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Learning Unit 4: Antibodies

Work through this learning unit in conjunction with Chapter 34 of your prescribed book.

Learning outcomes
At the end of this learning unit, you should be able to:

• Describe the characteristics of antibodies and the kinetics of their secretion.

• Compare the five classes of antibody.
• Discuss the mechanisms of action of antibodies
• Discuss the development and application of monoclonal antibodies.

4.1 Introduction to antibodies

This learning unit is based on Prescott’s Microbiology, Chapter 34, pp. 749 - 760. We previously
discussed how acquired/adaptive immunity is generated. In addition, we also discussed how antibodies
are produced from B cells. To this learning unit, we present the antibody structure, classes, their diversity,
kinetics, and monoclonal antibodies.

Antibodies are the secreted form of the B cell receptor. They are also known as immunoglobulins (Ig), and
consist of glycoprotein chains – two heavy chains and two light chains. There are 5 classes of human Ig
(lgA, IgG, IgM, IgD and IgE). All five main classes of immunoglobulins can occur as transmembrane
antigen receptors or secreted antibodies. Properties of these classes are shown on page 751 (table 34.2)
of the prescribed textbook. A brief review of this table shows us that:
• The heavy chains are assigned a Greek letter (alpha for IgA, delta for IgD, etc).
• IgG has the highest serum concentration of all these classes.
• IgM appears huge and has five times the valency in comparison to the other classes.
• IgG can cross the placenta and has a relatively long half-life in serum.
• IgG and IgM are associated with the classical complement pathway while IgA is associated
with the alternative pathway.
• IgE appears to be associated with allergies.

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4.2 Antibody structure

As shown in figure 9, all immunoglobulin molecules have a basic structure composed of four polypeptide
chains: two identical heavy chains and two identical light chains. The four chains are arranged in the form
of a flexible Y with a hinge region. This indicates the basic structure of the antibody: Each end of the
antibody has a different function – the sites for binding antigen and the other end of the antibody that are
recognized by cell receptors; heavy (H) and light (L) chains are connected by disulphide bonds and have
variable (V) and constant (C) regions with the antigen-binding site being associated with the V regions.
The amino ends of the chains are associated with the antigen-binding sites and the chains have loops (or
domains). The antigen binding regions, knows as the variable region or the V region varies extensively
between antibody molecules. The two heavy chains and the two light chains are identical, giving an
antibody molecule two identical antigen-binding sites. Thus, an antibody can bind simultaneously to two
identical antigens, thereby increasing the total strength of the interaction, which is called avidity. The
strength of the interaction between a single antigen-binding site and its antigen is called its affinity.

Figure 9. Antibody Structure. (a) A computer-generated model of immunoglobulin structure showing the
arrangement of the four polypeptide chains. (b) Heavy chains and light chains are connected to each other
by disulfide bonds. Each heavy and light chain pair to form an antigen-binding fragment (Fab). The
disulfide-linked heavy chains form the fragment (Fc). The Fc fragments are composed only of constant

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regions, whereas the Fab fragments have both constant and variable regions. (c) Both the heavy and the
light chains contain several homologous units of about 100 to 110 amino acids. Within each unit, called a
domain, disulfide bonds form a loop of approximately 60 amino acids. Interchain disulfide bonds also link
heavy and light chains together. All light chains contain a single variable domain (VL) and a single constant
domain (CL). Heavy chains contain a variable domain (VH) and either three or four constant domains (CH1,
CH2, CH3, and CH4). The variable regions (VH, VL), when folded together in three-dimensions, form the
antigen-binding sites.

Activity 4.1
What is the function of the Fc and Fab regions on an antibody?

4.3 Immunoglobulin function

The VH and the VL parts of the Ig heavy and light chains together make up the antigen binding site. These
regions fold according to the amino acids making up these parts of the Ig chains. Thus, when comparing
two similar, but different, antibodies, a slight variation in the amino acid composition of V regions will affect
the V region folding. This means that differently shaped antigens can interact with the V regions of slightly
different antibodies. In addition to the importance of shape and fit between antigen and antibody, note that
the interaction between antigen and antibody involves weak forces, such as hydrogen and hydrophobic
bonds, as well as electrostatic interactions.

The antibody molecule has two important functions: one is to specifically bind to a pathogen on its products
that have elicited the immune response; and the other function is to recruit other cells and molecules to
destroy the pathogen following antibody binding. For example, Binding of Ig can neutralize viruses and
marks pathogens for destruction by phagocytes and complement. The marking of pathogen is called
opsonization, leading to killing of the target cell (known as antibody-dependent cell-mediated cytotoxicity)

4.4 Immunoglobulin classes

The different classes of antibodies are adapted to function in different compartments of the body. Their
functional activities and distribution are summarized on page 751 (table 34.2) of the prescribed textbook.
IgG is by far the most abundant immunoglobulin in serum and accounts for around 80% of serum Ig. There
are four IgG subclasses, comprising mainly IgG1 (65%) and IgG2 (23%). Different subclasses contribute
to an increase in IgG function – for example, IgG1 and IgG3 bind to antigen and then their Fc regions are
recognised by neutrophils and macrophages resulting in phagocytosis of these cells. On the other hand,
IgG2 responds to, and is opsonic for, toxin molecules. IgG is the only immunoglobulin molecule with the
ability to cross the placenta and provide natural immunity in utero and to the neonate at birth. IgG is also
one of the two immunoglobulin classes that activate complement by the classical pathway.

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IgM – This Ig class accounts for only around 10% of immunoglobulins and is a massive molecule consisting
of five antibody units and is, thus, a pentameric molecule stabilized by a joining (J) chain. Note that within
the surface of B cells are found monomeric IgM molecules forming part of the B-cell receptors. An initial
infection is characterised by the secretion of IgM. Note the hexameric form of IgM mentioned on page 752
of the textbook. Can active complement by the classical pathway.

IgA – This Ig class consists of two antibodies forming a J-chain-linked dimer, and, together with a secretory
component, is found particularly associated with mucous membranes. This Ig class is very important in
immunity in the intestine where it binds to bacteria, viruses and parasites to prevent their binding to the
gut wall. This is called immune exclusion.

IgD – This appears to be a molecule involved in B cell activation.

IgE – This class of immunoglobulin is usually present at low concentrations but is associated with the
potent allergic response. This involves, particularly, mast cells and following the cross- linking of surface-
bound IgE molecules, histamine and other potent physiological mediators are released from the mast cells.

Activity 4.2
Describe the major functions of each immunoglobulin class.

4.5 Antibody Kinetics

Antibody kinetics refers to the study of antibody interactions, exploring the rates at which antibodies binds
to and dissociate from the target antigen. Thus, the production and secretion of antibody can be evaluated
with respect to time. As it has been mentioned before, monomeric IgM serves as the B-cell receptor for
antigen, and pentameric IgM is the first type of antibody secreted after B-cell activation. Following B-cell
activation, with the help of T-helper cells, IgM-secreting plasma cells may switch and produce another
antibody class, which can either be IgG, IgA, or IgE. This is known as antibody class switching and it will
not be discussed in detail in this learning unit. There are two types of antibody response that well describes
the antibody kinetics, which is the primary immune response and the secondary immune response.

4.5.1 Primary immune response

Following antigen exposure there may be a lag phase or latent period of perhaps days or weeks before,
particularly, IgM is secreted at relatively low concentration levels before relatively quickly declining in
concentration. Once B cells have differentiated into plasma cells, antibody is secreted and can be detected.

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During the primary antibody response, IgM appears first, then switches to another antibody class, usually
IgG (Figure 10). The affinity of the antibodies for the antigen’s determinants is low to moderate during the
primary antibody response. Antibody concentration is referred to as titer. The antibody titer rises
logarithmically to a plateau during the log phase. In the plateau phase, the antibody titer stabilizes. This is
followed by a decline phase, during which antibodies are naturally metabolized or bound to the antigen
and cleared from circulation.

4.5.2 Secondary immune response

As we have seen from the primary response that following exposure to an antigen the B cell differentiate
into plasma and memory cells. These memory cells which possesses specific immunological memory
primes the secondary immune response. After a second exposure to the same antigen, IgG in particular
is produced rapidly at high concentration levels for an extended period of time. Note that the antibody
affinity for the antigen is higher during the secondary antibody response.

Figure 10. Antibody production and kinetics. The four phases of a primary antibody response correlate to
the clonal expansion of the activated B cell, differentiation into plasma cells, and secretion of the antibody
protein. The secondary response is much more rapid, and total antibody production is nearly 1,000 times
greater than that of the primary response.

Activity 4.3
What is antibody kinetics?

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4.6 Antibody diversity

This section is part of the learning unit, but will not be discussed here , but please read and understand
the genetics of antibody diversity as indicated in the text and diagrams on pages 755 to 757 of the
prescribed textbook. Note the multiple antibody genes that contribute to antibody diversity, how they are
spliced together, how varying amino acid sequences are generated following antibody gene splicing and
how somatic mutations occur. All of these strategies have developed to increase variety within the proteins
making up the antibody molecule and, particularly, to increase variation within the antigen-binding site.

4.7 Action of Antibodies

As we have seen, the antigen-antibody interaction is a bimolecular association that exhibits exquisite
specificity. The interactions that occur in animals are essential in protecting the animal against the
continuous onslaught of microorganisms and their products, as well as cancer cells. This occurs partly
because the antibody coats the invading foreign material, marking it for enhanced recognition by other
components of the innate and adaptive immune systems. The mechanisms by which antibodies achieve
this are neutralization, opsonization, and immune complex formation.

• Neutralisation of small antigens such as toxins and even viruses. The binding of antibodies
to these antigens prevents their activity by, perhaps, preventing their entry into cells or by
marking the antigens for uptake by phagocytes followed by their destruction in the
phagolysosome.
• Marking of cells following attachment of antibodies is called opsonisation and accelerates/
facilitates the uptake of the foreign cells by phagocytes or their destruction following the
binding of complement proteins.
• immune complex formation. Remember that each antibody molecule has two antigen-
binding sites so that it is possible for each antibody molecule to interact with two separate
antigen molecules, thus crosslinking the antigens. As antigens normally have many
epitopes, many antibodies can bind antigens to form a massive immune complex. If the
antigen involved is soluble (such as protein or virus), the reaction resulting in the formation
of the immune complex is called a precipitin reaction. Likewise, an agglutination reaction
involves insoluble antigens such as cells.

Refer to page 758-760 To have a detailed explanation of the mechanism by which antibodies can
participate in host defense.

Refer to page 757 To have a detailed explanation of the clonal selection theory.
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What we have discussed above is when we have a proper B-cell activation that will lead to the production
of antibodies. It is also important to know what will happen when we have an improper B cell activation or
why cells do not respond to self-antigens. To have an insight, please study the section on ‘Acquired
Tolerance’ on page 760 of the prescribed textbook. Please note that Acquired Tolerance is part of your
scope and should be studied in depth.

Activity 4.4
1. Describe what is meant by combinatorial joining of V, D, and J gene segments.
2. Describe an immune complex.
3. Describe the three ways acquired immune tolerance develops in the vertebrate host.

4.8 Monoclonal antibodies.

This technology was developed by Kohler and Milstein in 1975.

It involves culturing of mammalian cells in vitro to produce antibodies of a single specificity. The cultured
cells are referred to as hybridoma cells as they are produced by fusing myeloma cells (with immortal
characteristics) with antibody-producing spleen cells. Individual cells are then isolated which produce
monoclonal antibodies of the desired specificity.

To produce monoclonal antibodies:

• Inoculate animals (rats or mice) with antigen.
• Wait for antibody production.
• Remove animal spleen and separate cells.
• Fuse cells with myeloma cells, using polyethylene glycol.
• Culture cells in HAT medium, containing hypoxanthine, aminopterin and thymidine.
• Myeloma cells are poisoned by aminopterin and spleen cells die naturally.
• Surviving hybridoma cells which produce antibodies relating to the original spleen cells
are tested for the desired antibody.
• If tested positive, the cells are cloned.

Applications of monoclonal antibodies:

• tissue typing
• identification of microorganisms
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• identification of tumour and other cell-surface antigens

• classification of leukaemias
• identification of T cell populations

Further activities may include:

• passive immunisation against (naturally occurring and bioterrorism) agents and toxins
• tissue and organ graft protection
• stimulation of tumour rejection
• delivery of anti-tumour agents

Activity 4.3
Please prepare a diagram indicating the process of preparing hybridoma cells and the selection of
monoclonal antibodies.

More readings on this topic

Refer to your prescribed textbook, Prescott’s Microbiology, Chapter 34, pp. 749 - 760.

Discussion forum on this topic

The following questions must be discussed on the myUnisa site. Please present your answers in the
discussion forum:
• How does toxin neutralization occur?
• How does antigen-antibody binding occur?
• How does the lag in IgM production compare during a primary and secondary response?
Contrast this with the lag seen in IgG production during a primary and secondary response.
What does this difference imply about the nature of the secondary response?

Self-assessment questions

1. Multiple Choice: Which of the following is not an antibody effector function?

A. Opsonization
B. Neutralization
C. Complement activation.
D. Linked recognition.
E. NK-cell cytotoxicity
F. Mast-cell degranulation
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2. Matching: indicate whether the following properties apply to IgA, IgD, IgE, IgG, and/or IgM.
A. First produced during humoral response
B. Monomeric (predominantly)
C. Dimeric (Predominantly)
D. Pentameric (Predominantly)
E. Contains a J chain
F. Capable of eliciting complement deposition
G. Most abundant in mucosal surfaces and secretions
H. Low affinity
I. Bound onto mast cells
3. Multiple Choice: Which of the following is the most abundant immunoglobulin class in
healthy adult humans and mice?
A. IgA
B. IgD
C. IgE
D. IgG
E. IgM
4. Matching: Match the immunoglobulin class to its main function
A. IgA i. Most abundant in serum and strongly induced during an immune response.
B. IgD ii. First one produced after B-cell activation.
C. IgE iii. Defense at mucosal sites.
D. IgG iv. Defense against parasites but also involved in allergic diseases.
E. IgM v. Function not well known; may serve as auxiliary BCR
5. Multiple Choice: Which of the following options is not a mechanism by which an antibody
can protect against a pathogen.
A. Neutralization
B. Co-stimulation of T cells
C. Opsonization
D. Complement activation/deposition

4.9 Conclusion
In this learning unit we have dealt with antibodies, in particular, their structures, classes, monoclonal
antibodies, and how their produced by B cells. An antibody molecule is made up of four polypeptide chains,
comprising two identical light chains and two identical heavy chains, and forms a flexible Y-shaped
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structure. Specific antibodies mediate the removal of soluble toxins and extracellular pathogens.
Antibodies do not interact with toxins and antigens only, but also with the Fc region of specific receptors
that are expressed by many types of phagocytes. Phagocytes also express receptors for the complement
proteins that are deposited on microbial surfaces, particularly in the presence of antibody. The classes of
immunoglobulins are defined by their heavy-chain C regions.

Sources consulted for this unit

Joanne M. Willey, Linda M. Sherwood, & Christopher J. Woolverton. 2017. Prescott’s Microbiology.
Tenth edition. Mcgraw-Hill Education. ISBN: 978-1-259-28159-4, MHID: 1-259-28159-0. New York.
(Prescribed book)

Kenneth Murphy & Casey Weaver. 2017. Janeway’s Immunology. 9th edition. Garland Science, Taylor &
Francis Group, LLC. ISBN: 978-0-8153-4505-3.USA and UK.