Download Antibodies - Molecular Immunology

Antigens/Antibodies
Chapters 4-6
Antigen vs. Immunogen
• Antigen
– Any substance that can
bind to an antibody or T
cell receptor
• Immunogen
– Any substance that can
elicit an immune
response
– All immunogens are
antigens
– Not all antigens are
immunogens, i.e.
haptens
Immunogenicity of Antigens
• Determined by
–
–
–
–
Foreignness
Molecular Size
Chemical Composition
Degradability
• Influenced by
– Host genotype
– Dose and route of
administration
– Presence of adjuvants
Epitopes
• Discrete sites on immunogens recognized by
antibodies or T cell receptors
• Macromolecules contain many epitopes with
different immunogenicities
• Immunogenicity can vary between individuals and
populations.
• Some epitopes are immunodominant
• Identification of B-cell
epitopes within the
HPV16 E7 protein.
• Overlapping peptides
were generated and
injected into mice.
• The antibody response to
each peptide was
measured by ELISA.
• Comerford, S.A. et al. J. Virol.
65:4681-4690, 1991
Epitopes
• Overlapping
• Non-overlapping
•
•
•
•
Allosteric
Linear
Conformational
Neoantigen
B and T Cell Epitopes Can Overlap
•Comerford, S.A. et al. J. Virol. 65:4681-4690, 1991
Antibodies
Objectives
• To discuss the general properties of all
immunoglobulins
• To describe the basic structure of immunoglobulins
• To relate immunoglobulin structure with function
• To define immunoglobulin hypervariable and
framework regions
• To define immunoglobulin classes and subclasses,
types and subtypes
• To describe the structures and properties of
immunoglobulin classes
Immunoglobulin (Ig)
• Immunoglobulins are glycoprotein molecules that are
produced by plasma cells in response to an immunogen
and which function as antibodies. The immunoglobulins
derive their name from the finding that they migrate with
globular proteins when antibody-containing serum is
placed in an electrical field.
Electrophoretic separation of serum proteins
General Functions
• Antigen Binding
• Effector Functions
GENERAL FUNCTIONS OF
IMMUNOGLOBULINS
• Antigen binding
– Immunoglobulins bind specifically to one or a few
closely related antigens.
– Binds to a specific antigenic determinant.
– Antigen binding is the primary function of antibodies
and can result in protection of the host.
– The “valency” of antibody refers to the number of
antigenic determinants that an individual antibody
molecule can bind.
– The valency of all antibodies is at least two and in
some instances more.
Effector Functions
• The immunoglobulins mediate a variety of these
effector functions:
– Fixation of complement - This results in lysis of cells
– Binding to various cell types:
• Phagocytic cells, lymphocytes, platelets, mast cells, and
basophils have receptors that bind immunoglobulins. This
binding can activate the cells to perform some function.
• Some immunoglobulins also bind to receptors on placental
trophoblasts, which results in transfer of the
immunoglobulin across the placenta. As a result, the
transferred maternal antibodies provide immunity to the
fetus and newborn
BASIC STRUCTURE OF
IMMUNOGLOBULINS
• Heavy and light chains
• Disulphide bonds
• Variable (V) and
constant (C) regions
• Hinge region
• Domains
• Oligosaccharides
• IgG structure
– 2 HEAVY chains = red and yellow
– 2 LIGHT chains = green and blue.
• 2 antiparallel beta sheets
– make up each of the
immunoglobulin domains (2 Ig
domains per L chain and 4
domains per H chain)
• Papain cleavage site.
– Papain cleavage gives
ONE Fc fragment (2
immunoglobulin domains from
each H chain) and
– TWO Fab fragments (each with 2
immunoglobulin domains from an
H chain and 2 immunoglobulin
domains from an L chain)
• each Fab fragment contains
ONE antigen binding site
plot of variability as a function of position along the sequence of the
amino acids that make up the heavy chain of Ig molecules.
STRUCTURE OF THE VARIABLE
REGION
• Hypervariable (HVR) or complementarity determining
regions (CDR)
– Comparisons of the amino acid sequences of the variable
regions of immunoglobulins show that most of the
variability resides in three regions called the HVR/CDR.
– Antibodies with different specificities (i.e. different
combining sites) have different complementarity
determining regions while antibodies of the exact same
specificity have identical complementarity determining
regions (i.e. CDR is the antibody combining site).
– Complementarity determining regions are found in both
the H and the L chains.
STRUCTURE OF THE VARIABLE
REGION
• Framework regions
• The FR regions form a beta-sheet structure which
serves as a scaffold to hold the HV regions in
position to contact antigen.
• Based on similarities and differences in the
framework regions the immunoglobulin heavy
and light chain variable regions can be divided
into groups and subgroups.
• Antibodies are divided into five major classes,
IgM, IgG, Iga, IgD, and IgE, based on their
framework/constant region structure.
HUMAN IMMUNOGLOBULIN CLASSES, SUBCLASSES,
TYPES AND SUBTYPES
• Immunoglobulin classes
The immunoglobulins can be divided into five different classes,
based on differences in the amino acid sequences in the constant
region of the heavy chains.
• All immunoglobulins within a given class will have very similar
heavy chain constant regions. These differences can be detected by
sequence studies or more commonly by serological means (i.e. by
the use of antibodies directed to these differences).
–
–
–
–
–
IgG - Gamma heavy chains
IgM - Mu heavy chains
IgA - Alpha heavy chains
IgD - Delta heavy chains
IgE - Epsilon heavy chains
Immunoglobulin Subclasses
• The classes of immunoglobulins can de divided into subclasses
based on small differences in the amino acid sequences in the
constant region of the heavy chains.
• All immunoglobulins within a subclass will have very similar heavy
chain constant region amino acid sequences. Again these
differences are most commonly detected by serological means.
• IgG Subclasses
–
–
–
–
IgG1 - Gamma 1
IgG2 - Gamma 2
IgG3 - Gamma 3
IgG4 - Gamma 4
heavy chains
heavy chains
heavy chains
heavy chains
• IgA Subclasses
– IgA1 - Alpha 1 heavy chains
– IgA2 - Alpha 2 heavy chains
IgG
•
Structure
– All IgG's are monomers (7S immunoglobulin). The subclasses differ in the number of
disulfide bonds and length of the hinge region.
Properties
• IgG is the most versatile immunoglobulin because it is capable of carrying
out all of the functions of immunoglobulin molecules.
• IgG is the major Ig in serum - 75% of serum Ig is IgG
• IgG is the major Ig in extra vascular spaces
• Placental transfer - IgG is the only class of Ig that crosses the placenta.
Transfer is mediated by a receptor on placental cells for the Fc region of
IgG. Not all subclasses cross equally well; IgG2 does not cross well.
• Fixes complement - Not all subclasses fix equally well; IgG4 does not fix
complement
• Binding to cells - Macrophages, monocytes, granulocytes and some
lymphocytes have Fc receptors for the Fc region of IgG. Not all subclasses
bind equally well; IgG2 and IgG4 do not bind to Fc receptors. A
consequence of binding to the Fc receptors on PMNs, monocytes and
macrophages is that the cell can now internalize the antigen better. The
antibody has prepared the antigen for eating by the phagocytic cells.
• IgG is a good opsonin.
IgG
•
•
•
•
•
•
•
•
Increases in:
Chronic granulomatous
infections
Infections of all types
Hyperimmunization
Liver disease
Malnutrition (severe)
Dysproteinemia
Disease associated with
hypersensitivity
granulomas, dermatologic
disorders, and IgG myeloma
Rheumatoid arthritis
•
•
•
•
•
•
Decreases in:
Agammaglobulinemia
Lymphoid aplasia
Selective IgG, IgA deficiency
IgA myeloma
Bence Jones proteinemia
Chronic lymphoblastic
leukemia
Antibodies
Continued
Reading
• Chapter 4 Kuby et al, Immunology 6th Edition
• Chapter 4 Abbas et al, Cellular and Molecular
Immunology 6th Edition.
IgM
• Structure:
– Is a pentamer (19S Ig) but it can
also exist as a monomer.
– In the pentameric form all heavy
chains are identical and all light
chains are identical.
– IgM has an extra domain on the
mu chain (CH4) and it has
another protein covalently
bound via a S-S bond called the J
chain. This chain functions in
polymerization of the molecule
into a pentamer.
Properties
• Third most common serum Ig.
• IgM is the first Ig to be made by the fetus and the first
Ig to be made by a virgin B cells when it is stimulated
by antigen.
• Pentameric structure, IgM is a good complement fixing
Ig - IgM antibodies are very efficient in leading to the
lysis of microorganisms.
• IgM is also a good agglutinating Ig - IgM antibodies are
very good in clumping microorganisms for eventual
elimination from the body.
• IgM binds to some cells via Fc receptors.
Surface IgM
Monomer
No J chain
Extra 20 aa in tail
Associated with two additional proteins
involved in signal transduction
IgA
• Structure
– Serum IgA is a monomer but IgA found in secretions is a dimer
– When IgA exits as a dimer, a J chain is associated with it.
– When IgA is found in secretions is also has another protein
associated with it called the secretory piece or T piece
– IgA is sometimes referred to as 11S immunoglobulin.
•
Unlike the remainder of the IgA which is made in the plasma
cell, the secretory piece is made in epithelial cells and is added
to the IgA as it passes into the secretions. The secretory piece
helps IgA to be transported across mucosa and also protects it
from degradation in the secretions.
Properties
• IgA is the 2nd most common serum Ig.
• IgA is the major class of Ig in secretions - tears,
saliva, colostrum, mucus. Since it is found in
secretions secretory IgA is important in local
(mucosal) immunity.
• Normally IgA does not fix complement, unless
aggregated.
• IgA can binding to some cells - PMN's and
some lymphocytes.
IgD
• Structure
– Exists only as a monomer.
• Properties
– IgD is found in low levels in
serum; its role in serum
uncertain.
– IgD is primarily found on B cell
surfaces where it functions as a
receptor for antigen.
– IgD on the surface of B cells has
extra amino acids at C-terminal
end for anchoring to the
membrane.
– It also associates with the Igalpha and Ig-beta chains.
– IgD does not bind complement.
IgE
• Structure
– IgE exists as a monomer and has an extra
domain in the constant region.
• Properties
– IgE is the least common serum Ig since it
binds very tightly to Fc receptors on
basophils and mast cells even before
interacting with antigen.
– Involved in allergic reactions - binding of
the allergen to the IgE on the cells results
in the release of various pharmacological
mediators that result in allergic symptoms.
– IgE also plays a role in parasitic helminth
diseases. Since serum IgE levels rise in
parasitic diseases, measuring IgE levels is
helpful in diagnosing parasitic infections.
Eosinophils have Fc receptors for IgE and
binding of eosinophils to IgE-coated
helminths results in killing of the parasite.
• d) IgE does not fix complement.
Isotypes of Light Chain
• Kappa (κ) and Lamda (λ), which are distinguished by their carboxyterminal constant (C) regions.
• An antibody molecule has either two κ light chains or two λ light chains,
but never one of each.
• In humans, about 60% of antibody molecules have κ light chains, and
about 40% have λ light chains.
• Changes in this ratio can occur in patients with monoclonal B cell tumors
because the neoplastic clone produces antibody molecules with the same
light chain.
• In fact, the ratio of κ-bearing cells to λ-bearing cells is often used clinically
in the diagnosis of B cell lymphomas.
• In mice, κ-containing antibodies are about 10 times more abundant than
λ-containing antibodies.
• Unlike in heavy chain isotypes, there are no known differences in function
between κ-containing antibodies and λ-containing antibodies.
Bringing together information
Natural Distribution and Production of
Antibodies
• Found in biological fluids throughout the body
• On surface of limited number of cells
• B cells are the only cells that synthesize
antibodies
Interesting facts
• A healthy 70-kg adult human produces about 2g3g of antibodies every day.
• Almost two thirds of this is IgA, which is produced
by activated B cells and plasma cells in the walls
of the gastrointestinal and respiratory tracts and
actively transported into the lumens.
• The large amount of IgA produced reflects the
large surface areas of these organs.
• Antibodies that enter the circulation have limited
half-lives.
• After exposure to an antigen, much of the initial antibody
response occurs in lymphoid tissues:
– the spleen,
– lymph nodes,
– mucosal lymphoid tissues,
• Secreted forms of antibodies accumulate in the plasma (the
fluid portion of the blood), in mucosal secretions, and in
the interstitial fluid of tissues.
• Secreted antibodies often attach to the surface of other
immune effector cells, such as mononuclear phagocytes,
natural killer (NK) cells, and mast cells, which have specific
receptors for binding antibody molecules.
Looking at Structure of Ig Molecules
• Breakthrough came when researchers discovered
that:
– multiple myeloma patients produced biochemically
identical antibody molecules
• Led to an extremely powerful technique for
producing monoclonal antibodies, described by
Georges Köhler and Cesar Milstein in 1975.
– They developed a method for immortalizing individual
antibody-secreting cells from an immunized animal by
producing "hybridomas," each of which secreted
individual monoclonal antibodies of predetermined
specificity.
Antigen-antibody complexes are held together by non-covalent
forces (therefore, antigen binding by antibody is reversible)
Affinity of Igs
• The strength of the binding between a single combining site of an
antibody and an epitope of an antigen is called the affinity of the
antibody.
• The affinity is commonly represented by a dissociation constant
(Kd), which indicates how easy it is to separate an antigen-antibody
complex into its constituents.
• A smaller Kd indicates a stronger or higher-affinity interaction
because a lower concentration of antigen and of antibody is
required for complex formation.
• The Kd of antibodies produced in typical humoral immune
responses usually varies from about 10-7M to 10-11M.
• Serum from an immunized individual will contain a mixture of
antibodies with different affinities for the antigen, depending
primarily on the amino acid sequences of the CDRs.
Avidity of Igs
• Polyvalent antigens will have more than one copy of a particular
determinant.
• Although the affinity of any one antigen-binding site will be the
same for each epitope of a polyvalent antigen, the strength of
attachment of the antibody to the antigen must take into account
binding of all the sites to all the available epitopes.
• This overall strength of attachment is called the avidity and is much
greater than the affinity of any one antigen-binding site.
• Thus, a low-affinity IgM molecule can still bind tightly to a
polyvalent antigen because many low-affinity interactions (up to 10
per IgM molecule) can produce a single high-avidity interaction.
Antigen-Ig Interactions
• Specificity
– antibodies generated in response to the antigens of one
microbe usually do not react with structurally similar self
molecules or with the antigens of other microbes
– biochemical constituents of all living organisms are
fundamentally similar
– However, some antibodies produced against one antigen
may bind to a different but structurally related antigen.
– This is referred to as a cross-reaction.
– Antibodies that are produced in response to a microbial
antigen sometimes cross-react with self antigens, and this
may be the basis of certain immunologic diseases
Antigen-Ig Interactions
• Diversity
– The ability of antibodies in any individual to specifically
bind a large number of different antigens is a reflection of
antibody diversity
– Total collection of antibodies with different specificities
represents the antibody repertoire.
– The genetic mechanisms that generate such a large
antibody repertoire occur exclusively in lymphocytes.
– They are based on the random recombination of a limited
set of inherited germline DNA sequences into functional
genes that encode the V regions of heavy and light chains
as well as on the addition of nucleotide sequences during
the recombination process
Antigen-Ig Interactions
• Affinity Maturation
– The ability of antibodies to neutralize toxins and infectious microbes is
dependent on tight binding of the antibodies. Tight binding is achieved by
high-affinity and high-avidity interactions.
– Generation of high-affinity antibodies involves subtle changes in the structure
of the V regions of antibodies during T cell-dependent humoral immune
responses to protein antigens.
– These changes come about by a process of somatic mutation in antigenstimulated B lymphocytes that generates new V domain structures, some of
which bind the antigen with greater affinity than did the original V domains
– Those B cells producing higher-affinity antibodies preferentially bind to the
antigen and, as a result of selection, become the dominant B cells with each
subsequent exposure to the antigen.
– Called affinity maturation, results in an increase in the average binding affinity
of antibodies for an antigen as a humoral immune response evolves.
– An antibody produced during a primary immune response often has a Kd in
the range of 10-7 to 10-9M and in secondary responses, the affinity increases,
with a Kd of 10-11M or less.
The Immunoglobulin Superfamily
• Many of the cell surface and soluble molecules that mediate
recognition, adhesion, or binding functions in the vertebrate
immune system share partial amino acid sequence homology and
tertiary structural features that were originally identified in Ig heavy
and light chains.
• Features are found in many molecules outside the immune system
that also perform similar functions.
• These diverse proteins are members of the Ig superfamily
(sometimes called the Ig supergene family).
• A superfamily is broadly defined as a group of proteins that share a
certain degree of sequence homology, usually at least 15%.
• The conserved sequences shared by superfamily members often
contribute to the formation of compact tertiary structures referred
to as domains.