Antibody Structure and Function PDF

Chapter 4.
Immunoglobulin Structure
and Function
1
2
- Light chains consist of 2

domains (C and V).
- Heavy chains have 4-5

domains (depending on
the class of antibody)
*
Heavy chain= 446 aa
1. Functional Regions
2. Types of chains
3. Constant &
Variable regions
4. Glycoprotein
- Each heavy and light chain

is made up of a number of
domains (= Ig folding or Ig
domains).
Light chain= 214aa
- Each domain is about 110

amino acids in length and
contains an intrachain
disulfide bond between
two cysteines about 60
amino acids apart.
Basic Antibody Structure
-150,000 molecular weight
Multiple myeloma = cancerous plasma cells

Monomer = 150,000
1
2
3
4
What is the difference?
- Constant (C) and

Variable (V) regions
RECAP:
Pepsin
100,000 MW
(Fab)2
Papain
2 (45,000)
1 (50,000)
- The Fc region plays NO role in antigen binding.
Mercaptoethanol
2 (50,0000)
2 (25,000) 2 H + 2 L
- Papain breaks antigen molecules into 2 Fab fragments

and an Fc fragment.
- Pepsin breaks antibody molecules into an F(ab)2
fragment and a VERY SMALL pFc fragment.
- Mercaptoethanol treatment results in 2 heavy and 2
light chains
- Complexes of antibodies cross-linked by antigen are
called immune complexes.
2 Fab + Fc
1. Constant region - amino

acid sequence in the Cterminal regions of the H and
L chains is the same.
1
1
2. Variable region - amino

acid sequence in the Nterminal regions of the H and
L chains is different. This
region provides antibodies
with unique specificity.
3. Hyper-variable regions are
regions within the variable
regions (greater
specificities).
Summary
Molecule consists of Constant and Variable regions
for both Light and Heavy chains (CH, VH, CL, VL)
Ig molecule made of domains
Domains ~ 110 aa
Each antigen-binding site is made up of the Nterminal domain of the heavy and the light chains
IgM and IgE possess 4 CH domains (CH1-CH4)
while IgG, IgA and IgD have 3 CH domains (CH1CH3). Hinge region is missing.
Hypervariable regions in the Variable regions of
both H and L chains.
Figure 3.3
-Within the variable

domains are three
regions of extreme
variability.
Complementarity-Determining Regions, or CDRs.
These are referred to as

the hypervariable
regions.
These regions of the
variable domains
actually contact the
antigen.
They therefore make up
the antigen-binding
site.
These regions are also
called the
complementaritydetermining regions, or
CDRs.
Heavy Chain
Light Chain
H chain CDRs
- A simulated antigenbinding site showing

how the CDRs form
points of contact with
the antigen.
RECAP:
- Antibodies are comprised of repeating 110 aa units referred to
as domains or Ig folds.
- The C-terminal domains are constant from antibody to

antibody (within a class).
- The constant region domains are responsible for all functions
of antibody other than antigen binding (opsonization, ADCC,
complement activation) Biological Function!
L chain CDRs
- The N-terminal domains are variable from antibody to antibody

and are referred to as variable domains.
- The variable domains contain 3 hypervariable regions - the
CDRs.
- The CDRs of the V domains in both H and L chains make up
the antigen-binding site.
Antibody-Mediated Effector
Functions
Binding to Antigen
OPSONIZATION: FcR in Macrophages and
neutrophils
COMPLEMENT ACTIVATION: IgG and IgM
ADCC NK cells trough FcR
CROSSING EPITHELIAL LAYERS IgA (but
also IgM)
CROSSING PLACENTA- IgG
Fc receptors enhance phagocytosis of foreign cells/particles

coated with IgG
Antibody made in response to foreign cells (cells/viral
particles/bacteria etc) will bind to those cells.
Macrophages (and neutrophils) possess receptors for the Fc
region of IgG.
Binding of macrophage Fc receptors to antibody bound to
cells/particles facilitates and increases phagocytosis of
cells/particles.
ADCC - Antibody-dependent cellular cytotoxicity - mediated by IgG

Antibody made in response to
foreign cells (cells/viral
particles/bacteria etc) will
bind to those cells.
Cells of the innate immune
system (neutrophils,
eosinophils, macrophages,
NK cells) possess receptors
for the Fc region of IgG.
These cells bind to antibody
on the surface of foreign cells
and release lytic compounds
lysis.
Monomer,
Dimer,
and
Pentamer
Kuby Figure 14-12
Structural Variants of the Basic Immunoglobulin Molecule
Relative abundance in normal serum:
Different heavy chains can be used

There are five major types of heavy chain --> five major classes
(isotypes) of antibody
- gamma --> IgG (in humans 4 subclasses: IgG1, IgG2, IgG3, IgG4)
- mu --> IgM
- alpha --> IgA (in humans, 2 subclasses: IgA1, IgA2)
- delta --> IgD
- epsilon --> IgE
IgG
8 - 16 mg/ml
IgA
1.4 - 4 mg/ml
IgM
0.5 - 2 mg/ml
IgD
0.003 - 0.04 mg/ml
IgE
17 - 450 ng/ml (<0.0005 mg/ml)

IgD
The function of antibody varies depending on which heavy chain is

used.
IgM
IgE
IgA
IgG
IgA
IgM
IgD
IgE
IgG
IgG
IgM
IgA
IgD
IgE
-Most abundant in
secondary responses
-Crosses placenta (FcRn)
-Complement activation
-Binds to FcR in
phagocytes
Figure 3.15a
Crosses placenta
Complement Activator
Fc binding
Crosses placenta
Fc binding
Crosses placenta
- Best Complement activation

- First Ab produced in
neonate
- First antibody
produced after challenge
- Mucosal transport
(to some degree)
- Monomer on B cells
- J chain: polymeric
- Dimer in mucosal secretions

- Mucosal transport
- Monomer in circulation
- J chain (polymeric) and Secretory components
Secretory Component
Role of IgE in allergic reactions

IgE antibodies mediate the immediatehypersensitivity (allergic) reactions that
are responsible for symptoms of hay
fever, asthma, hives and anaphylactic
shock.
IgE binds to Fc receptors on the
membranes of blood basophils and
tissue mast cells.
IgD
- Role unknown
- Present on the
surface of
MATURE
B cells Marker!!
Cross-linkage of receptor-bound IgE

molecules by antigen (allergen)
induces degranulation of basophils
and mast cells.
A variety of pharmacologically active
mediators present in the granules
are released, giving rise to allergic
manifestations
SUMMARY
- IgA and IgM are secreted across epithelial surfaces
- IgG, IgD and IgE can be found only within the
body - in serum or lymph.
- IgA and IgM are also found in serum and lymph BUT
IN ADDITION can also be found in secretions such as
mucous secretions, saliva and tears.
- The IgA and IgM found in external secretions differs
from that found in serum by the presence of an
additional component referred to as the "secretory
component".
Antigenic Determinants on
Immunoglobulins
Abs are glycoproteins and themselves very
immunogenic
Epitopes on immunoglobulins are divided
into:
ISOTYPIC
ALLOTYPIC
IDIOTYPIC
- This component is acquired as the IgA or IgM is

transported across the epithelial cell barrier.
**
Constant region determinants that define each antibody

class and subclass
The function of antibody varies depending on which heavy chain
is used.
Allelic variation (Allotypes): IgG of a particular class may be

slightly different between individuals (e.g. variation in the
IgG amino acid sequence)

Note: This type of variation has no effect on antibody
function.
RECAP - Sequence variation in antibodies:

1. Different light changes - no significant
functional effect
2. Different heavy chains - very significant
functional effect - isotypic variation
3. Allelic variation between individuals - no large
functional effect - allotypic variation
Generated by variation in amino acid sequence in the VH and
VL. Most exactly, in the CDRs in the V regions
Variation in the antigen binding site (Idiotypes)
4. Variation in the antigen-binding site - idiotypic

variation
Remember: Idiotype = Ag binding site
B Cell Receptor (BCR):

- Short cytoplasmic tail (328 aa) .signaling?
- Signaling through a
homodimer, Ig- and Ig-
- Ig molecule + Ig-/Ig- is
the BCR
- The homodimer molecule
is member of the Ig
superfamily group
Ig Superfamily
Divergence from a common gene ancestor coding
for 110 aa.
A member MUST have a typical Ig domain or
fold 110 aa with an intra chain disulfide bond
50-70 aa apart.
Most members do not bind Ag!! Then, they must
facilitate interaction with surface proteins
You must know members with roles in: a) immune
function, b) Receptor/Signal transduction, and c)
Adhesion
Immune Function
Receptors
Neonatal
Monoclonal Antibodies
Kohler & Milstein 1975
Fusion of normal, activated B cell and
plasmacytoma (cancerous plasma cell)
Hybrid: immortal, secrete Ab, hypoxanthine
RESULTS:
Plasmacytoma VS B cell
Plasmacytoma:
Cancerous plasma cell (Immortal)
Does not secrete Abs
Lacks HGPRT
Spleen B cell
Hybrid
**
Plasmacytoma
Normal spleen B cell

Limited life span
Secretes Abs
Possess HGPRT
Die in culture
Immortal, Secretes
Lacks HGPRT
Ab, Possess
hypoxanthine (HGPRT)
1
2
Applications?
3
4
Diagnosis
Research
Treatment
Affinity VS Avidity
Affinity (polyclonal Ab) = high because
of multiple epitopes
Avidity (monoclonal Ab) = low affinity but
high avidity because of strong epitopeAb interaction
IgG - Most abundant Ig of internal body fluids (serum, extracellular fluids) - combats microorganisms and
toxins within the body tissues.
The End
IgA - Most abundant Ig in mucous secretions - protects external surfaces of the body
IgM - The first class of antibody produced during an immune response. Present both in internal body
fluids and in secretions.
IgD - Functions not well defined. Found mostly on the B cell plasma membrane
IgE - Increases during parasitic infections. Causes symptoms of allergy.
Complement
fixation by
classical
pathway
Ability to cross
the placenta
Binds to mast
cells and
basophils
Binds to
macrophages
and polymorphs
IgG
IgA
IgM
IgD
IgE
++
+++
++
+++
+++

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Chapter 4.

- Light chains consist of 2

- Heavy chains have 4-5

- Each heavy and light chain

Light chain= 214aa

- Each domain is about 110

Basic Antibody Structure

-150,000 molecular weight

Multiple myeloma = cancerous plasma cells

What is the difference?

- Constant (C) and

- The Fc region plays NO role in antigen binding.

- Papain breaks antigen molecules into 2 Fab fragments

1. Constant region - amino

2. Variable region - amino

-Within the variable

Complementarity-Determining Regions, or CDRs.

These are referred to as

- A simulated antigenbinding site showing

- The C-terminal domains are constant from antibody to

- The N-terminal domains are variable from antibody to antibody

Fc receptors enhance phagocytosis of foreign cells/particles

ADCC - Antibody-dependent cellular cytotoxicity - mediated by IgG

Kuby Figure 14-12

Structural Variants of the Basic Immunoglobulin Molecule

Relative abundance in normal serum:

Different heavy chains can be used

0.003 - 0.04 mg/ml

17 - 450 ng/ml (<0.0005 mg/ml)

The function of antibody varies depending on which heavy chain is

- Best Complement activation

- Dimer in mucosal secretions

Role of IgE in allergic reactions

Cross-linkage of receptor-bound IgE

- This component is acquired as the IgA or IgM is

Constant region determinants that define each antibody

Allelic variation (Allotypes): IgG of a particular class may be

IgG amino acid sequence)

RECAP - Sequence variation in antibodies:

4. Variation in the antigen-binding site - idiotypic

Remember: Idiotype = Ag binding site

B Cell Receptor (BCR):

Normal spleen B cell

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