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Topic (7): Antibodies and Antigens
INTRODUCTION
Antibodies (Abs) are one of the three classes of molecules able to differentiate between
antigens [Ags] (the other two are T-cell receptor [TCR] and major histocompatibility complex
[MHC]) {Figure 1}. Antibodies have a very special reputation among these antigen-
recognition molecules because they have the broadest range of antigen specificities and they
possess a bigger affinity (strength of binding) to antigens compared to the two others.
An antigen (Ag) is any substance that is specifically recognized and bound by the antigen
receptors of T or B lymphocytes. The antigen that is used to immunize the host and induce
antibody production is called the cognate (i.e. related) antigen. An epitope (antigenic
determinant) is a small region of an immunogenic molecule that binds to an antigen receptor.
While B and T cells carry receptors that are similar from the functional point of view, in that
they both bind antigen, these receptors recognize epitopes in fundamentally different ways.
The TCR recognizes an epitope composed of an antigenic peptide presented in association with
an MHC molecule, while B cell receptors recognize epitopes on macromolecules in solution,
or on macromolecules bound to cell surfaces.
Almost every kind of
organic molecule
(including proteins,
carbohydrates, lipids, and
nucleic acids) can be an
antigen. Proteins and
polysaccharides have the
size and properties
necessary to be the most
prevalent antigens to
induce an immune response.
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IMMUNOGLOBULIN (ANTIBODY STRUCTURE)
Electrophoresis is a technique used for characterizing proteins. Antibodies were identified in
the third peak (the γ-peak) of electrophoretically fractionated serum globulin proteins, thus they
are also termed gamma globulins. Basic structure of an Ab responsible for certain functional
features was inferred using digestion by two different enzymes. If papain was used as the
proteolytic enzyme, the antibody molecules were reproducibly cleaved into three parts. Of
these, two were identical to each other and retained the ability to bind antigen. The third
fragment did not bind antigen and tended to crystallize. The first two fragments Fab (fragment,
antigen-binding), and the third fragment Fc (fragment, crystalline). If another enzyme, pepsin,
was used in for proteolytic cleavage, the results were different: the Fc’ fragment was unstable
and degraded quickly, while the two Fab fragments were bonded to each other. These doublets
of Fab fragments F(ab’)2 (Figure 2).
Each Ab molecule consists of four polypeptide chains: Two heavy chains that are identical
to each other and have a molecular weight of approximately 55 to 70 kDa. The other two chains
are also identical to each other and have a molecular weight of approximately 24 kDa and they
are called antibody light chains. Each light chain is attached to a heavy chain, and the two
heavy chains are attached to each other through covalent disulfide-bonds.
Each heavy and each light chain has its own variable (V) and its own constant region (C). This
macromolecule consisting of four polypeptide chains is called a monomeric antibody molecule
because, antibodies of some isotypes form multimeric aggregates, in which several monomeric
molecules are joined together.
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The entity that can physically bind an epitope (the antibody variable region), is the heavy chain
variable region and the light chain variable region that are juxtaposed in space (Figure 3).
Now, we will discuss the concept of antibody domain (Figure 3). A domain is a portion of an
Ab chain that has the length of about 110 amino acids and a sphere-like shape of the fold. The
Ab domains follow each other along the length of individual heavy and light chains. The light
chains always have two Ig domains, of which the N-terminal domain is the variable region of
the light chain (or the VL region), and the C-terminal domain is its C-region (or the CL region).
The heavy chains consist of four or five domains, of which the N-terminal domain is the
variable region of the heavy chain (or the VH region), and the remaining three or four domains,
comprise the heavy chain constant region (or the CH region) and are called the CH1, CH2, CH3
and CH4 (in IgM and IgE) domains.
The portion of Ab between the CH1 and CH2 domains is very flexible and forms the so-called
hinge, the region where the VH and the CH1 domains together with the VL and CL domains that
are attached to them form an angle, allowing the variable regions to deviate in space from the
longitudinal axis.
The Ab interaction with epitopes is always mediated through portions of the VH and VL
domains. The interaction with complement and Fc receptors (to confer the Ab effector
functions), is mediated through the CH2 domains.
At this point we need to understand… What gives each Ab molecule a particular specificity to
interact with a sole specific epitope?
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Within the V domains of both the L and H chains are three short hypervariable regions that
exhibit extreme amino acid variability. These regions of five to seven amino acids are
responsible for the diversity that allows the total repertoire of Abs to recognize almost any
molecule in the universe of antigens. The antigen-binding sites are formed by 3D
juxtaposition of the three hypervariable regions in the VL domain with those in the VH domain.
Because these sequences result in a structure complementary to an antigenic epitope, the
hypervariable regions are also called complementarity-determining regions (CDRs). [Figure
4]. The CDR loops contribute to unique regions within the antigen-binding site that form the
paratope of the Ab which binds specifically to the corresponding epitope of the antigen.
In contrast to the variability and hypervariability of the V domains, the C domains exhibit very
little variation among antibodies, which is expected for a structure that carries out the same
effector action in response to a wide variety of antigens. So, what are these effector functions
of Abs?
1. Neutralization of pathogens and interference with pathogen attachment.
2. Activation of the complement system with subsequent lysis of the microbe.
3. Opsonization and aiding in phagocytosis.
4. Aiding NK cell killing through ADCC (do you still remember that?).
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So, these functions are carried out by the CH domains and the structure of these CH domains
will determine the particular function of each Ab group that can be classified as separate classes
or isotypes.
There are five classes of human Ab heavy chains, called: α, γ, δ, ε, and µ corresponding to the
five isotypes of Abs, which are called IgA, IgG, IgD, IgE, and IgM,. The amino acid differences
in these different chains can affect the size, charge, solubility, and structural features of a
particular Ab, which in turn influence where an Ig goes in the body and how it interacts with
surface receptors and other molecules. A mechanism called isotype switching occurs later in
the life span of a B cell clone that allows its individual members to produce Ab of different
constant region sequences. All of these differences can influence how a given antibody will
clear its Ag.
For the light chain constant region, two classes are present that are referred to as κ (kappa) or
λ (lambda) chains. In an individual human’s repertoire of Ab-producing B cells, 60% produce
Abs with κ light chains, and 40% produce Abs with λ chains.
IMMUNOGLOBULIN ISOTYPES: STRUCTURE AND FUNCTION
I. IgM: Monomeric IgM is always the first form and isotype of Ig generated by naïve B cells.
Following its initial activation by Ag, the naïve B cell proliferates and differentiates, and its
progeny produce the pentameric secreted form of IgM. All other Ab isotypes (except IgD) are
generated by isotype switching, a process that occurs only late in a primary response. Thus, it
is IgM that is expressed first in any primary immune response, and those that are synthesized
first in the newborn (which has just begun to encounter antigens on its own). The detection of
increased IgM levels in an adult indicates a recent exposure to a novel antigen. It is also the Ab
produced in response to Ti Ags that can activate a B cell in the absence of T cell help.
IgM comprise only about 5–10% of normal serum Ig. However, the low absolute numbers of
IgM are balanced by the number their binding sites. Because of its pentameric nature, the IgM
displays 10 Fab sites that can theoretically bind to a pathogen.
IgM are large pentamers of total molecular mass 970 kDa with J-chain (joining chain, a small
polypeptide that regulates the multimerization of IgM and IgA) and they are generally
concentrated in the blood. IgM is the most efficient isotype in activating complement. IgM is
not an isotype prominent in either opsonization or ADCC.
II. IgD (the enigma): IgD is the second Ig isotype to be synthesized by a B cell, and first
appears on its surface early in B cell development. Recent studies have shown that IgD plays
an important role in the regulation of tolerogenic and protective B cell responses at mucosal
sites of antigen entry, including the respiratory route. This mucosal homeostasis may augment
immune adaptations following post‐natal exposure of the mucosa to airborne and food antigens,
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including allergens. An early dysregulation of these adaptation processes could contribute to
the pathogenesis of common disorders such as allergies by disrupting mucosal homeostasis.
III. IgG: This Monomeric isotype is the most common in the circulation and tissues. In the
blood of normal adult, 70–75% of serum Ig is IgG. It can be classified into four subclasses with
the following proportions of total IgG in serum: IgG1 (67%), IgG2 (22%), IgG3 (7%), IgG4
(4%). These subclasses have minor amino acid differences in the CH domains, however, these
minor differences confer different half-lives and effector functions for the different subclasses.
The diffusibility and high serum concentration make IgG the most prevalent Ab in the
extracellular fluid. IgG is a key opsonin and is important in ADCC. The IgG1 and IgG3
subclasses are particularly good opsonins and mediators of ADCC because FcγRs bind IgG1
and IgG3 antibodies with high affinity. This is related to the length of the hinge region in the
CH2 domain of the Ig structure, since IgG3 has an extended hinge, and IgG4 has a very short
hinge. IgG is an important activator of complement (though not as efficient as IgM). IgG3 is
the most efficient complement-activating IgG followed by IgG1 and IgG2. IgG4 cannot fix the
complement. In addition, the only Abs to cross the placenta is of IgG class. Maternal IgG1,
IgG3, and IgG4 molecules have been found to readily cross the placenta via binding to neonatal
Fc receptor (FcRn), whereas IgG2 crosses the placenta with much lower efficiency.
IV. IgA: 85–90% of IgA antibodies are found in the secretory form in the external secretions
of the body, which include tears, saliva, mucous secretions of the gastrointestinal, urogenital,
and respiratory tracts and breast milk. Secretory IgA antibodies are of enormous importance
because they facilitate antigen removal right at the mucosal surface, the most common site of
initial pathogen attack. The remaining 10–15% of IgA antibodies occurs in blood. There are
two subclasses, designated IgA1 and IgA2 that differ in content by 22 amino acids, 13 of which
are located in the hinge region and are deleted in IgA2. The lack of this region appears to make
IgA2 more resistant to some bacterial proteases that are able to cleave IgA1. Hence IgA2 is the
predominant form in secretions at mucosal surfaces, while IgA1 is mainly found in serum.
IgA2 is present as a dimer with the two monomers held together by a J chain. A secretory
component (SC), is later attached to the FC region around the hinge portion of α chains of the
dimeric IgA. This protein, is derived from epithelial cells found in close proximity to the
plasma cells. Secretory IgA has antiviral activity through neutralization. IgA also plays a role
in the elimination of helminth worms, in that IgA-coated parasites can be dispatched by ADCC
carried out by eosinophils bearing FcαR. IgA does not have the ability to fix the complement.
V. IgE: IgE is present in the serum at the lowest concentration of all Ab isotypes. IgE
antibodies do not cross the placenta, cannot fix complement, and do not function as opsonins.
The serum concentration of IgE rises dramatically in response to worm infections. IgE
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antibodies are also responsible for the symptoms experienced in allergic reactions such as hay
fever, and more severe conditions such as asthma and anaphylactic shock. Shortly after
synthesis, IgE attaches to basophils and tissue mast cells by means of specific surface proteins,
termed high-affinity FCε receptors. When two adjacent IgE molecules on a mast cell bind
specific antigen, a cascade of cellular events is initiated that results in degranulation of the mast
cells with release of vasoactive amines such as histamine and heparin.
ANTIGENS AND ITS INTERACTION WITH ABS AND B CELLS
B cells provide Ag receptors that can recognize soluble or cell-bound Ags without prior
modification (compared to T cells that fail to do so, and need MHC presentation of a modified
Ag). Thus, the B lymphocyte population is able to recognize a broad range of molecules,
including proteins in their native or denatured conformations, lipids, carbohydrates and nucleic
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acids. However, most B cells cannot proceed beyond recognition of the antigen without T
cell help. The term T cell help is used to describe the cooperation between helper T cells and
B cells that is necessary for most B cell responses. T cell help takes the form of direct
intercellular T-B cell contacts, and the binding to the B cell of specific cytokines produced by
a T cell responding to the same antigen. Without T cell help, the majority of B cells are unable
to achieve complete activation. Antigens that bind to B cell receptors but cannot activate B
cells without T cell help are called T-dependent (Td) antigens. For some non-protein Ags,
interaction between the B cell receptors and the Ag alone is sometimes sufficient to activate
the B cell. These Ags, which are often polymers, are called T-independent (Ti) antigens,
because no T cell help is required for lymphocyte activation. Ti antigens generally are large
polymeric proteins or polysaccharides (and sometimes lipids or nucleic acids) whose structure
is composed of repetitive elements, as occur in many bacterial and viral products and structural
elements.
Ti antigens are only a small fraction of the immunogens that attacks the host. A high proportion
of the molecules making up a pathogen are proteins of unique amino acid sequences that lack
the large repetitive structures needed to cross-link BCRs and trigger B cell activation.
Obviously a Td antigen must contain protein, since it must supply at least one peptide that can
act as a T cell epitope. The immunogenicity of a Td antigen also depends on other physical
properties such as its foreignness, conformation, and molecular complexity. The nature of the
antigenic challenge must also be taken into account (i.e. the immunogenicity of a particular
molecule may be affected by its dosage and its route of entry or administration). Finally, host
factors may influence the immunogenicity of a molecule. Each step in the immune response is
controlled by the expression of the host’s genes, and subtle allelic differences in the genetic
constitution of an individual can alter the type, as well as the intensity, of the immune response
to a given immunogen.
END of TEXT
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References