CHAPTER 3

ANTIBODY STRUCTURE I
See APPENDIX: (3) OUCHTERLONY ANALYSIS; (6), EQUILIBRIUM DIALYSIS;
(7) CROSS-REACTIVITY
Electrophoretic separation of serum proteins identifies the GAMMA-GLOBULIN
fraction as containing the majority of antibodies. Three terms which are often
confusingly interchanged are defined and distinguished (GAMMA-GLOBULIN,
IMMUNOGLOBULIN, ANTIBODY), as are two terms describing antibody/antigen
binding, AFFINITY and AVIDITY.
All antibodies are made up of one or more IgG-like subunits, each of which has
exactly two antigen-combining sites. The affinity of these sites for their antigen (defined
as the Keq of the binding reaction) is highly heterogeneous in any normal immune
response. While the avidity of an antibody (its ability to form stable complexes with
antigen) does depend on its intrinsic affinity, it also increases dramatically with an
increasing number of combining sites per antibody.

In order to determine the structure of antibodies, we first must have a way of isolating
these molecules in relatively pure form. Well begin by describing the general process of
serum fractionation, then go on to analyze the nature of antigen-antibody binding.
The many components of normal serum can be separated from one another by various
means:
Salt precipitation. Ammonium sulfate [(NH4)2SO4] as well as a variety of other salts
can be used to precipitate serum components; different proteins will precipitate at different
concentrations of salt, providing a convenient means of separating them. The fraction
containing most of the antibody activity generally precipitates at relatively low salt, at about
30-40% of saturated ammonium sulfate. This is a very widely used experimental method for
fractionation of serum components (and proteins in general).
Ethanol precipitation. Ethanol can also be used to precipitate serum components,
which come out of solution at different concentrations and under different conditions of ionic
strength, pH and temperature. This is a more elaborate procedure to carry out than salt
fractionation, but is the basis for Cohn Fractionation, which in modified form remains a
standard procedure for preparing serum protein fractions for clinical use more than sixty
years after its original description in the 1940s.
Electrophoresis. Different serum proteins migrate at varying rates in an electric field,
a property which can be used to separate them. While this procedure can be adapted for use
on a preparative scale, it is most commonly used for analysis.

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A typical pattern generated by electrophoresis of a serum sample (e.g. on a filter paper strip)
is shown in Figure 3-1.

albumin

globulins

1 2

ELECTROPHORESIS OF NORMAL SERUM

Figure 3-1
Several important points emerge from this pattern:
1) Most serum proteins carry a negative charge, and therefore tend to migrate from the
point of origin (labeled "O") toward the anode, the positively charged electrode.
2) Four major peaks are seen in this example; these are named (from the anodal, or
positive, side) the albumin peak (which is by far the largest), followed by four globulin
peaks, 1- and 2-globulin, -globulin and -globulin.
3) This pattern is deceptively simple; serum actually contains hundreds of known proteins.
Thus, "-globulin" is not a single protein, but a mixture of many components which all
happen to migrate in a particular region on electrophoresis.
4) Most (but not all) antibodies migrate in the -globulin region.
5) The -globulin peak is markedly broader than the others, reflecting the high degree of
heterogeneity of the antibodies it contains. This heterogeneity is so great that some
antibody molecules in fact migrate in the positions characteristic of -globulin or globulin.
6) The -globulin peak is generally centered near the origin, labeled "O"; this reflects the
fact that antibodies as a group are relatively neutral, i.e. less highly charged than most
other serum components.

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Three easily confused terms are all commonly used to refer to antibody molecules,
gamma-globulins, immunoglobulins and antibodies. To avoid this confusion let's explicitly
define each of them:
GAMMA-GLOBULIN -- Any molecule which migrates in the gamma-globulin peak
on electrophoresis. Most, but not all, antibodies are in this category, although the term is
often used to refer to antibodies in general. (Other serum components migrate in this region
as well; therefore, strictly speaking, not all gamma-globulins are antibodies.)
IMMUNOGLOBULIN -- A family of molecules (to which all antibodies belong) with
similar structures and physical properties. We shall see that these involve homologous amino
acid sequences, similar "domain" structures and similar quarternary structures (the ways in
which different polypeptide chains are joined into a larger functional unit).
ANTIBODY -- A molecule belonging to the Immunoglobulin family, with binding
specificity for a particular antigen. While all antibodies are immunoglobulins, most but not
all antibodies are gamma-globulins.
Note that our definition of "antibody" requires knowledge of the binding specificity
of the molecule. If one is dealing with an "antibody" molecule whose specificity is
not known, or is irrelevant, it is more accurate to refer to it simply as an
"immunoglobulin". (Common usage of these terms varies considerably, however.)
ANALYSIS OF THE ANTIBODY COMBINING SITE:
VALENCY, AFFINITY AND AVIDITY
If we immunize a rabbit with DNP-BSA, we can obtain an antiserum which contains
antibodies to both the hapten and the carrier protein. This antiserum will precipitate DNPBSA in addition to DNP-KLH (Keyhole Limpet Hemocyanin, an unrelated protein carrier).
If we attach DNP to SRBC (sheep red blood cells) or to latex particles, we can show that the
antiserum is capable of showing agglutination (and possibly hemolysis in the case of SRBC).
We can use these antibodies to the DNP hapten in order to learn about antibody
structure and function. Specifically, we will ask two questions:
1)
2)

How many hapten molecules can a single antibody molecule bind (i.e how many
combining sites does it have, or what is its valency)?
What is the strength of binding of the hapten to its combining site(s) on the
antibody molecule (i.e. what is the affinity of the combining site)?

We have previously made the prediction that in order for an antibody molecule to be
capable of precipitation or agglutination it must have at least two combining sites, in order to
permit cross-linking of the antigen into large, insoluble complexes. We can determine the
actual number of combining sites of our anti-DNP antibodies, as well as their affinity, by
several techniques; one of them, EQUILIBRIUM DIALYSIS, is discussed more fully in
APPENDIX 6, and we will use the results of such an analysis as the basis for our discussion
below.

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RABBIT IgG ANTIBODIES HAVE TWO HAPTEN-COMBINING SITES

The structure of rabbit IgG antibodies represents the basic structure of all antibodies
and we can show by equilibrium dialysis that each anti-DNP antibody molecule can bind
exactly two DNP molecules. Thus, our minimum prediction of at least two combining sites is
fulfilled. Other kinds of antibodies can be shown to have more than two combining sites
(IgM and some IgA), but we will see that such antibodies are always made up of multiple
units of the basic "IgG-like" structure, each of which bears precisely two combining sites.
CONVENTIONAL ANTIBODIES ARE HETEROGENEOUS
WITH RESPECT TO AFFINITY
The DNP hapten is bound to each combining site by non-covalent forces, and the
strength of this binding is measured by the equilibrium constant of the binding reaction,
known as the AFFINITY. The antiserum we describe above contains anti-DNP antibodies
with many different affinities, typically ranging from 105 to 1010. (Antibodies certainly exist
with affinities outside this range, but such values are difficult to determine accurately due to
technical limitations.)
This antibody heterogeneity is a hallmark of the immune response, and has many
practical and theoretical implications (see discussions of Clonal Selection and Affinity
Maturation [Chapter 7], and Isotype Switching [Chapter 9]). The broadness of the gammaglobulin peak on serum electrophoresis (which we have already described) is one
consequence of this heterogeneity; in fact, a sharp, narrow gamma-globulin peak
(representing a homogeneous protein) is a pathological sign of a myeloma or other
monoclonal gammopathy. However, homogeneous antibodies known as HYBRIDOMAS,
or MONOCLONAL ANTIBODIES can be generated experimentally, and are important in
many research and clinical applications (see APPENDIX 13).
ANTIBODY AVIDITY: ABILITY TO FORM STABLE COMPLEXES WITH ANTIGEN
AFFINITY is a thermodynamically defined term representing the strength of
interaction of a single combining site with its hapten. Naturally produced antibodies always
have two or more sites, however, so that affinity does not tell the whole story with respect to
antigen-binding. A bivalent anti-DNP antibody, for example, can simultaneously bind to two
DNP haptens on a single BSA molecule, resulting in a much more stable complex than if it
only bound to a single site.
AVIDITY, on the other hand, is the term used to describe the ability of an antibody to
form stable complexes with its antigen. Avidity, of course, depends partly on affinity; all
other things being equal (which they rarely are), one IgG antibody with a higher affinity for
DNP than another will also have a higher avidity. However, various other factors also play a
role, such as the number and spacing of the epitopes on the antigen, the distance between the
combining sites on the antibody, and properties such as the "flexibility" of the particular
antibody molecule.
Avidity does not have a formal thermodynamic definition, and is most commonly
used only in a relative context (by demonstrating that one antiserum may exhibit a higher or
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lower avidity than another). Nevertheless, in discussing the interaction of an intact antibody
(which is at least bivalent) with a conventional antigen (which is almost always highly
multivalent), one must almost always think in terms of "avidity" rather than "affinity". This
is of particular importance when considering the biological effectiveness of antibodies which
have more than two combining sites, such as serum IgM and some IgA.

CHAPTER 3, STUDY QUESTIONS:

1.

Define the terms ANTIBODY, IMMUNOGLOBULIN and GAMMA-GLOBULIN.

2.

How is EQUILIBRIUM DIALYSIS carried out, and what can it measure?

3.

Define and distinguish antibody AFFINITY and AVIDITY.

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