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Antibody Structure and Function
1
Antibody Structure and
Function
n Definition: Antibodies (Abs) are recognition
proteins produced by plasma cells (derived from
activated B cells) found in serum and other body
fluids of vertebrates that react specifically with the
antigen (Ag) that induced their formation.
n Antibodies belong to a class of proteins called
Immunoglobulins (Igs)
n Definition of Ig: A family of globular proteins that
has in common the Ig fold as a structural domain.
These proteins include Ab molecules and molecules
having antigenic determinants in common with Abs.
n What molecules other than Abs are Igs?
Abs
Ig
Myeloma proteins
2
Antibody Structure and
Function
l
Myeloma proteins are found in individuals who
develop a type of cancer call multiple myeloma.
l
These proteins have the characteristic structure
of Abs but arise from a disease state rather than
an immune response.
l
A myeloma protein is an Ig whose Ag-binding
specificity is unknown but all the protein
molecules are IDENTICAL.
n Importance ?
Structure of Igs
n Why is it important to understand the chemical
structure of Igs?
n Reason: Because the chemical structure of Igs
determines three important functions.
3
Antibody Structure and
Function
l
Versatility in Ag binding
t
t
t
l
105 to 109
Even to artificial Ags that do not exist in nature
How can Ab molecules be so diverse to allow a
response to this vast array of determinants? -And have a common structure.
Specificity
Why are Abs specific? That is, why don't Abs react
with unrelated determinants?
l
Biological effector mechanisms
How do Abs elicit these effector mechanisms?
All these properties are explained by determining
the CHEMICAL STRUCTURE of Igs
4
Antibody Structure and
Function
Electrophoresis studies by
Tiselius & Kabat
Abs = γ−globulins
Studies showed that the MW of Abs = 150 to 1000 kD
5
Antibody Structure and
Function
Edelman & Porter's Work
Fragmentation Studies Using Chemicals and
Enzymes
Chain Structure of Ig
Rabbit IgG (150,000 mw)* (Edelman)
reducing &
IgG =========> subunits of IgG
denaturing
agents
Heavy (H)
+
(50,000 mw)
Light (L) chain
(25,000 mw)
*Found in equimolar amounts = 2 H + 2 L chains
6
Antibody Structure and
Function
PAPAIN (Porter):
papain
IgG =========> Fab
+ Fc
(50 kD)
(50 kD)
n Fab = fragments Ag binding
n Fc = fragment crystallizable
n Molar ratio of Fab/Fc was 2:1, therefore there
are two Ag-binding sites on each IgG
molecule
PEPSIN (Nisonoff):
pepsin
IgG =========> F(ab')2
(100,000 mw)
n F(ab')2 fragments have both Ag-binding sites
held together by disulfide bonds but
missing Fc
7
Antibody Structure and
Function
n STRUCTURE: IgG composed of 2 L and 2 H
chains, which are linked by interchain disulfide
bonds and noncovalent interactions.
Chemicals
L
Papain
Fab
H
H
L
Fc
Fab
Pepsin
n In any Ab molecule, the 2 L chains are
IDENTICAL and the 2 H chains are
IDENTICAL.
ALL Igs HAVE THIS BASIC STRUCTURE!
n Our purpose for looking at Ig structure: Three
reasons (mentioned earlier)
n To do this, we have to look more closely at Ig
structure: Ig fine structure
8
Antibody Structure and
Function
Ig L chains
Amino acid sequence comparisons of Bence-Jones κ L
chains led to a startling finding
NH2
1
110
214 COOH
CL
VL
Ig H chains
1
113
446
VH
VL
CH
CL
CH
VH
VH
VL
CH
CL
n IMPLICATION: V regions of L and H chains explain
versatility of Ab binding to 105 to 109 different antigenic
determinants. Igs binding different determinants have
different VL and VH regions.
9
Antibody Structure and
Function
n Analogy:
l
l
l
P-S-Y-C-H O-L-O-G-Y
P-H-Y-S-I O-L-O-G-Y
I-M-M-U-N O-L-O-G-Y
Closer Look at Ab Fine Structure
n Further amino acid sequencing of Ig chains showed
areas or “hotspots” of increased variability in the V
region.
n These hotspots are called hypervariable regions
or complementarity-determining regions (CDRs)
n Less variable amino acid residues/regions between
CDRs are called framework residues
10
Antibody Structure and
Function
Wu and Kabat Plot
FR = Framework residues
CDRs = Complementarity-determining regions; also
called hypervariable regions
11
Antibody Structure and
Function
Domains
n Igs are not linear chains of proteins -- they are
globular proteins.
n Each Ig chain has homology units called DOMAINS.
l Domains are regions of compact globular
structure; for example, IgG domains = light
chain: V & C and heavy chain: V, Cγ1, Cγ2,
Cγ3, & hinge region (IgM and IgE have an
additional heavy chain C domain but no hinge
region domain)
l Two types of Ig-like domains: V and C
t
l
These domains are the prototype of the Ig
Superfamily domain structure, also called the Ig
fold. What is this Ig fold?
All Ig domains contain two layers of β-pleated
sheet with 3 or 4 strands of antiparallel
polypeptide chain (a "sandwich" structure). This
characteristic structure is the Ig fold.
t
Significance: holds loops out from sandwich
12
Antibody Structure and
Function
The Ig Superfamily
n The Ig superfamily is characterized by their 3D
structure because all the members contain at least
one Ig fold.
n The Ig fold, so-called because it is a single-stranded
β-sheeted barrel composed of a sandwich of two
extended protein sheets roughly 110 amino acid
residues long.
n The two antiparallel sheets (purple) are connected
with one disulfide bridge (yellow).
Side-view of Ig fold
13
Human Ab Light Chain Folding Into Ig Domains
CL domain
VL domain
b strands
COOH
Disulfide
bridge
CDRs
NH2
Location of CDRs in VL domain
CDR1 CDR2 CDR3
Light chain
28-34
89-97
50-56
CL domain
14
3D Structure of Complete IgG
VH domain
CL domain
CH1
VH domain
Antigen-binding site
Antigen-binding site
VL domain
Carbohydrate chain
VL domain
CH2
Heavy chains
CH3
(a)
VH
Antigenbinding
site
VL
CH1
CL
S
VL
S
CH2
CH2
Carbohydrate
CH3
(b)
VH
Antigenbinding
site
15
Antibody Structure and
Function
Summary of Ig Fine Structure
n Roughly, the first 110 amino acid residues at the
NH2 termini of both L and H chains comprise the V
region (the Ag-binding site).
n Within the V region, some areas are more variable
than others.
l
Areas of greatest variability are called Hypervariable
Regions or Complementarity-Determining Regions
(CDRs)
Responsible for the shape of the Ag-binding site of Igs
l
Amino acid residues between CDRs are called
Framework Residues
n L and H chains are divided into homology units,
domains, which have in common the Ig fold.
n The COOH termini contain the C regions.
16
Ig Domains
Antigen-binding
clefts
Light
chains
NH2
Fab
S
S
S
Fab
S
VL
S
S
S
S
CL
VH
S
S
S
S
S
S
Variable
region
S
S
S S
S
S
S
S
CH1
S S
Hinge
region
Heavy
chain
S S
CH3
S S
S S
CH2
Carbohydrate
S S
COOH
Fc
Biological
activity
17
Antibody Structure and
Function
All Known Ab-Mediated EFFECTOR
FUNCTIONS Are Ascribed to the Fc Region
n They can be divided into three generic categories:
l
l
l
Activation of the classical complement pathway
Interaction with effector cells
t For example: Mφs, NK cells, or mast cells
Compartmentalization of Abs (where Abs localize)
n There are three main ways Abs contribute to
immunity:
l
l
l
Neutralization
Opsonization
Complement activation
n Abs protect the host by:
l
l
l
l
l
binding to bacterial toxins and viruses, leading to their
neutralization
enhancing phagocytosis of microorganisms
activation of complement, leading to lysis of cells
localizing at mucous membrane surfaces, they bind
to organisms and prevent colonization
binding to Fc receptors on cells to mediate ADCC
18
Antibody Structure and
Function
Human Ig Classes and Subclasses
n There are five major groups of human Igs, called
Immunoglobulin Classes, they include:
IgG, IgA, IgM, IgD, and IgE
?
How do you know what class an Ig belongs to?
n Igs are divided into classes on the basis of the
antigenic determinants they possess on their
heavy chains.
n Concept: Abs can be IMMUNOGENIC
19
Antibody Structure and
Function
Heavy (H) Chains of Igs
n Abs specific for H chain antigenic determinants
define the Ig class an Ab belongs to
Human Ig
= Ab against
human Ig
n There are 5 antigenic types of H chains in humans.
The antigenic determinant that defines the H chain
is:
H chain type
Ig class
1.
γ
IgG
2.
α
IgA
3.
µ
IgM
4.
δ
IgD
5.
ε
IgE
20
Antibody Structure and
Function
n Ab specific to a particular H chain will react only with
that chain and not other H chains.
H chains always γ
n IgG
L chains can be κ or λ
n Similar for other Igs
n Ig subclasses
l
l
Even within a class of Ig (e.g. IgG) the H chains have
minor antigenic variations.
Examples: IgG H chain has 4 variations, therefore
IgG is divided into:
H chain
γ1
γ2
γ3
γ4
IgG
IgG1
IgG2
IgG3
IgG4
21
Antibody Structure and
Function
n IgA H chain has 2 variations, therefore IgA is
divided into:
H chain
IgA
α1
IgA1
α2
IgA2
LIGHT (L) CHAINS OF Igs
kappa (κ)
n Light chains
lambda (λ)
n An Ab molecule will have either κ or λ L chains but
not both together.
n κ and λ chains are found on all classes of Igs.
22
Antibody Structure and
Function
n Three categories of antigenic determinants
found on Igs:
l
l
l
Isotypic
Allotypic
Idiotypic
n These determinants define isotypes,
allotypes, and idiotypes of Igs.
n Isotypic antigenic determinants: determinants
present on some Igs of ALL members of a species.
The antigenic differences that characterize the class
and subclass of H chains and the type and subtype
of L chains are called isotypes. Thus, isotype
refers to an Ab class.
l
Each normal individual expresses all the
isotypes, characteristic of the species.
23
Antibody Structure and
Function
n Allotypic antigenic determinants: determinants
present on the Igs of only SOME members of a
species, because these epitopes are encoded by
alleles (or alternative genes) at a single locus. The
Igs are called allotypes.
They are due to
polymorphisms within species in the amino acid
sequence of Ab isotypes.
l Example:
The IgG1 of one person is not
necessarily absolutely indentical to the IgG1 of
another person.
n Idiotypic antigenic determinants: determinants
that are common to Igs having specificity for the
same epitope on Ag molecules.
(idiotypic
determinants
reflect
the
Ag-bining
site
structure)
l
Example: two Igs both IgG1 with κ L chains.
t
Both have the same allotypic markers; one Ab is
specific for Ag epitope A, the other is specific for
Ag epitope B.
The difference in chemical
structure of Abs needed to bind these epitopes,
define their idiotypes.
24
Antibody Structure and
Function
Structure of Human Ab Classes When in Their
Soluble Form
(As part of the B-cell antigen receptor complex: their carboxyl
termini
have
hydrophophic sequences, they are
monomeric, and they contain two identical Ig-α/Ig−β
heterodimers)
25
Antibody Structure and
Function
Human Ab Classes
n Immunoglobulin G (IgG): the predominant class of Abs
found in humans.
l IgG is the only Ab that can cross the placenta from
mother to fetus, and is responsible for immunologic
protection of an infant during its first few months after
birth.
l IgG is:
t the primary Ab induced by protein Ags, the main
Ab in the secondary Ab response (has higher
affinity for antigen) and
t the main Ab involved in neutralization (any
isotype can), opsonization, ADCC, complement
activation, and precipitation reactions.
n Immunoglobulin A (IgA): is the predominant Ab found in
human bodily secretions such as saliva, tears, and nasal
mucous.
l It is a monomeric molecule in the blood.
l In bodily secretions, it is found as a dimer composed
of two Ab molecules, a secretory component, and a
joining (J) chain; therefore, this form is called
secretory IgA.
l It can bind to 4 epitopes.
l It can activate the alternative complement pathway.
26
Antibody Structure and
Function
n Immunoglobulin M (IgM): is present, as a monomer,
along with IgD on the surface of mature B cells.
l In blood, IgM normally exists in clusters of five
(pentamer), connected at the bottoms of the Y.
l The monomers are held together by disulfide bridges
to a polypeptide called the J chain.
l IgM can bind to 10 epitopes.
l It is prominent in the primary Ab response to most
Ags.
l IgM is the main Ab:
t induced by polysaccharide Ags and
t involved
in agglutination and complementmediated cytolytic reactions.
n Immunoglobulin E (IgE):
is involved in allergic
reactions, because it naturally binds to certain cells
causing release of compounds that mediate these
reactions.
n Immunoglobulin D (IgD): is the predominant Ab class
found on Ag-naive B cells.
l It may be involved in B-cell growth and maturation.
l It is found in trace amounts in human blood.
27
Antibody Structure and
Function
Monoclonal Abs
n Heterogeneous response vs. need for
homogeneous Ab
n Homogeneous Ab:
l
l
l
Specific for a single determinant of the Ag
Same class and subclass
All the Ab would bind the determinant with the
same affinity
Why search for homogeneous Ab?
28
Antibody Structure and
Function
n Studies of the interaction between epitopes
and the Abs’ Ag-combining sites
l
Use myeloma proteins
t
l
l
Problem: Is the Ag-binding site of the myeloma
protein really an accurate representation of Ab
produced during an immune response?
Limit cross-reactivity of Ab
Standardization of diagnostic reagents
29
Antibody Structure and
Function
n In 1975, Kohler and Milstein developed
homogeneous
Ab
against
any
antigenic
determinant in almost unlimited quantity (1984
Nobel Prize)
n Two problems to overcome:
l
l
Ab-forming cell = production of specific Ab but
it is a terminal cell
Myeloma cell = Ab of unknown specificity but
it is an immortal cell
n Fused with a myeloma cell that had two unusual
properties:
l
l
It did NOT secrete Ig
Metabolically deficient
t
t
Lacked the enzyme Hypoxanthine-Guanine
Phosphoribsyl Transferase (HGPRT−)
It cannot use the exogenous hypoxanthine to
synthesize purines.
30
Antibody Structure and
Function
Hybrid-myeloma
HYBRIDOMA
Resulting fusions
Plasma cell
FUSION
=
Myeloma cell
How to select for proper hybrid?
n Culture cells in a selective medium
(Hypoxanthine-Aminopterin-Thymidine; HAT) for
a few weeks
n Grow-up selected clone of desired Ab specificity =
Monoclonal Ab
31
Conventional Antisera vs. Monoclonal
Antibody Production
CONVENTIONAL ANTIBODY
PRODUCTION
MONOCLONAL ANTIBODY
PRODUCTION
Antigenic
determinants Repeated antigen
inoculation
Antigen
Remove
spleen
The mouse produces B cells
specific for each
antigenic determinant.
FUSION
Spleen cells
Myeloma cells
Hybrid-myeloma cells
Antiserum
(contains a mixture of antibodies
specific for each antigenic
determinant)
Clone 1
Grow up
individual cells
into clones
Clone 2 Clone 3
Individual populations of antibodies
32
Development of Monoclonal Antibodies
Repeated antigen
inoculation
Fusion
Remove spleen
Nonsecretor myeloma cells
(HAT sensitive)
Culture may contain:
1
2
Spleen cells grown in
HAT medium
(HAT resistant)
3
Culture in HAT
medium
5
1
2
3
Test for positive cultures
(contain antibody against antigen)
4
5
Clone antibody producers
Spleen cells (end cells, thus
will die with time)
Myeloma cells (killed by
HAT medium)
Fused spleen cells (same
as no. 1)
Fused myeloma cells (same
as no. 2)
Fused spleen cells—myeloma
cells (hybrid cell = hybridoma;
lives forever [myeloma cells]
and produces antibodies
[spleen cells or plasma
cells])
CELLULAR DNA SYNTHESIS
1
2
De novo pathway (endogenous sources)
Salvage pathway (exogenous sources)
H
HGPRT
Ribonucleotide
De novo
DNA
Ribonucleotide
TK
T
4
33
Transfectomas: the production of
hybrid antibody molecules
IN VITRO MANIPULATIONS
Human H
Mouse H
chain gene
chain gene
Leader
Leader
VDJ
VDJ
1.
CH1
2.
INTRODUCTION INTO
E. coli plasmid
CH1
Hinge
Hinge
CH2
CH2
CH3
CH3
+
L chain gene
Proliferating E. coli
containing the plasmid
Mouse–human
recombinant
heavy chain
gene
3.
TRANSFECTION
+
E. coli
spheroplast
4.
Myeloma cell
TRANSFECTANT
Selection
EXPANSION
Tissue culture
HUMAN–MOUSE
RECOMBINANT
ANTIBODY
Ascites