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CHAPTER 2
ANTIGEN/ANTIBODY INTERACTIONS
See APPENDIX (1) THE PRECIPITIN CURVE; (2) LABELING OF ANTIBODIES
The defining characteristic of HUMORAL immune responses (which distinguishes
them from CELL-MEDIATED responses), is their ability to be transferred by serum, and
the proteins within serum which are responsible for such immunity are ANTIBODIES.
We can formulate intriguingly circular definitions for antibodies and ANTIGENS, and
note that the universal property of antibodies is their ability to specifically bind their
cognate antigens. The consequences of such binding, however, can vary considerably,
depending on the nature of the particular antigen and antibody involved.
We distinguish the PHYSICAL and the BIOLOGICAL PROPERTIES of
antibodies, and the properties of ANTIGENICITY versus IMMUNOGENICITY, and
introduce the concept of ADJUVANTS, substances which are capable of increasing
immunogenicity.
We'll begin by defining three important terms:
ANTIBODY - The molecule present in serum and other body fluids which mediates
humoral immunity, and which can bind specifically to an antigen. Serum which
contains antibodies (directed against one or more antigens) is termed an antiserum.
ANTIGEN - A molecule which can be specifically bound by an antibody (typically a
protein or carbohydrate recognized as "foreign").
EPITOPE (= “antigenic determinant” = "antigenic specificity") - The minimum
target structure on an antigen which is bound by a particular antibody molecule. A
particular antigen molecule may (and generally does) bear many different epitopes or
“determinants”, each of which can be a target for antibody binding.
(NOTE: Antibodies themselves can serve as antigens; human antibodies, for instance, are
"foreign" to rabbits, and can elicit rabbit antibodies to human antibody molecules. As we
will see later, the use of antibodies as antigens has been an extremely powerful tool for
understanding antibody structure and genetics.)
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DEFINING HUMORAL IMMUNITY
Experimentally defining a humoral immune response involves demonstrating that such
immunity can be transferred by serum (or other fluids). The example below (Fig, 2-1)
illustrates some key features of humoral immunity.
Live
Pneumococcus
DIES
Naive
Killed
Pneumococcus
Naive
Immune
Live
Pneumococcus
wait
2
weeks
TRANSFER
SERUM
SURVIVES
Immune
"Active Immunity"
Live
Pneumococcus
SURVIVES
Naive
"Passive Immunity"
Figure 2-1
If a mouse is injected with a sufficient dose of live Pneumococcus bacteria, it will die
of infection within a few days. If, however, it has previously been injected with killed
organisms, not only does it not succumb to infection, but it will survive a subsequent
injection of a normally lethal dose of this organism; such a mouse has been immunized, and is
therefore said to be immune to Pneumococcus. Although not illustrated here, we can further
demonstrate that this resistance is specific – the immune mouse will retain normal
susceptibility to some other organism to which it had not previously been exposed. Such
specificity establishes that the immunity we see is a result of the mouse’s adaptive immune
response.
Question: Does this resistance represent humoral immunity?
To find out, we take serum from the immune mouse and inject it into a non-immune
recipient, then inject a lethal dose of Pneumococcus. We find that this recipient survives this
treatment; serum from an immune mouse transfers immunity to a naïve recipient. This
demonstrates that immunity to this organism is mediated by humoral immunity. (NOTE:
This does not, however, mean that resistance to all bacterial infections is mediated by
humoral immunity. As we will see in Chapter 12, transferring serum from a mouse which is
immune to another bacterium, Listeria (which is an intracellular pathogen), does not confer
resistance to naïve recipients; such immunity is therefore not humoral.)
This illustration also serves to define two distinct modes of adaptive immunity,
namely ACTIVE IMMUNITY and PASSIVE IMMUNITY. Immunization of the mouse in
the second line of Fig. 2-1 results in a state of "active" immunity; the animal's own immune
system is responsible for resistance to the subsequent bacterial challenge. On the other hand,
transfer of serum, as in line 3 above, results in a state of "passive" immunity in the recipient;
such immunity is the result of the presence of transferred antibody (see below). The animal's
own immune system does not participate at all, and this immunity lasts only as long as
sufficient levels of antibody are present.
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The substance present in immune serum which is responsible for transferring
immunity is antibody. In addition to transferring resistance to infection, these serum
antibodies can carry out a variety of other functions. For example, if immune serum is mixed
with a suspension of Pneumococcus, the bacteria will be seen to rapidly "clump" together.
This effect is known as agglutination, and is one of the many ways in which antibodies can
be detected and quantitated.
The various effects that antibodies may exhibit can be generally categorized as
physical effects, which depend only on the physical nature of the antibody and antigen, or
biological effects, which additionally depend on the particular biological properties of the
target antigen or other biologically active molecules which are involved.
PHYSICAL EFFECTS OF ANTIBODY
Agglutination. "Clumping" of a particulate antigen, e.g. bacteria or SRBC (sheep red
blood cells). Agglutination of red blood cells is a technique which has been widely
used in clinical and basic research as well as in the clinical laboratory, and is called
HEMAGGLUTINATION. Many soluble antigens can be made effectively particulate
by coating them onto SRBC or latex or other particles; the resulting clumping by
antibody is known as passive agglutination.
Precipitation. Interaction of antibody with a soluble antigen to form an insoluble
complex, e.g., with BSA (bovine serum albumin).
In liquid - the precipitate can be recovered by centrifugation and analyzed (see
APPENDIX 1, THE PRECIPITIN CURVE). If either the antigen or antibody is
radioactively labeled (see APPENDIX 2, LABELLING OF ANTIBODIES), it can be
used in a RadioImmunoPrecipitation (RIP) assay, first developed in the 1950s.
In agarose - if the antigen-antibody interaction takes place in a semi-solid medium
such as agarose, the resulting precipitate can be easily visualized. This is of special
significance in a configuration known as Ouchterlony Analysis (see APPENDIX 3,
OUCHTERLONY ANALYSIS).
Precipitation and agglutination are both consequence of cross-linking of antigens by
antibody into large complexes. The ability of antibodies to carry out this process implies that
each antibody can bind at least two antigen molecules, and that it can only occur if the
antigen molecule has two or more epitopes (“determinants ") which can be recognized by that
antibody.
Binding. If an antigen is bound to a solid matrix (latex particles or a plastic dish, for
example), and if the antibody is labeled in some way (with a visible, radioactive or
enzyme molecule), binding of the antibody to its antigen can be easily and sensitively
measured.
If a radioactive label is used, the assay is called a solid-state
RadioImmunoAssay (RIA). With an enzyme-based label, on the other hand, it
becomes an Enzyme-Linked ImmunoSorbent Assay (ELISA). These solid state
assays (particularly ELISA's) have largely replaced precipitation and agglutination
assays in a wide variety of clinical and research applications.
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BIOLOGICAL EFFECTS OF ANTIBODY
Protection from infectious disease. We have already seen in the Pneumococcus
example (Figure 2-1) how this manifestation of antibody can be assayed by transferring
serum from one animal to another.
Immobilization. An antibody directed against components of the flagellae of motile
bacteria or protozoa can cause these flagellae to stop moving. This results in the loss of
the organisms' ability to move around, and this loss of motility can be detected by
microscopic examination.
Cytolysis. If the target antigen is an integral component of the membrane of certain
sensitive cells, antibodies may cause disruption of the membrane and death of the cell.
This requires the participation of a collection of other serum components collectively
known as COMPLEMENT (see Chapter 5), and binding of these components to
antibodies is referred to as “Complement Fixation”.
If the antigen target is a red blood cell, this effect is known as hemolysis, which can
be readily detected visually. In the case of a bacterial cell target, the effect is referred
to as bacteriolysis.
If the target is a nucleated cell the effect is referred to as cytotoxicity, and may be
measured by release of a radioactive label incorporated into the cell (such as 51Cr),
exclusion of "vital" dyes such as Trypan Blue, or any of several other measures of cell
viability.
Opsonization. If the target antigen is particulate (e.g. a bacterium, or an antigencoated latex particle), bound antibodies may greatly increase the efficiency with which
the particles are phagocytosed by macrophages and other "scavenger" cells. This
improvement of phagocytosis is known as opsonization, and may be facilitated even
further by the presence of complement. As will be discussed later, opsonization is the
result of antibodies’ increasing the degree to which antigenic particles will "stick" to
phagocytic cells. This phenomenon has therefore been referred to as immune
adherence, and depends on the presence in the membranes of white blood cells of
specific receptors either for antibody (FcR, or "Fc-receptors") or for complement (CR,
or "complement receptors"), both of which will be discussed later (see Chapter 14, for
example).
ONE COMMON DEFINING PROPERTY OF ANTIBODIES:
ALL ANTIBODIES EXHIBIT SPECIFIC BINDING TO ANTIGEN
Different antibodies may show various combinations of effects; some antibodies may
precipitate but not interact with complement (and therefore not show cytolysis), some may be
opsonizing but not be capable of agglutination. The single common feature of all antibodies,
however, is that of specific recognition and binding to antigen. All other effects, physical or
biological, are secondary consequences of this specific binding. The structure of antibodies
and the basis of their ability to specifically bind antigen are the subjects of the next two
chapters (Chapters 3 and 4).
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ANTIGENS, IMMUNOGENS AND HAPTENS
We have been discussing "antigens" as molecules (1) which can elicit antibody
production upon injection into an appropriate host; and (2) to which these antibodies can then
bind. The difference between these two properties is an important one which we will now
make explicit by defining two related but distinct terms:
IMMUNOGEN. A molecule which can elicit the production of specific antibody upon
injection into a suitable host.
ANTIGEN. A molecule which can be specifically recognized and bound by an
antibody.
These definitions imply that an immunogen must be an antigen, but an antigen is not
necessarily an immunogen. Let’s illustrate this in the following table:
Substance
Molecular weight
Immunogen?
Antigen?
1) BSA
2) DNP
3) DNP10-BSA
"68,000"
~200
"70,000"
4) "clarified" BSA
68,000
yes
no
DNP - yes
BSA - yes
no (see Note)
yes
yes
yes
yes
yes
If we take a conventional preparation of purified bovine serum albumin (BSA) and
inject it into a mouse (line 1 in the table above), the mouse will produce antibodies which
will bind to BSA. BSA is therefore both an immunogen and an antigen.
If we take the small organic molecule dinitrophenol (DNP) and inject it into a mouse
(line 2), no antibodies will be produced which can bind DNP. DNP is therefore not
immunogenic; we will deal with its antigenicity shortly.
We can chemically couple DNP molecules to the protein BSA, yielding DNP-BSA. If
we inject this material into a mouse (line 3), we see that antibodies to BSA are elicited (as we
would expect), but also find antibodies which will bind specifically to the DNP groups on
BSA; we can further demonstrate that these anti-DNP antibodies will also bind free DNP (or
DNP coupled to any other molecule). Therefore, DNP-BSA is both immunogenic and
antigenic (with respect to both the DNP groups and the BSA itself), and the free DNP is also
antigenic, even though we have shown it is not immunogenic. DNP is an example of a
HAPTEN, a small molecule which is not immunogenic unless it is coupled to a larger
immunogenic CARRIER molecule, in this case BSA. (Such a hapten/carrier system will be
used in Chapter 14 to illustrate the mechanisms of cell interactions required to generate
humoral immune responses).
We can further demonstrate that the immunogenicity of BSA depends on the presence of
aggregates of BSA molecules. If we take a sample of our BSA and centrifuge it at high speed
we can remove any aggregated material, leaving behind only single, monomeric BSA
molecules in solution. If we immediately inject this "clarified" BSA into a mouse we find
that it does not elicit the production of antibodies (as seen in line 4); this monomeric BSA is
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therefore not immunogenic (nor can it serve as an effective carrier for a hapten). It is still
antigenic, however, which we can show by reacting it with the anti-BSA antibodies which we
made against the non-clarified BSA (in line 1, for example).
(NOTE: "Clarified" BSA not only fails to induce antibody formation, but can induce a state
of TOLERANCE to BSA, defined as the specific inability of the mouse to respond to
subsequent injections of normally immunogenic BSA. The mechanism of such tolerance will
be discussed in Chapter 18.)
REASONS FOR LACK OF IMMUNOGENICITY
Substances may lack immunogenicity for a variety of reasons:
1) Molecular weight too low. Haptens, for example, are not immunogenic until they are
coupled to a high molecular weight carrier. There is no simple cutoff for required
molecular weight, however; we have already seen that even the 68,000 mw of BSA is
not sufficient to be immunogenic unless the molecules are aggregated into even larger
complexes.
2) Not foreign to host. The adaptive immune system normally responds only to "foreign"
substances. A sheep, for instance, will normally not make antibodies against its own
red blood cells (SRBC), although SRBC are highly immunogenic in mice. (The basis
of normal SELF-TOLERANCE is covered in Chapter 18).
3) Some molecules are intrinsically poor immunogens for reasons which are not well
understood. Lipids, in general, are poor immunogens, probably because they do not
have a structure rigid enough to be stably bound by antibodies. Nucleic acids are also
relatively weak immunogens, although they are nevertheless common targets for
antibodies present in various autoimmune diseases (discussed in Chapter 19)
HOW TO INCREASE IMMUNOGENICITY: ADJUVANTS
(See also CHAPTER 22)
An ADJUVANT is any substance which, when administered together with an antigen,
increases the immune response to that antigen. One of the most widely used adjuvants (in
animals but not in humans) is FREUND'S ADJUVANT, which consists of mineral oil, an
emulsifying agent, and killed Mycobacterium (the organism which causes tuberculosis). A
solution of the desired antigen in water or saline is homogenized with this oil mixture,
resulting in a water-in-oil emulsion which is injected into the recipient. Its powerful
adjuvant properties result from several factors:
1) The antigen is released from the emulsion over an extended period of time, causing a
continuous and more effective stimulation of the immune system. (Antigen given in
soluble form will typically be cleared in a matter of hours or days, whereas it can
persist for weeks or months in a depot created by the adjuvant.)
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2) The Mycobacteria contain substances which non-specifically stimulate the immune
system, resulting in a higher level of response to the specific antigen. One of these
substances which has been extensively studied is Muramyl Dipeptide (MDP).
Although Freund's Adjuvant is not used in humans, other forms of adjuvant can be
used, such as alum precipitation of antigen, by which a soluble antigen is precipitated
together with aluminum hydroxide, resulting in particles of the salt coated with antigen. A
soluble antigen is thus converted to a particulate form, and again is released from the mixture
over an extended period of time. Substances such as purified MDP and others are also being
used to develop effective adjuvants which are less toxic, and therefore potentially usable in
humans (see Chapter 22)
CHAPTER 2, STUDY QUESTIONS:
1.
Define ANTIBODY, ANTIGEN, IMMUNOGEN and HAPTEN.
2.
How would you determine if a particular immune response is a HUMORAL
response?
3.
Describe assays which could be used to measure AGGLUTINATION,
PRECIPITATION, HEMOLYSIS and OPSONIZATION.
4.
Describe two antibody assays which require no antibody function other than specific
binding to an antigen.
5.
Define and distinguish ACTIVE versus PASSIVE immunity.
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