the
antigen-presenting cell, allowing the
antigen to enter the cytosol. Thus, the

Freund's adjuvant with MDP

Oilinwater emulsion
with muramyldipeptide (MDP),
a constituent of mycobacteria

Similar to complete
Freund's adjuvant

antigen-presenting cell can stimulate a

response to the viral protein, much as a
virus infecting these cells would stimulate
an antiviral response.

Ill

Delayed release of antigen;

enhanced macrophage uptake

Alum (aluminum hydroxide)

Aluminum hydroxide gel

Alum plus

Bordetella pertussis

Aluminum hydroxide gel

with killed B. pertussis

Delayed release of antigen;

enhanced uptake by
macrophages; induction
of co-stimulators

Immune stimulatory
complexes (ISCOMs)

Matrix of Quil A
containing viral proteins

Delivers antigen to cytosol;

allows induction of cytotoxic
T cells

Appendix I: The Immunologist's Toolbox

analyzing the specific antibody produced in a humoral immune response.

This is most conveniently achieved by assaying the antibody that accumu
lates in the fluid phase of the blood or plasma; such antibodies are known as
circulating antibodies. Circulating antibody is usually measured by collecting
blood, allowing it to clot, and then isolating the serum from the clotted blood.
The amount and characteristics of the antibody in the resulting antiserum are
then determined using the assays we describe in Sections A-5-A-11.
The most important characteristics of an antibody response are the specifi
city, amount, isotype or class, and affinity of the antibodies produced. The
specificity determines the ability of the antibody to distinguish the immuno
gen from other antigens. The amount of antibody can be determined in many
different ways and is a function of the number of responding B cells, their rate
of antibody synthesis, and the persistence of the antibody after production.
The persistence of an antibody in the plasma and extracellular fluid bathing
the tissues is determined mainly by its isotype or class (see Sections 5-12 and
10-14); each isotype has a different half-life in vivo. The isotypic composition
of an antibody response also determines the biological functions these anti
bodies can perform and the sites in which antibody will be found. Finally, the
strength of binding of the antibody to its antigen in terms of a single antigen
binding site binding to a monovalent antigen is termed its affinity; the total
binding strength of a molecule with more than one binding site is called its
avidity. Binding strength is important, because the higher the affinity of the
antibody for its antigen, the less antibody is required to eliminate the antigen,
because antibodies with higher affinity will bind at lower antigen concentra
tions. All these parameters of the humoral immune response help to deter
mine the capacity of that response to protect the host from infection.
Antibody molecules are highly specific for their corresponding antigen, being
able to detect one molecule of a protein antigen out of more than 108 similar
molecules. This makes antibodies both easy to isolate and easy to study, and
invaluable as probes of biological processes. Whereas standard chemistry
would have great difficulty in distinguishing between two such closely related
proteins as human and pig insulin, or two such closely related structures
as ortho- and para-nitrophenyl, antibodies can be made that discriminate
between these two structures absolutely. The value of antibodies as mole
cular probes has stimulated the development of many sensitive and highly
specific techniques to measure their presence, to determine their specificity
and affinity for a range of antigens, and to ascertain their functional
capabilities. Many standard techniques used throughout biology exploit the
specificity and stability of antigen binding by antibodies. Comprehensive
guides to the conduct of these antibody assays are available in many books on
immunological methodology ; we will illustrate here only the most important
techniques, especially those used in studying the immune response itself.
Some assays for antibody measure the direct binding of the antibody to its
antigen. Such assays are based on primary interactions. Others determine
the amount of antibody present by the changes it induces in the physical state
of the antigen, such as the precipitation of soluble antigen or the clumping
of antigenic particles; these are called secondary interactions. Both types of
assay can be used to measure the amount and specificity of the antibodies
produced after immunization, and both can be applied to a wide range of
other biological questions.
Because assays for antibody were originally conducted using antisera from
immune individuals, they are commonly referred to as serological assays,
and the use of antibodies is often called serology. The amount of antibody is
usually determined by antigen-binding assays after titration of the antiserum
by serial dilution, and the point at which binding falls to 50% of the maximum
is usually referred to as the titer of an antiserum.