30/12/2011 1 Antibody Structure & Function School of Chemical & Life Sciences (Chemical) CLB335 Molecular Biotechnology Antibodies • Main mediator of humoral immunity • Belong to a family of globular proteins called immunoglobulins (secreted form of immunoglobulins) • Secreted soluble proteins produced by plasma cells when B cells bind to specific antigens Structure of Antibody • Y-shaped molecule with two identical heavy polypeptide chains (termed H chains) and two identical light polypeptide chains (termed L chains) held together by covalent disulphide bond • Each light chain is about 25 kilodalton (kD) while each heavy chain is 55 to 70 kD depending on the class of immunoglobulin Light chains (depicted in green) Heavy chains (depicted in blue) 3-dimensional structure of IgG obtained from X-ray crystallography Structure of Antibody • Light chains exist in 2 distinct forms called kappa (κ) and lambda (λ) which are present in different ratios depending on the type of species (e.g. mouse has 95% κ while human has 60% κ) • Heavy chains exist in 5 distinct forms called γ, μ, α, δ and ε which give rise to the 5 classes of immunoglobulins and their different biological functions • Both light chains and heavy chains consist of two distinct regions – one constant region (C) on the C-terminal half of the chain and a variable region (V) on the N-terminal half
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30/12/2011
1
Antibody
Structure & Function
School of Chemical & Life Sciences (Chemical) CLB335 Molecular Biotechnology
Antibodies
• Main mediator of humoral immunity
• Belong to a family of globular proteins called immunoglobulins
(secreted form of immunoglobulins)
• Secreted soluble proteins produced by plasma cells when B cells
bind to specific antigens
Structure of Antibody
• Y-shaped molecule with two identical heavy polypeptide chains
(termed H chains) and two identical light polypeptide chains
(termed L chains) held together by covalent disulphide bond
• Each light chain is about 25 kilodalton (kD) while each heavy
chain is 55 to 70 kD depending on the class of immunoglobulin
Light chains
(depicted in green)
Heavy chains
(depicted in blue) 3-dimensional structure of IgG
obtained from X-ray crystallography
Structure of Antibody
• Light chains exist in 2 distinct forms called kappa (κ) and
lambda (λ) which are present in different ratios depending on the
type of species (e.g. mouse has 95% κ while human has 60% κ)
• Heavy chains exist in 5 distinct forms called γ, μ, α, δ and ε
which give rise to the 5 classes of immunoglobulins and their
different biological functions
• Both light chains and heavy chains consist of two distinct
regions – one constant region (C) on the C-terminal half of the
chain and a variable region (V) on the N-terminal half
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Structure of Antibody
• Variable regions (V) of light and heavy chains are about 110
amino acids long and denoted by VL and VH respectively
• Constant region (C) on the light chain is also about 110 amino
acids long and while that for the heavy chain is about 330 or 440
amino acids long depending on the class of immunoglobulins; they
are denoted by CL and CH respectively
• The variable region is the part of the molecule that binds to the
epitope of an antigen and give rise to its specificity
Hypervariable Regions
• The variable regions of the light and heavy chains can be further
divided into the hypervariable and framework regions
• Each hypervariable region contains three highly variable amino
acid sections known as the complementary-determining regions
(CDRs)
• Each CDR is about 10 amino acid residues in length and are
denoted by CDR1 (from the N-terminal end), CDR2 and CDR3
• The complementary-determining regions (CDRs) are located
around amino acid residues 30, 50 and 95; and form protruding
loops on the surface of the domains which are complementary to
the configuration of the antigen that binds to the antibody (i.e. the
CDRs determine the specificity of the antibody)
Framework Regions
• The complementary-determining regions (CDRs) are flanked by
relatively constant regions with little variability in the amino acid
sequence known as the framework regions, which accounts for
about 85% of the variable region
Hypervariable
regions of light
chain
Variable regions
of light chain
Variable regions of
heavy chain
Hypervariable
regions
Hypervariable
regions of
heavy chain
Antigen-binding
site
Globular Domain
• In addition to the inter-chain disulphide bonds that hold the L and
H chains together, intra-chain disulfide bonds also exist within
the chain forming loops of peptide chains
• Both the light and heavy chains contain repeating segments of
approximately 110 amino acids that fold independently into a
compact globular structure known as domains.
• Each light chain consists of 2 domains – 1 variable (VL) and 1
constant (CL) domain
• Each heavy chain consists of 4 or 5 domains – 1 variable
domain (VH) and 3 or 4 constant domains (CH1, CH2, and CH3) (Both μ and ε chains have one variable and four constant domains)
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Globular Domain
• Variable domains are responsible
for antigen binding while the
constant domains of the heavy
chains (excluding CH1)
determines the other biological
properties of the antibody
Light Chains
Heavy
Chains
Antigen-binding Site
CH1
CH2
CH3
CH1
CH2
CH3
CL CL VH VH
VL VL
Hinge Region
• The hinge region is a short segment of
amino acids (predominantly cysteine and
proline residues) found between the CH1
and CH2 regions of the heavy chains
• The hinge region confers flexibility to the
antibody for binding with antigens as it
allows variability of the angle between the
arms of the Y-shaped antibody as
well as rotational flexibility of
each individual arm
Functions of Antibody Functions of Antibody
• Opsonization
Microorganisms or other foreign particles coated with antibodies are
especially susceptible to phagocytosis as the antibodies bind to the Fc
receptors of phagocytes such as dendritic cells, macrophages and neutrophils
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Functions of Antibody
• Immune Complex Formation
Cross-linking of antigens and antibodies
can result in large aggregates known as
immune complexes. Precipitation can
occur if the complexes become large enough.
An agglutination reaction can also occur if the immune complex involves
the cross-linking of cells or particles.
• Virus & Toxin Neutralization
Antibodies can neutralize viruses and toxins by direct binding to these
antigens (which will be cleared by the complement pathway) and prevent
their adherence to surface receptors on target cells
Functions of Antibody
• Complement Fixation
Complement pathway
(classical) is triggered
by the binding of C1q
(one of the three proteins
forming the complex C1)
to antibodies attached
on the surface of
pathogen
C1q is only activated
when it binds at least
two Fc pieces
Immunoglobulins
• Group of glycoproteins present in the serum and tissue fluid of
all mammals
• Can be expressed as secreted or membrane-bound forms
• Membrane-bound form are present on the surface of B cells
where they act as receptors for specific antigens
• Activation (binding) of B cells by antigens result in the
development of plasma cells which secrete antibodies (secreted
form of immunoglobulins)
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History of Immunoglobulins Tiselius and Kabat discovered γ-globulin (IgG)
from the electrophoresis of rabbit serum
1940
1990
1950
1960
1970
1980 Cesar Milstein and George Köhler created hybridoma
cells to produce monoclonal antibodies (Mab)
Rodney Porter and Gerald Edelman received the
Nobel Prize for elucidating the chemical structure
of antibody
IgE was isolated from pollen proteins by the Japanese
scientist couple Teruka and Kimishige Ishizaka.
Immunoglobulins
• Immunoglobulins can be classified into 5 different classes with
distinct characteristics. They are IgG, IgA, IgM, IgD and IgE.
• The heavy (H) chains are structurally and functionally distinct
for each class and are designated by Greek letters that
correspond to the immunoglobulin class.
No. Immunoglobulin Class
(Isotype) Heavy Chain
1 IgG γ (gamma)
2 IgA α (alpha)
3 IgM μ (mu)
4 IgD δ (delta)
5 IgE ε (epsilon)
Immunoglobulins IgG
• Biological & Structural Properties
Molecular weight ≈ 150,000 Da with two γ heavy chains (≈ 50,000 Da each)
and two lights chains (≈ 25,000 Da each) held together by disulfide bonds
Contains 4 subclasses IgG1, IgG2, IgG3 and IgG4 in the approximate
proportions of 65, 25, 5 and 5% respectively
Constitute 15% of the total serum proteins in humans and 75% of the total
serum antibodies
Has the longest half life of all Ig isotypes – approximately 23 days
Only isotype that can pass through the human placenta and provide
immunity to the fetus in utero
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IgG
• Biological Functions
Major antibody synthesized during secondary immune response
Activation of complement (classical pathway)
Neutralization of toxins and venom by blocking the active sites (passive
immunization)
Agglutination (clumping) and immobilization of microorganisms
Opsonization of microorganisms to enhance phagocytosis
Involve in antibody-dependent cell-mediated cytotoxicity (ADCC) where the
antibodies coated on the target cells bind with receptors on natural killer cells
ADCC
• Triggered by the interaction of antibody (IgG) with the CD16
receptor on the surface of NK cells
• Mechanism of attack is similar to that of cytotoxic T cells which
involve the release of cytoplasmic granules containing perforin
and granzymes, inducing apoptosis
IgM
• Biological & Structural Properties
First immunoglobulin to be produced upon initial exposure to an antigen
(elevated levels of IgM usually indicate recent infection or immunization)
Macroglobulin of high molecular weight (900,000 Da)
Pentameric molecule with 5 units of four chain structure (two L and two H
chains joined together by disulfide bonds between their Fc portions and by a
polypeptide chain called the J chain)
Mainly found in serum due to its large size
Relatively short-lived with a half life of approximately 5 days
Does NOT pass through the human placenta and presence in cord blood
indicates acute fetal infection
High avidity but low affinity to antigens due to presence of multiple binding
sites
Structure of IgM
Antigen binding sites
μ heavy chain
Light
chain
= disulfide bond
J chain
All the Fc portions
are joined together
by disulfide bonds
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IgM
• Biological Functions
Efficient agglutinating agent due to its pentameric form – macromolecular
bridges can be formed between epitopes on distant molecules
Most efficient initiator of complement (classical pathway)
Primary antibody (isohemagglutinin) against the red blood cell antigens of the
ABO blood groups – responsible for the clumping (agglutination) of red blood
cells if the recipient of a blood transfusion receives blood that is not compatible
with their blood type
IgA
• Biological & Structural Properties
Major immunoglobulin found in external secretions
such as saliva, mucus, sweat, gastric fluid and tears
Molecular weight ≈ 165,000 Da with two heavy α
chains and two lights chains
Relatively short-lived with a half life of
approximately 5.5 days
Contains 2 subclasses IgA1 and IgA2
Present in small amount in serum as a monomer
(one four-chain unit) but found predominantly as a
dimer in mucous secretions known as secretory IgA
(sIgA)
Secretory IgA (sIgA) contains two IgA monomers
connected by a J chain and contains a secretary
component which protects the dimeric IgA from
proteolytic cleavage
IgA
Dimeric IgA are produced by plasma cells in the lamina propria (connective
tissue adjacent to mucosal surfaces)
Dimeric IgA can bind to the polymeric immunoglobulin receptor on the
(basolateral) surface of epithelial cells before being taken up into the cell via
endocytosis
The receptor-IgA complex passes through the cellular compartments before being
secreted into the lumen containing a portion of the receptor known as the
secretory component
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IgA
• Biological Functions
Primary immunologic defense against local infections in areas such as the
respiratory or gastrointestinal tract
Protects mucosal surface
Activates complement via the alternative pathway
Efficient antiviral agent and protects mucosal surfaces via immune exclusion
and intracellular neutralization mechanisms
Immune exclusion involves the inhibition of contact between pathogen and the
mucosal epithelial cell surface
Intracellular neutralization involves the inhibition of key viral replication
steps (such as removal of capsid) from the interaction between sIgA and the
endocytosed virus
IgA Protection Mechanisms
Immune
Exclusion
Intracellular
Neutralization
B Cell Maturation IgD
• Biological Properties & Functions
Present in trace amounts in serum
Monomer with a molecular weight ≈ 180,000 Da with two heavy δ chains and
two lights chains
Co-expressed with IgM on the surface of mature B cells
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IgE
• Biological Properties & Functions
Mainly found in the linings of the respiratory and gastro-intestinal tract
Molecular weight ≈ 200,000 Da with two heavy ε chains and two lights
chains
Present in the least abundance and has the shortest half life (2 days) among
the 5 classes of immunoglobulins
High affinity to receptors on mast cells and basophils – trigger Type I
hypersensitivity reactions (allergy) when IgE bind to these cells
Plays an important role in parasite immunity through the activation of
eosinophils
IgE-Mediated Hypersensitivity
Hypersensitivity
Immunity Against Parasitic Worm Summary
Properties IgG IgM IgA IgD IgE
Structure Monomer Pentamer Dimer Monomer Monomer
Molecular Weight
(Da) 150,000 900,000 400,000 180,000 200,000
Half Life (days) 23 5 6 3 2
Serum
Concentration
(mg/ml)
12 1.2 2 0.03 0.00004
Placental Transport Yes No No No No
Activate
Complement
Yes
(Classical)
Yes
(Classical)
Yes
(Alternate) No No
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Antigenic Determinants
• Immunoglobulins are glycoproteins which can function as
antigens themselves
If a rabbit is immunized with human antibodies, the antibodies will be
recognized as a foreign antigen by the rabbit’s immune system resulting in
the production of anti-human antibodies (anti-serum)
• Antigenic determinants of immunoglobulins can be classified into
three categories – isotypic, allotypic and idiotypic
Isotypic Determinants
• Isotypic determinants are the antigenic specificities that
distinguish the different classes and subclasses of immunoglobulins
present in all normal individuals of a given species
• Isotypic determinants are located on the constant region of the
heavy chain which determine the five classes of immunoglobulins
(IgG, IgA, IgM, IgD and IgE) and their effecter functions
• Antibodies to isotypes are used for the quantitation of
immunoglobulin classes and subclasses in the diagnosis of various
immunodeficiency diseases (e.g. B cell leukemia)
Allotypic Determinants
• Allotypic determinants are the antigenic specificities that
distinguish immunoglobulins of the same class between different
groups of individuals in the same species
• Allotypic differences arise from genetic variations of the alleles
encoding the antigenic determinants and occur mostly in the
constant regions of the heavy chain
• Antibodies to allotypic determinants may be produced by a
pregnant mother in response to paternal allotypic determinants in
the fetal immunoglobulins (may be used for paternity testing)
IgG1
Person 1
IgG1
Person 2
Idiotypic Determinants
• Idiotypic determinants are the antigenic specificities exclusive to
each individual immunoglobulin molecule
• Idiotypic determinants are located at the hypervariable regions of
the light and heavy chains, i.e. the antigen binding sites
• Anti-idiotypic antibodies stimulate B cells to make antibody and
can be used as a vaccine against highly dangerous pathogens
Mouse IgG1
against antigen A Mouse IgG1
against antigen B
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Polyclonal vs. Monoclonal Antibodies
• Polyclonal antibodies are
heterogeneous mixtures of antibodies
produced from multiple clones of
plasma cells, with different specificity
for the various epitopes on an antigen
• Monoclonal antibodies are identical
antibodies produced from a single
clone of plasma cells with specificity
for one particular epitope on an
antigen
Polyclonal Antibodies
Monoclonal Antibodies
History of Monoclonal Antibodies
Köhler and Milstein developed the hybridoma technology which allows
monoclonal antibody to be produced
First antibody Orthoclone OKT3® approved for therapeutic use
First chimeric antibody ReoPro® produced by Centocor was approved
by FDA
First humanized antibody Zenapax® was approved by FDA for the
prevention of acute kidney transplant rejection
First human antibody Humira® produced by Abbott Laboratories was
approved by FDA for the alleviation of symptoms associated with
rheumatoid arthritis
Vectibix® produced by Amgen became the first antibody produced from
transgenic humanized mouse to be approved for therapeutic use
1975
1986
1994
1997
2002
2006
Hybridoma Technology
• Monoclonal antibodies are typically produce from hybridoma
cells made by fusing myeloma (cancerous) cells with spleen cells
from a mouse that has been immunized with the desired antigen:
Antigen is injected into mouse to stimulate the production of antigen-specific
(antibody-producing) plasma cells
Plasma cells are isolated from the mouse’s spleen and fused with myeloma
cells using polyethylene glycol which change the membrane’s permeability
Cells are grown in HAT (hypoxanthine, aminopterin and thymidine) medium
which is selective for fused cells.
Surviving cells are cultured and supernatants from each clone are placed into
antigen-coated wells to identify the cell producing the specific antibody
Identified hybridomas producing the desired antibody are grown in large