Serology: Antibodies and Antigens
Serology: Antibodies and Antigens
Authors: Compiled by Dr RW Worthington
Licensed under a Creative Commons Attribution license.
TABLE OF CONTENTS
Introduction ................................................................................................................... 2
Antibody and antigen.................................................................................................... 2
Antigens ................................................................................................................................ 2
Antibody ................................................................................................................................ 5
Antibody and antigen interaction .............................................................................. 13
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Serology: Antibodies and Antigens
INTRODUCTION
When any foreign substance gains access to an animal’s body or a mucosal surface the immune
system attempts to exclude or destroy it. The defence mechanisms include innate and acquired
immune defences.
The innate defence mechanisms include the mechanical barriers to the entrance of foreign
substances such as skin and mucosal surfaces, production of bactericidal substances, the action of
natural commensual flora, production of lysozyme, complement and acute phase proteins, the action
of phagocytes and natural killer cells etc.
The acquired immune mechanisms depend on the activation of the immune system by antigen. The
immune defences are broadly divided into the production of antibody by the stimulation of Blymphocytes and the activation of T-lymphocytes to produce a cellular response. Foreign substances
that can stimulate the immune system are called antigens. The immune system cells of the body are
able to distinguish antigens that are foreign and those that are “self” or part of their own tissues and
must not be attacked by the defence system. This text will be limited mainly to a discussion on the use
of serology for the diagnosis of animal diseases and topics relevant to that subject. Serology is that
branch of immunology involved with the detection of antibodies or the use of antibodies as reagents to
detect antigens particularly those of infectious agents.
ANTIBODY AND ANTIGEN
Antigens
Antigens are substances that are foreign and will stimulate the immune system to respond to their
presence. The immune system is primarily a defence mechanism against pathogenic organisms and
some toxins, but may also be activated by harmless substances. Bovine serum albumen injected into
a rabbit is not toxic or pathogenic but will stimulate the production of antibody. There is no apparent
benefit to the production of antibody against harmless substances; it probably merely reflects the fact
that the immune system recognises foreign rather than harmful substances.
Antigens are usually large molecules such as proteins or polysaccharides but in principle any
substance may act as an antigen. Antigenic particles may contain many different antigens. A single
virus or bacterium usually contains several different antigenic molecules on its surface. Antigenic
molecules are always large; protein molecules with molecular weight of less than 6,000 are poorly or
non-antigenic. Polysaccharide molecules have to be considerably larger than this to be antigenic.
However, the immune system does not recognise an entire molecule but small distinctive parts of a
molecule called epitopes or antigenic determinants.
individual epitopes not whole macromolecules.
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Antibodies are therefore, directed against
Serology: Antibodies and Antigens
If a bacterial or viral particle is presented to the immune system, antibodies will be made primarily
against the accessible epitopes of the antigens presented on the surface of the organism. In addition
to this products that are released from the interior of the cell may also be antigenic and stimulate
antibody production.
Structures on the surface of bacteria such as fimbriae and flagellae are
composed of proteins and are usually good antigens that elicit a strong antibody response. Other
surface molecules such as lipopolysaccharide on the surface of gram-negative bacteria and protein
spikes on viruses are also strongly antigenic. Although there may be many potential epitopes some
induce a greater response than others and most antibody will be formed against the most reactive
epitopes that are known as immunodominant epitopes. Some bacteria produce potent exo-toxins that
are strong antigens and antibodies are readily formed against epitopes on these toxins.
Many antibodies have protective functions and will help to inactivate the invading organisms or toxins,
while others do not appear to provide any protection. In some diseases, particularly the diseases like
brucellosis where the organism is an intracellular parasite, the presence of high levels of antibody
does not result in destruction of the organism, and animals remain carriers of infection for many years
whilst also having high levels of antibody circulating in their blood. The functions of the various
antibodies are discussed below.
Proteins and polypeptides are made up of long chains of amino acids. All the characteristics of a
protein are determined by the sequence of the approximately 20 amino acids found in them. They
have enormous variability in structure and function, varying from insoluble hair and keratin to highly
soluble haemoglobin, essential enzymes to potent toxins, structural molecules, contractile myosin
fibres etc. The primary amino acid sequence determines all the structural and functional
characteristics of a protein and is coded for by a single gene. Once the primary sequence of amino
acids has been coupled together the inherent thermodynamic properties of the molecule determine
how it will fold itself up into a fixed three-dimensional structure. Some parts of protein molecules may
form alpha helices consisting of strings of amino acids wound up in a spiral and other parts may have
a beta pleated structures consisting of straight lengths of amino acid chains bent backward and
forward to form a pleated pattern. Other sections of the proteins may consist of a variety of more
random connecting pieces. The overall shape of the molecule is called its conformation or tertiary
structure and determines the function and the spatial arrangement of the molecule that allow it to
interact with other proteins such as antibodies, enzymes or structural proteins. Small parts of the
protein structure that are of an immunologically distinct shape and are presented in such a way as to
be accessible to antibody act as distinctive epitopes. Sequential epitopes are composed of short
sections of the primary sequence of the antigen. Conformational epitopes consist of small structures
of distant parts of the primary sequence of the antigen, which due to the folding of the molecule are
closely associated to form an immunologically distinctive structure.
Repeated sequences of amino acids occur occasionally in proteins but they are the exception rather
than the rule and for this reason protein molecules do not usually contains repeats of the same
epitope on a molecule. However, viruses, bacteria, protozoa fungi etc. have many repeated structures
on their surfaces consisting of one or several different molecules so that particular epitopes are
repeated often on these infectious particles. Some large protein molecules consist of more than one
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Serology: Antibodies and Antigens
peptide or protein chain held together by a number of weak bonding forces or in some cases by a
covalent bond which forms between two cysteine molecules that have been oxidised to form a
disulphide bond. Repeated epitopes will be found in complex proteins containing more than one
identical sub-unit.
Figure 1-1 Formation of covalent disulphide bonds between cysteine molecules
Because disulphide linkages are strong covalent bonds they are very important in holding the
individual units of large macromolecules together or in stabilizing protein conformations through
formation of stable intracranial linkages. Disulphide bonds can
be broken by reducing them with reagents such as mercaptoethanol or dithiothreitol, which contain
sulphydryl moieties.
PrSSPr + 2RSH  2PrSH + RSSR
Protein molecules consisting of sub-units held together by disulphide bonds can be disrupted by
reduction with mercaptoethanol and the chains separated by physical means such
chromatography, electrophoresis and centrifugation.
as
Proteins in which sub-units are held together by weak bonding forces can be disrupted by hydrogen
bond disrupting agents such as strong solutions of urea. These reagents will also disrupt the
conformation of single chain proteins. Another reagent commonly used by immunologists that disrupts
the secondary and tertiary structure of proteins is the detergent sodium dodecyl sulphate (SDS). If an
antigen has been denatured and its conformation destroyed, only sequential epitopes will generally be
available, as the denaturing agent will disrupt many conformational epitopes.
Proteins that occur in different animal species but have the same function and have evolved from the
same gene usually have similar structures and matching sequences of amino acid homology. For
example serum albumen molecules in all mammalian species have similar amino acid sequences,
structure and function. Proteins from related species will therefore often have identical or similar
epitopes as well as some different epitopes that have become altered or deleted or have evolved
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Serology: Antibodies and Antigens
during evolution. These proteins will therefore elicit a mixture of similar and different antibodies when
injected into a host animal.
Polysaccharide molecules often consist of many repetitions of small sequences of sugars. They
therefore contain many repeated antigenic epitopes.
Antibody
Antibodies are proteins that are produced by plasma cells that evolve from B-lymphocytes that have
been stimulated by contact with a matching antigen. The genomes of individual B-lymphocytes are rearranged during their maturation by mixing and matching small regions on the precursor cell genome
to create up to 1011 unique genes each capable of forming a single antibody molecule. A library of
1011 B-lymphocyte types each coding for a pre-designed antibody are available to be used for the
production of antibody when stimulated by a particular antigen.
Antigen in circulation is taken up by phagocyctic processing cells that digest the antigen into small
pieces and present the pieces, in a fold of a major histocompatibility antigen on the phagocyte cell
surface, to a matching B-lymphocyte. The B-lymphocyte recognizes a particular antigen because preformed antibody that it has produced is present on its surface and interacts with the antigen fragment
(epitope) on the presenting cell. The interaction of the B-lymphocyte with the fragment of antigen
stimulates the cell to generate a clone of antibody producing cells that all produce identical antibody.
Memory cells are also produced that remain in circulation and respond quickly in large numbers to
future invasions of the same antigen.
The above is an extremely simplified and abbreviated account of what is known about this process. It
does not touch on the actual molecular mechanisms involved, the complexities of the cell structures
and types, or that part of the immune response that involves T-cells, the production of lymphokines
and killer cells and many other details. Interested readers should consult one of the available
immunology texts.
Antibody structure
Antibodies belong to a group of proteins collectively known as immunoglobulins. There are
five different classes or isotypes of immunoglobulins IgA, IgD, IgE, IgG and IgM. The basic
structure
Figure
of
an
immunoglobin
molecule
0-2
is
represented
diagrammatically
in
and
Figure 0-2. The molecule consists of two heavy chains and two light chains held together by
disulphide bonds. At the amino terminal end of both heavy and light chains there is a variable
region where the amino acid sequence is different in each antibody.
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Serology: Antibodies and Antigens
Figure 0-1 Diagrammatic structure of a basic immunoglobulin molecule.
Figure 0-2 Space fill model of a basic immunoglobulin molecule.
The amino acid sequences in the two variable regions of the light chains (V L) of any molecule
are identical but differ from those of the heavy chains that are also identical to each other.
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The variable region or domain consists of about 110 amino acids in both heavy and light
chains. The remaining carboxyl terminal domain of the light chain is a constant region (CL)
that is identical in all light chains of the same type and sub-type. There are two types of light
chains -  (kappa) and  (lambda) chains. All  chain constant regions are identical, as are
the constant regions of  chains. Each immunoglobulin molecule contains one type of light
chain.
There are five different types of heavy chains that give rise to the five immunoglobulin
classes.  (gamma) heavy chains are found in IgG molecules,  (mu) chains are found in IgM,
 (alpha) chains are found in IgA,  (delta) chains are found in IgD and  (epsilon) chains in
IgE. In each molecule there are two identical heavy chains. The heavy chain has a variable
domain (VH) of about the same size as the light chain variable domain and a variable number
of constant domains (CH1, CH2,…….etc.) depending on the class of antibody. For IgE there
are four constant domains and for the other classes there are three.
Further mutations on heavy chains have led to small variations of amino acid sequence within
the same animal species. These variations have resulted in the formation of various subclasses of immunoglobulins within species.
Classes and sub-classes of immunoglobulins in various animal species. Details taken from (IR Tizzard,
Veterinary Immunology 9th edition ELSEVIER) IgG1.
Species
Immunoglobulins
IgA
IgD
IgE
IgG
IgM
Human
IgA1 IgA2
IgD
IgE
IgG1, IgG2, IgG3, IgG4
IgM1,IgM2
Cattle
IgA
IgD
IgE
IgG1, IgG2,IgG3
IgM
Sheep
IgA1, IgA2
IgD
IgE
IgG1, IgG2, IgG3
IgM
Pig
IgA
IgD
IgE
IgG1, IgG2a, IgG2b, IgG3, IgG4
IgM
Horse
IgA
IgD
IgE
IgG1, IgG2, IgG3, IgG4, IgG5, IgG6
IgG7
IgM
Dog
IgA
IgD
IgE
IgG1, IgG2, IgG3, IgG4
IgM
Cat
(IgA1, IgA2)?
?
IgE
IgG1, IgG2, IgG3, IgG4?
IgM
Mouse
IgA1, IgA2
IgD
IgE
IgG1, IgG2a, IgG2b, IgG3
IgM
Immunoglobulins are themselves antigenic and antibodies can be raised against them. For
example antibodies can be raised against bovine immunoglobulins by injecting them into a
different species of animal such as a rabbit. Antisera against immunoglobulins have been
used to identify the various sub-classes of immunoglobulins.
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Differences between immunoglobulins can be of the following types:
Isotypes are different types of immunoglobulins that are present in all healthy members of a
species e.g. in humans there are genes for 1, 2, 3, 4, 1, 2, , ,  and chains.
Allotypes are variations between individual within the same species, e.g. an allotype, called
G3m(bc) that occurs in some people is a variant of IgG3 with a phenylalanine at position 436
of the heavy chain.
Idiotypes are variations due to mutations that occur in the variable domain of an
immunoglobulin. They are specific for individual cell clones.
The variable regions of the heavy and light chains form a structure that will match and bind
with a particular epitope. This structure is called the paratope or antigen-binding site of the
molecule. There is also a hinge region in the heavy chain close to where the light and heavy
chains are joined by disulphide bonds that ensure that the molecule is not completely rigid.
Each antibody has at least 2 paratopes and can bind at least two epitopes. For this reason
antibodies can cross link antigen molecules. The amino acid sequence of some parts of the
variable regions of both heavy and light chains show only small sequence differences
between different antibodies but in other regions known as hyper variable marked differences
occur. There are three hyper variable regions in the light chain and three in the heavy chain.
The six hyper variable regions are so arranged in the paratope that they form the contact
points between antigen and antibody and their structure is therefore responsible for the
accurate fit of antibody and antigen (Error! Reference source not found.).
The carboxy terminal part of the immunoglobulin molecule is called the Fc region and is the
main but not the only region where carbohydrate is bound to antibodies. It is also the region
where complement interacts with antibody.
IgG
IgG is the major immunoglobulin found in serum and body fluids and is the main
immunoglobulin class detected in most serological tests. Its structure is typical of that
described for a basic immunoglobulin and it has a molecular weight of around 150,000. IgG is
the main antibody produced in the later part of a response to an antigenic challenge and
remains in the serum for the longest period following an immune response. Different subclasses of IgG apparently have different functions and for example in cattle IgG1 binds
complement efficiently but IgG2 does not. The heavy chains of IgG are  chains and contain
variable and three constant domains. IgG can bind complement, precipitate or agglutinate
antigens and neutralise toxins or bind to virus particles and prevent them from multiplying. In
cattle IgG1 is excreted in the milk instead of IgA.
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IgM
IgM is a very large molecule having consisting of five basic immunoglobulin molecules held
together by disulphide bonds between the units. It also has an additional small protein called
a J chain attached to it. The heavy chains are of the  class and contain one variable and
three constant domains. The first response of an animal during a disease is predominantly
one of IgM antibodies. The IgM levels later decline and IgG becomes the dominant antibody.
The molecule is extremely efficient in precipitation and agglutination reactions due to its
multiplicity of paratopes able to bind and cross-link epitopes. It can also fix complement but is
more easily inactivated by heat than IgG and therefore tends to become inactivated in the
serum inactivation procedure of a complement fixation test. The molecule is also highly
susceptible to sulphydryl reagents and can be selectively inactivated by mercaptoethanol or
similar reagents. The antibody is mainly confined to the blood.
IgA
IgA is also known as secretory antibody since it is the main antibody type found in secretions
in the nasal mucosa, conjunctiva gut, urinary tract, mammary gland and respiratory tract. It is
present in the serum at low concentrations where it may occur as a dimer or monomer. Its
function is therefore presumed to be to provide immunity at this level and to prevent the entry
of infectious agents at these sites. IgA found in these secretions is always present as a dimer
consisting of two basic immunoglobulin units held together by a J chain, and a secretory
component. The secretory component is a protein that attaches to the dimer by weak bonding
forces as well as by a disulphide bond in the C2 region. The J chain is attached by a number
of disulphide bonds. The heavy chains are of the  type and contain one variable and three
constant domains.
IgD
This immunoglobulin class makes up less than 1% of the immunoglobulin in the serum and is
unstable and has a short biological half-life. Its function is unclear. It’s heavy chains consist
are of the  type and have one variable and three constant domains.
IgE
IgE is present in extremely low levels in serum. It is found attached to the membranes of mast
cells by its Fc part. In the presence of antigen it cross links antigen on the cells surface thus
damaging the cell membrane and causing it to release of a number of reactive substances
from granules in the mast cells, thus triggering an anaphylactic reaction. Its heavy chains are
of the  type.
Antibodies measured in serological tests for the diagnosis of infectious diseases belong
almost exclusively to the IgG and IgM classes.
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Polyclonal and monoclonal antibodies
When an animal is immunised with a particular antigen there are usually several epitopes on
the antigen that can trigger an immune response. The immune system therefore responds by
producing a variety of different antibodies with specificity for different epitopes. In addition
there may be a number of different paratopes that can fit each epitope. Therefore, a number
of different antibodies with varying paratopes and affinity for the same epitope may be
produced. Furthermore antibodies of different classes and sub-classes may be produced.
Each of these different types of antibodies will be produced from a clone of cells that has
arisen from a single activated B-lymphocyte. The type of serum generated by this type of
immunisation will therefore be a polyclonal antiserum.
Polyclonal sera generally exhibit a high degree of antigen specificity. However, they may
cross react with other antigens that contain identical or similar epitopes capable of fitting,
albeit with lower affinity, a paratope of one of the antibodies in the serum.
A monoclonal antibody (MAb) preparation is one that contains only antibody produced by a
single clone of cells. All the antibody molecules in the preparation are therefore identical and
epitope specific. However, they may cross react with epitopes that are similar enough to fit
their paratope. All molecules in a monoclonal antibody preparation will be of the same class
and sub-class and idiotype.
The first MAbs that became available for study were those produced by myelomas. Myeloma
cells are malignant antibody producing cells that are all derived from a single malignant cell.
Patients with myelomas produce massive amounts of monoclonal antibody, but the antigens
for which they are specific are unknown. However, they were valuable research tools and a
great deal was learned about the structure of antibodies from the study of myeloma
immunoglobulins.
In 1975, Milstein and Kohler made a major breakthrough when they developed a technique
for producing monoclonal antibodies directed against selected antigens. To understand the
process it is necessary to understand something about the synthesis of purine and pyrimidine
bases in multiplying cells. All organisms must produce purines and pyrimidines and their
activated derivatives adenosine triphosphate (ATP), guanosine triphosphate (GTP), uridine
triphosphate (UTP), Cytosine triphosphate (CTP), and thymidine triphosphate (TTP), in order
to make nucleic acids as they multiply. Purines and pyrimidines can be synthesised by living
cells from simple starting materials including phosphoribosyl pyrophosphate (PRPP) and
glutamine for purines, and aspartate and carbomyl phosphate for pyrimidines. This synthesis
from new products is often called de novo synthesis. In these biosynthetic pathways several
steps are catalysed by enzymes that require folic acid as a co-enzyme. Therefore, a
competitive inhibitor, aminopterin that is a structural analogue of folic acid, can be used to
block the pathways.
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However it is apparently efficient for the body to re-cycle the breakdown products of nucleic
acid catabolism rather than use the de novo synthesis pathways. The salvage pathways are
alternative pathways for the synthesis of purine and pyrimidine phosphates. When DNA and
RNA are catabolized the breakdown products include the purine bases hypozanthine,
adenine and guanine. These purine bases are salvaged and used to synthesize the ATP and
GTP required for the synthesis of DNA and RNA. The enzymes hypozanthine-guanine
phosphoribosyl transferase and adenine phosphoribosyl transferase are essential for this
process. In cell culture medium containing aminopterin to block the normal de novo synthesis
of purines, the required purine ribosyl phosphates (PRPP) can be synthesized if hypozanthine
is provided in the medium. The reactions can be simplistically represented as follows:
Hypozanthine + PRPP ---- IMP----AMP ---- ADP ---- ATP

GMP ---- GDP ---- GTP
In the pathways for pyrimidine synthesis only the formation of TMP is blocked by aminopterin.
However, TMP can be synthesised from thymine in a reaction catalysed by thymine kinase.
HAT medium contains hypozanthine and thymidine as purine and pyrimidine precursors and
aminopterin to block the de novo synthesis of purines. It will only support cell growth if the
cells can produce hypozanthine phosphoribosyl transferase and thymidine kinase.
Kohler and Milstein immunised mice and then harvested their spleen cells. B-lymphocytes will
not normally grow in tissue culture but die within a few days. Spleen cells were then cocultivated with mouse myeloma cells in the presence of a cell-fusing agent, polyethylene
glycol. The myeloma cells used were able to grow indefinitely in tissue culture, but were
selected mutants that did not produce immunoglobulin and were defective in the enzyme
hypozanthine phosphoribosyl transferase. After fusion of the cells the cultures were grown on
HAT medium. Unfused B-lymphocytes died off during culture and unfused myeloma cells
were unable to grow in HAT medium as they did not produce hypozanthine phosphoribosyl
transferase and also died off. Fused cells (hybridoma cells) able to grow indefinitely in culture
as they had acquired a hypozanthine phosphoribosyl transferase gene by fusing with
lymphocytes. Hybridoma cells were then cloned and supernatant culture fluid from cloned
cells was tested to see whether they were producing antibody to the correct antigen.
Once hybridoma cells have been produced they can be grown in tissue culture and antibody
harvested from their culture supernatants. The yield of MAb obtained in this way is low and
much higher yields can be obtained by injecting the cells into the peritoneal cavity of mice
where they develop into tumours. As the tumours grow ascites fluid accumulates in the
mouse peritoneal cavity and can be harvested. The ascites fluid contains high concentrations
of MAb. Today it is ethically more acceptable to use tissue culture systems for the
propagation of the cloned B cells as the ascites method can result in pain and discomfort in
the mice.
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Figure 0-3 A general representation of the method used to produce monoclonal antibodies.
The ability to produce monoclonal antibodies has radically transformed the practice of
serology, enabling tests of greatly improved specificity to be developed. A great
number of MAb reagents are now available commercially and new hybridomas are
being produced probably daily in laboratories around the world. Nearly all monoclonal
antibodies presently available are of mouse origin although rat cells have also been
used. For serological tests there are few disadvantages to having to use MAbs from
mice and in several tests particularly competitive ELISAs it is an advantage to have
mouse antibodies that can be easily distinguished from the antibodies from another
species, with which they are competing.
MAb reagents react in an absolutely
consistent manner since all antibody molecules are identical, directed against the
same epitope and all have the same affinity for that epitope. Successive batches of
antibody prepared from the same hybridoma will produce identical antibodies. On the
other hand no two preparations of polyclonal serum are identical as they contain
mixtures of antibodies of varying specificity, affinity, isotype and allotype directed
against a multiplicity of epitopes. In order to get a constant reference reagent of
polyclonal serum, a large amount of pooled serum from several animals is usually
aliquotted and stored for future use over a long period of time.
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Serology: Antibodies and Antigens
ANTIBODY AND ANTIGEN INTERACTION
A fundamental concept that defines the interaction between antibody and antigen is that antibodies
are bivalent (or polyvalent in the case of IgM and IgA) in that each antibody has at least two identical
antigen binding sites. These sites consist of paratope clefts that recognise and bind to epitope
structures in a highly but not absolutely specific manner. The binding site of the antibody has a
complementary shape to the antigen epitope that it binds to and can be imagined as a pocket into
which the antigen epitope fits. X-ray crystallography studies from which sophisticated models have
been constructed show the interaction between some antibodies and antigens is indeed one where
there is a matching fit between paratope and epitope.
Space fill models that show the respective antibody and antigen binding sites. a) Represents the whole
antibody. b) Represents one of the Fab fractions that is bound to an antigen, indicating the contact areas
called the paratope on the antibody and the epitope on the antigen. c) Represents a wireframe model of
the antibody variable domain with the hyper variable areas in the paratope highlighted. d) and e)
Represent a frontal view of the paratope on the antibody indicating the various hyper variable areas.
The affinity of the interaction between the antibody and the antigen epitope depends on the closeness
of fit between the two structures. The most important sites where antibody and antigen come into
contact and must fit each other well are the parts if the paratope that represent the hyper variable
regions. The interaction is strengthened by the alignment of structures that allow weak bonding
interactions such as hydrogen, electrostatic and hydrophobic bonds to form. The totality of the weak
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bonds and the goodness of fit of the binding site account for the affinity of binding between the two
molecules.
Interaction between antibody and antigen is defined by the characteristics of affinity and avidity.
Affinity relates to the strength of binding between a specific antibody and its complementary epitope.
It can best be measured for a monoclonal antibody preparation. A polyclonal antiserum will contain
antibodies belonging to different antibody classes and antibodies with varying affinity and specificity
for different epitopes, but an antibody against an immunodominant epitope may predominate. The
antigen generally contains several different epitopes. Avidity is a measurement of the average affinity
of binding between the mixture of serum antibodies and an antigen.
Covalent bonding does not occur between antibody and antigen. Therefore it is possible to separate
antibody and antigen by procedures such as changing the pH, increasing the ion strength of the salt
solution or buffer in which they are dissolved or suspended, using hydrogen bond disrupting agents
such as urea or guanidine or chaotrophic agents like isothiocyanate.
Antibody preparations specific for particular antigens can be isolated by reacting a serum with an
antigen collecting and washing the complex (precipitate or agglutinate), disrupting it with a suitable
disruptive agent and then separating antibody from antigen by suitable methods such as column
chromatography, electrophoresis, centrifugation etc.
In practice it is easier to covalently bind the antigen in question to an insoluble matrix such as
cellulose or Sephadex, which is packed into a column. The antiserum is then passed through the
column and the antibody binds to the immobilised antigen. The column can then be washed free from
contaminants and the antibody discharged from the column with a suitable antibody/antigendisrupting reagent. The same process can be used to isolate antigen if a suitable antibody preparation
is covalently bound to the insoluble matrix. This technique is known as affinity chromatography.
Although it has been very valuable in the past for producing antibody preparations that react
specifically with particular antigens, it yields mixtures of antibodies of differing affinity, specificity and
antibody types that react with different epitopes on a large antigen molecule. Such preparations have
been used to increase the specificity of tests but the development of the technique for the production
and use of monoclonal antibodies has to a large extent superseded this type of technology.
Monoclonal antibody preparations consist of single molecule types, with specificity for a particular
epitope thus making them superior reagents for providing highly specific serological tests.
Configurations of tests using monoclonal and polyclonal antisera are discussed elsewhere.
The complexity of sera raised against complex antigens makes the precise study of antigen antibody
interactions difficult. In his classical studies Karl Landsteiner used small molecular weight haptens as
antigens. A hapten is a small molecule that is not antigenic on its own, but when bound to a large
molecular weight antigenic carrier becomes antigenic. Covalently binding a small molecular weight
hapten to a large carrier molecule such as a protein, in effect creates defined epitopes on the protein.
In this way Landsteiner was able to produce antisera with affinity for defined small molecules. For
example antibody can be produced against benzene ring structures coupled to a protein. When an
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antibody was raised against an amino benzene ring with a sulphonate molecule in the meta position,
this antibody cross-reacted, but less strongly, with a benzene ring with a sulphonate in the ortho
position and rather weakly when the sulphonate was in the para position. The antibody also crossreacted weakly with benzene rings with arsenate or carboxylate moeities in the meta position.
Antigen/antibody interactions are therefore not absolutely specific, but cross-reactions only occur
between antibodies and epitopes with similar structures.
An antibody generated against a specific antigen may cross react with heterologous molecules
containing identical or closely related epitopes. Cross-reactions most often involve antigens from
phylogenetically related species. For example for several commonly used tests (ELISA,
immunoblotting, immunoperoxidase tests etc), it is very useful to produce antisera against
immunoglobulins from different species of animals. By injecting sheep IgG into a rabbit an antiserum
can be raised that will react with sheep IgG. However, it will also cross-react with IgG from cattle,
deer, other ruminants and even more distantly related mammals.
Cross-reaction between an antiserum and IgG preparations from a range of animal species can be
quite extensive particularly between IgG preparations from closely related species of animals. They
occur because the protein structure is similar having several identical epitopes or epitopes that have
been slightly altered, by the substitution of a few amino acids during the evolution of the species. If an
antiserum with high specificity for bovine IgG is required, it is necessary to inject bovine IgG into a
closely related species such as a goat. The goat will make antibody only against bovine epitopes that
do not occur in goats but will not make antibody against the epitopes that its immune system
recognises as self. The antiserum will therefore cross-react minimally with IgG from species other
than cattle, but will still not be entirely species specific. Even a defined monoclonal antibody
preparation against a particular type of immunoglobulin from one species, could react with similar
types of immunoglobulin from another species if it has specificity for an epitope that is common to
more than one species. To achieve absolute specificity a monoclonal antibody with specificity for an
epitope that is unique to a particular species is required.
In working with infectious diseases the same principle applies and it is not uncommon to get some
cross-reactions between closely related species of organism. For example antisera made against the
lipopolysaccharides of one smooth Brucella species will cross-react with all smooth Brucella species,
but will cross-react minimally with rough Brucella species that have a different surface structure. The
specificity of smooth Brucella abortus antisera can be improved by absorbing serum prepared against
Brucella abortus with Brucella melitensis organisms, to remove the cross- reacting antibodies.
However, even this absorbed serum will still react strongly with both Brucella abortus and Brucella
suis. Cross- reactions may also occur with bacteria of a different genus when they produce similar
antigenic molecules. Yersinia enterocolitica type 09 produces a polysaccharide antigen on its surface
that has epitopes that are identical in structure to those found on Brucella abortus polysaccharide.
Therefore, sera produced against Brucella abortus will cross-react with Yersinia enterocolitica strain
09 but not with other strains of Yersinia enterocolitica. In this case and other cases where the
antigenic molecules are identical the reaction is in fact not a cross-reaction, but a specific one.
However, from a diagnostic point of view it is commonly regarded as a non-specific reaction because
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Serology: Antibodies and Antigens
a test result that identifies the wrong disease is considered to be a false positive result. The specificity
of serological tests is an important practical issue in diagnostic testing and it is important to
understand the theoretical basis for specific and non-specific reactions as well as the epidemiological
implications of specificity, which are described later.
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