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L8 – Antibody: Structure
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An antibody molecule can act as both the antigen receptor on the
surface of B-cells and as soluble molecules capable of fighting
infection
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The N-terminal end of each of the
arms of the Y forms an antigen-binding structure
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Therefore, the basic antibody unit has an approximate
molecular weight of 150kDa
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These classes are defined on the basis of their heavy chain
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There are also two alternative versions of the light chain: κ and λ
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There are two sub-classes of IgA: IgA1 and IgA2
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The number of inter-chain disulphide bonds
varies amongst different immunoglobulin molecules
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This Fc fragment provides the biological
characteristics of the immunoglobulin class related to the particular
heavy chains
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Detailed structure of the antibody molecule
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Each of the light chains consists of one variable and one constant
immunoglobulin domain, designated VL and CL, respectively
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Amino acid analysis shows that the N-terminal portions of the heavy
and light chains have variable compositions when different
immunoglobulins are compared
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Certain sequences in the variable region show quite remarkable
diversity and these hypervariable sequences have been localized to
three areas on the light chain and 3 on the heavy chain
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There
are 3 CDRs on the heavy chain and 3 on the light chain and these
regions make up the antigen combining site
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In addition, non-covalent interactions between VL and VH and CL and
CH1 on each Fab arm hold them firmly together
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There are also intra-chain disulphide bonds within each of the domains
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The arrangement is stabilised by a
disulphide bond linking the two sheets
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Hypervariable regions
Variability, for a given position, is defined as the ratio of the number of
different residues found at that position compared to the frequency of the
most common amino acid
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Different Ig classes
There are 5 classes of antibodies:
• IgG
• IgA,
• IgM
• IgD
• IgE
in order of abundance in the circulation
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There are five types of mammalian heavy chain
denoted by the Greek letters: γ, α, µ, δ and ε for IgG, IgA, IgM, IgD and IgE
respectively
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Any of these 5 classes of antibody can use either kappa or lambda light
chain
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IgG is monomeric
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In IgG, the hinge region tends to be exposed and
sensitive to proteases that cleave the molecule
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The CH2 domains
of IgF are held apart by masking of the hydrophobic regions by carbohydrate
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Sites on
the CH2 and CH3 domains bind to the Fc receptors on macrophages and
polymorphs and NK cells
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The CH2 domain provides the site for fixation of the complement component
C1q, which initiates the classical complement pathway and also controls the
catabolic rate, which determines the half-life
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When present in the circulation, IgA
is monomeric
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IgM
IgM is one of the two classes of antibodies that has an extra constant domain
CH4
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Pentameric IgM can adopt a star or staple
configuration
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Therefore, IgM is largely intravascular
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IgD
IgD is a monomer and primarily found on the surface of B-cells as a BCR
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IgE
A monomeric antibody found in very low concentrations in the circulation
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Antigens binding to
IgE cross-links IgE Fc receptors and triggers an acute inflammatory reaction
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L9 – Antibody: Genetics
A unique genetic recombination process, whereby a relatively small number of
genes can be utilized to encode millions of different proteins, generates the
antigen specific receptors present on lymphocytes
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Both types of antigen
receptors consist of variable and constant regions
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(Note: the mechanisms of somatic hypermutation and class
switching are not seen in the generation of TCR diversity)
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The human immunoglobulin gene loci
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The heavy chain variable region is generated from the joining of three
gene segments, variable (V), diversity (D) and joining (J) gene
segments
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The heavy chain gene segments are located on chromosome 14
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The
functional heavy chain repertoire is formed from 40VH, 27 DH and 6 JH
gene segments
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Downstream of the J gene segment is the constant (C)
gene segment, which specifies the class of antibody
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It is
composed of 40 Vκ gene segments and 5 Jκ gene segments followed
by a single Cκ gene segment downstream of the J gene segments
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The λ light chain gene segments are located on chromosome 2
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In this

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case, a Cλ gene segment follows each Jλ gene segment
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TCR genes on other chromosomes

A given B cell would use one out of the 40VH, one out of the 27DH and one
out of the 6JH gene segments chosen more or less at random
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Variability will be increased by the use of different light chain sequences by
different B-cells
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Therefore, the repertoire of antibodies with
different specificities produced will reach over a million, however, variability is
further brought about by additional mechanisms such as recombinatorial
inaccuracies and N-nucleotide addition
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When B-cell precursors differentiate into
fully-fledged B-cells, V(D)J gene recombination of the immunoglobulin genes
is one of the first things that is going to occur
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Before
this however, the genes are in a germ-line configuration
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One of the possible VH gene segment is then spliced to the
recombined DHJH segment
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The recombined DNA is transcribed and a primary RNA
transcript is produced, which is then spliced further bringing together the
variable and constant regions and processed into mRNA
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At the light chain locus, somatic recombination occurs with V and J gene
segments only
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Therefore,
CDR1 and 2 can only be one of 40 possible sequences, however, CDR3 has
much more variability as it is a combination of one out of 40 V, one out of 27D
and one out of 6 J gene segments
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Upstream of the flanking sequence is the TATA box, which is found in
the promoter region and binds RNA Polymerase II
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Association with enhancers will increase the rate of transcription and
the promoters are relatively inactive

V(D)J recombinase
• The actual process of V(D)J recombination is mediated by V(D)J
recombinase, a complex comprised of lymphocyte-specific enzymes
and some enzymes that are ubiquitously expressed
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• RAG-1 and RAG-2 are encoded by recombination-activating genes and
are referred to as recombinases
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• Efficient recombination occurs between segments with a 12-nucleotide
spacer and a 23-nucleotide spacer
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• On the heavy chain gene locus, RSS are found downstream of each VH
gene segment, on both sides of the DH gene segment and then
upstream of each JH gene segment
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Mechanism
• RAG-1 and RAG-2 bind to the RSS and the high-mobility group (HPG)
proteins bend the double-stranded DNA to bring together the two
RSSs
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• This leads to the creation of a hairpin loop containing intervening
sequences, which is repaired by non-homologous end-joining
machinery
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• This happens when a D gene segment is joined to a J gene segment
and then subsequently when V joins to DJ
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These are
referred to as recombinatorial inaccuracies or junctional inaccuracies, a
mechanism that creates further diversity
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Following the cut,
sometimes the enzyme terminal deoxynucleotidyl transferase (TdT) picks up a
small number of nucleotides at random and adds them to the ends of the cut
DNA before it is religated
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Following activation, the immunoglobulin gene undergoes somatic
mutation that is far greater than the normal rate of mutation
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The area of this mutation is very precisely targeted to the
hypervariable regions, but can also occur in framework regions
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The enzyme responsible for somatic
hypermutation is known as activation-induced cytidine deaminase (AID),
which is also very important for class-switch recombination
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This gives
rise to antibodies of the same antigen specificity but different effector
functions
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It is known
that the heavy chain exons for IgD, IgG, IgE and IgA are located downstream
of the heavy chain exon for IgM
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This is because IgM and
IgD are always produced together
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• The DNA between the two S regions is removed
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Once class-

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switching has occurred, you cannot go back to produce and antibody
upstream of the new constant exon due to the loss of that DNA
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L10 – Antibody: Function
Antibodies are found in two different forms
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The other form is the secreted antibody, which is secreted by
plasma cells derived from B-cells
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Epitopes come in many different forms, as do antibody-combining
sites
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The area of the antigen that

comes into contact with the antibody is known as the footprint and range from
4 to 10nm2
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Approximately 80-90% of
antibodies recognise discontinuous epitopes on the antigen
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Only when the molecule is correctly folded do these three
amino acids come into close proximity with each other and are able to interact
with the antigen-combining site of the monoclonal antibody
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However, some antibodies do recognise what is referred to as a linear
or continuous epitope
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On the contrary, epitopes can be partially
linear in nature with additional discontinuous amino acids contributing towards
the epitope
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In general, for most antibody-antigen interactions, there are usually about 1520 amino acids that are involved in the binding of antibody to antigen
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However, affinity maturation of the
immune response can occur, resulting in amino acids that previously
contributed the least to the binding energy being made to fit better
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These are
all non-covalent and therefore, antibody antigen binding is a reversible
interaction
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• Electrostatic forces are due to the attraction between oppositely
charged ionic groups on the side chains of the opposing amino acids
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• Hydrogen bonding can occur in hydrophilic groups on opposing side
chains as seen in the interaction between the amino acids serine and
tyrosine
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• Hydrophobic interaction can occur for example between leucine and
isoleucine
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• Van der waals forces depend on the interaction of the electron clouds
around the opposing amino acids
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If Ka is large, then the
equilibrium is far to the right and the formation of the Ab-Ag complex is
favoured
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A high affinity means the more effective the
antibody will be at dealing with infection
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This is particularly pronounced for secretory IgA,
which being a dimer, has 4 antigen binding arms and for IgM, which being a
pentamer, has 10 antigen binding arms
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Bonus effect of multivalent binding
Some antigens are soluble
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This has an advantage to it as the effect of
neutralization is enhanced
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However, unlike the innate response, the adaptive response is highly
specific for individual antigens and exhibits immunological memory
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On subsequent encounter with the same antigen, a
secondary immune response is mounted, which is much more potent
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Innate responses remain
the same upon numerous encounters with the same pathogen
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Antibody production is much faster during the secondary immune
response; more antibodies are produced and most of the antibodies
class switch to IgG, IgA or IgE
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IgM antibody production during the secondary immune response is
similar to that seen in the primary immune response but the production
of IgG antibodies in the secondary immune response is much greater
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Vaccination involves priming an immune response to an inoculus
antigen, thereby allowing the immune system to produce memory cells
and upon a subsequent encounter, a secondary immune response is
generated upon the first encounter with the actual pathogen
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There are two types of memory T-cells, central memory T-cells reside
in the lymph nodes and express the chemokine receptor CCR7,
whereas effector memory T-cells are present in other body tissues and
express other chemokine receptors (and not CCR7), as well as
adhesion molecules that direct them to particular locations
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If however, they express the α4β7-intergrin and CCR5 chemokine
receptor, they are home to mucosa
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After a short period of time, class switching
occurs and IgG of the same antigen specificity is produced
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But the heavy chain V(D)J
sequence now gets placed next to a different constant region gene segment
in a process referred to as CSR
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Switch from membrane to secreted antibody
Antibodies can either be secreted or they can be membrane-bound through
differential splicing of the pre-mRNA transcript encoding a particular
immunoglobulin
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If
transcription continues through to the membrane exons, then the Cµ4 can be
spliced to the M sequences resulting in the M2 poly-A addition signal being
utilised
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However, if transcription termination or cleavage occurs in the intron between
Cµ4 and M1, the Cµ4 poly-A addition signal is used and the secreted form is
produced
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However, antibody usually acts to focus other
components of the immune response onto the pathogen
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In order to combat this, the cytokine IFN is produced in
response to viral infection and sets up a state in the surrounding
environment that inhibits viral replication
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For example, in malaria, response against the plasmodium
protozoal parasite very much depends on the stage of the life cycle of
the parasite
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• IgM, IgG and IgA class of antibodies are particularly good at direct
neutralisation of antigens
• IgM antibodies are good agglutinins of bacteria
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• IgG antibodies can opsonize bacteria to make them more susceptible
to phagocytosis
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CR1 binds C3b, which can bind to
antibody-antigen complexes (including IgM or IgG antibodies) and
these immune complexes are picked up in the circulation by
erythrocytes, where they will go to the liver and spleen, where resident
macrophages will take up these immune complexes and destroy the
antigen
• IgM and IgG also plays a role in antibody-dependent cellular
cytotoxicity, the cell that is involved in killing via ADCD is called a K
cell
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• Mast cells and basophils have an FcεR, which is recognised by IgE
and therefore, IgE can stimulate the release of inflammatory mediators
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For example, nematode worms are expelled from the gut by
IgE-stimulated mast cell mediated inflammatory responses
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It
is present as a monomer on B-cell surfaces
Secreted pentameric IgM acts mainly in blood and is the first antibody
class to be produced in the immune response
Activates complement
It is a powerful agglutinin due to having 10 antigen-binding arms
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IgA
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IgD
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IgE
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Has 4 subclasses:
o IgG1 – most abundant antibody in the blood; activates
complement and enhances phagocytosis; can cross the
placenta
o IgG2 – activates complement
o IgG3 – activates complement and enhances phagocytosis; can
cross the placenta
o IgG4 – can cross the placenta
IgG is unique among the 5 classes of antibody such that is can cross
the placenta and help the foetus against potential infections  
Stimulated production of IgG can occur under the influence of the
cytokines IL-4, IL-5, IL-6, IL-13 and IFN-γ
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