ANTIBODIES

Quiz
Antibodies are considered as effector molecules
for
A. Cellular immunity
B. Humoral Immunity
Quiz
Antibodies are present in
A. Soluble form
B. Cell bound form
C. Both A and B
D. None of the above
ANTIBODIES
Antigen binding proteins
• Present on the B-cell membrane and
• Secreted by plasma cells
ANTIBODIES: Membrane Bound
Confers
• Antigenic specificity on B cells

Antigen-specific B-cell proliferation is elicited by

• Interaction of membrane antibody with antigen
ANTIBODIES: Secreted
Circulate in blood and serve as the effectors of
humoral immunity
• Searching out Ag
• Neutralizing Ag
• Marking Ag for elimination
ANTIBODIES: Epitopes of Ag
Immune system responds by producing
• Abs to several epitopes on Ag
• Requires recruitment of several clones of B cells
Quiz
Antibodies are having
A. Epitope
B. CDR
C. Both A and B
D. None of the above
ANTIBODIES: Monoclonal
Specifically binds a single antigenic determinant

Monoclonal antibodies make up

• Polyclonal and heterogeneous serum Ab
Quiz
Monoclonal antibodies are having
A. Similar CDRs
B. Different CDRs
C. Both A and B
D. None of the above
Antibodies Are Heterodimers
Antibody has a common structure of four peptide
chains
Two identical light (L) chains
• Polypeptides of about 25,000 molecular weight
Two identical heavy (H) chains
• Larger polypeptides of molecular weight 50,000 or
more
Light Chain Bound to Heavy Chain
By disulfide bond and
By non-covalent interactions
• Salt linkages
• Hydrogen bonds
• Hydrophobic bonds

Form a heterodimer
Antibodies Are Heterodimers
Non-covalent interactions and disulfide bridges
link
Two identical heavy and light (H-L) chain
combinations to each other
Basic four-chain (H-L)2 antibody structure
• Dimer of dimers
Variable Regions
First 110 AA of N-terminal region of
• Light or heavy chain
Varies greatly among Abs of different specificity
Segments of highly variable sequence
V regions:
• VL in light chains and
• VH in heavy chains
Quiz
CDR of Antibodies are formed by
A. VL region
B. CL region
C. VH region
D. CH region
E. Only A and C
Complementarity-determining Regions
(CDRs)
V regions on both light and heavy chains,
• Constitute the antigen-binding site of the
antibody molecule
Constant Regions
Regions of relatively constant sequence
• Beyond the variable regions

C regions
• CL on the light chain
• CH on the heavy chain
Constant Regions
Antibodies are mostly glycoproteins

Sites of attachment for carbohydrates are

restricted
• Constant region
Antibodies: Papain Digestion
Brief digestion of IgG with the enzyme papain

Produced three fragments

• Two of which were identical fragments
• Third was different
Antibodies: Papain Digestion
The two identical fragments (each with a MW of
45,000), had antigen-binding activity
• Called Fab fragments (“fragment, antigen
binding”)
Antibodies: Papain Digestion
The other fragment (MW of 50,000) had no
antigen-binding Activity was
• Called Fc fragment (“fragment, crystallizable”)
Antibodies: Pepsin Digestion
Generated a single 100,000-MW fragment
composed of two Fab-like fragments
• Designated the F(ab)2 fragment, which
• Binds antigen
Antibodies: Pepsin Digestion
The Fc fragment was
• Not recovered from pepsin digestion
• It had been digested into multiple fragments
Antibodies: Mercaptoethanol Reduction
Mercaptoethanol reduction and alkylation, a
• Irreversibly cleaves disulfide bonds
Antibodies: Mercaptoethanol Reduction
Seperating
• Two 50,000-MW polypeptide chains, designated
as heavy (H) chains
• Two 25,000-MW chains, designated as light (L)
chains
Quiz
Pepsin digestion of antibodies will produce
A. 2 Fab
B. 1 Fc
C. Both A or B
D. None of the above
Quiz
Papain digestion of antibodies will produce
A. 2 Fab
B. 1 Fc
C. Both A or B
D. None of the above
Antibody Sequencing: Difficulty
Insufficient amounts of homogeneous protein
• Heterogeneity of serum antibodies made them
unsuitable for sequencing
Antibody Sequencing: Multiple Myeloma
Pure immunoglobulin obtained from multiple
myeloma patients made sequencing possible
• Multiple myeloma is a cancer of antibody-
producing plasma cells.
• Its protein-synthesizing machinery and
secretory functions are not altered
Antibody Sequencing: Light Chain
Light-chain sequencing revealed that
immunoglobulins have constant and variable
regions
• There were two light chain types, kappa (κ) and
lambda (λ).
Antibody Sequencing: Light Chain
• In humans, 60% of the light chains are kappa and
40% are lambda,
• In mice, 95% of the light chains are kappa and
only 5% are lambda.
Antibody Sequencing: Light Chain
A single antibody molecule contains only one light
chain type
• Either kappa or lambda
• Never both
Antibody Sequencing: Heavy Chain
Heavy-chain sequencing revealed five basic
varieties of heavy chains
• The remaining part (except the 110 AA) of the
protein revealed five basic sequence patterns
Antibody Sequencing: Heavy Chain
They correspond to five different heavy-chain
constant (C) regions
• Each of these five different heavy chains is called an
isotype
Quiz
Antibodies light chains are having
A. Kappa chain
B. Lambda chain
C. Either A or B
D. None of the above
Immunoglobulin Fine Structure
• Diversity in the Variable-Region Domain Is
Concentrated in CDRs
• CDRs Bind Antigen
• Conformational Changes May Be Induced by
Antigen Binding
• Hinge Region
• Constant-region Domains
Diversity in the Variable-Region Domain
Is Concentrated in CDRs
• A large number of VL and VH domains revealed
that the sequence variation is concentrated in a
few discrete regions of these domains.
• Maximum variation is seen in those portions of
the sequence that correspond to the loops that join
the beta-strands.
Diversity in the Variable-Region Domain
Is Concentrated in CDRs
• These regions were originally called hypervariable
regions in recognition of their high variability.
• these areas are now more widely called
complementarity determining regions (CDRs).
CDRs Bind Antigen
• Confirmed directly by high-resolution x-ray
crystallography of antigen-antibody complexes.
• More residues in the heavy-chain CDRs appear to
contact antigen than in the light-chain CDRs.
Conformational Changes May Be
Induced by Antigen Binding
• Binding of antigen induces conformational changes
in the antibody, antigen, or both.
• This conformational change results in a closer fit
between the epitope and the antibody’s binding
site.
HINGE REGION
• The , , and α heavy chains contain an extended
peptide sequence between the CH1 and CH2
domains that has no homology with the other
domains.
• This region, called the hinge region, is rich in
proline residues and is flexible, giving IgG, IgD, and
IgA segmental flexibility.
CONSTANT-REGION DOMAINS
• The heavy chains in IgA, IgD, and IgG contain
three constant-region domains
• and a hinge region, whereas
• The heavy chains in IgE and IgM contain four
constant-region domains and no hinge region.
CONSTANT-REGION DOMAINS
• IgA, IgD, IgG IgE, IgM
• CH1/CH1 CH1/CH1
• Hinge region CH2/CH2
• CH2/CH2 CH3/CH3
• CH3/CH3 CH4/CH4
Quiz
Antibodies having hinge region are
A. IgE
B. IgM
C. Both A and B
D. None of the above
Antibody Classes and Biological Activities
Vary by heavy chain AA sequence
• Different class-specific structural and functional
properties
Immunoglobulin G (IgG)
Most abundant (About 80%)
Consists of
• two -heavy chains and
• two κ or two λ light chains
Immunoglobulin G (IgG)
Four subclasses in humans
according to decreasing serum
concentration
• (IgG1, IgG2, IgG3 and IgG4)
Differ in heavy chain sequence
Immunoglobulin G (IgG): Types
Mostly differ in
• Size of the hinge region
• Number and position of the inter-chain
disulfide bonds between heavy chains
Immunoglobulin G (IgG): Functions
• Cross placenta and protect foetus (IgG1, IgG3 and
IgG4)
• Effective complement activator (IgG1, IgG2, IgG3)
Immunoglobulin G (IgG): Functions
• Bind with Fc receptor on phagocytes and mediate
Opsonization (IgG1, IgG3 and IgG4)
Quiz
Secretory Antibodies are
A. IgE
B. IgM
C. IgG
D. IgA
E. Both B and D
Immunoglobulin M (IgM)
• 5% - 10% of total serum
immunoglobulins
• Average serum
concentration is 1.5 mg/ml
Immunoglobulin M (IgM)
Monomeric IgM are
exposed as membrane
bound antibody on B-cells

Secreted as pentameric by
plasma cells
Immunoglobulin M (IgM):
Five monomer units are held together by disulfide
bonds that
• Link their carboxyl-terminal heavy chain domains
(C4/C4) and (C3/C3) domains

Antigen binding region is outside

Immunoglobulin M (IgM):
Fc regions in the centre of the pentamer
• Ten antigen-binding sites on the periphery of the
molecule
Immunoglobulin M (IgM):
Because of steric hindrance
• Only 5 or fewer molecules of larger antigens can
be bound simultaneously
Immunoglobulin M (IgM): J chain
Fc regions are bound by J (Joining) chain inside

Disulfide-bonded to the carboxyl-terminal

cysteine residue of
• Two of the ten chains
Immunoglobulin M (IgM): J chain
Required for polymerization of the monomers to
form pentameric IgM

• Added just before secretion of the pentamer

Immunoglobulin M (IgM): Functions
• First Ab produced during primary response
• First Ab synthesised by neonates
• Higher valency because of structure
Immunoglobulin M (IgM): Functions
• More efficient for viral particles and RBCs
• Lower amount is sufficient to neutralize viral
infection and clumping of RBCs (Agglutination)
Immunoglobulin M (IgM): Functions
• Activates complement because of close Fc regions
• Diffuses less because of large size, so less in
intracellular tissue fluids
Immunoglobulin M (IgM): Functions
J-Chain allows it to bind on receptors of secretory
cells
• Transported across epithelial lining
• Enters the external secretions
• Present on mucosal surface
Called a secretory Ig (along with IgA)
Quiz
Highest amount of Secretory Antibodies are of
A. IgE
B. IgM
C. IgG
D. IgA
E. IgD
Immunoglobulin A (IgA):
10% -15% of total Abs in serum
Predominant in external secretions
• Breast milk
• Saliva
• Tears
• Mucus of the bronchial, genitourinary, and digestive
tracts
Immunoglobulin A (IgA):
IgA is highest produced Ab (5 – 15g per day)

Exists primarily as monomer

• Polymeric (dimers, trimers, and some tetramers)
forms are seen with J-chain
Immunoglobulin A (IgA):
External secretions are called Secretory IgA (dimer
or tetramer) consisting
• A J-chain polypeptide and
• A polypeptide chain called secretory component
Immunoglobulin A (IgA):
Secretory component is derived from the receptor
that is
• Responsible for transporting polymeric IgA
across cell membranes
Immunoglobulin A (IgA): Function
• Works at entry site of pathogen (mucous
membrane)
• Binds to virus or bacteria (Antigen)
• Prevents them attaching the mucosal cell

IgA-Antigen complex is eliminated by respiratory

track cells
Immunoglobulin A (IgA): Function
Defence against bacteria such as
• Salmonella
• Vibrio cholerae
• Neisseria gonorrhoeae

Viruses such as polio, influenza, and reovirus

Quiz
Non-secretory Antibodies are
A. IgE
B. IgG
C. IgD
D. All of the above
Quiz
Antibodies involved for allergic reactions are
A. IgE
B. IgM
C. IgG
D. IgA
E. IgD
Immunoglobulin E (IgE):
• Low concentration
• Mediate the
hypersensitivity
reactions
Immunoglobulin E (IgE): Function
• Binds with Fc receptor of basophil and mast cell
• Induces cell to release the granules
• Process is known as degranulation
Immunoglobulin E (IgE): Function
• Gives rise to allergic symptoms
• Mast cell locally release mediator for the anti-
paracytic defence
Immunoglobulin D (IgD)
• Found on membrane of
B-cells
• Low levels are found
(0.2%)
• No biological effector
function has been
identified
Antigenic Determinants on Ig
• Isotype
• Allotype
• Idiotype
Isotype
Constant-region determinants
Define
• Each heavy-chain class and subclass
• Each light-chain type and subtype

Within a species
Isotype
Encoded by
• A separate constant region gene

All members of a species carry the same

• Constant-region genes
Isotype
Different species
• Inherit different constant-region genes
• Express different isotypes
Allotype
Multiple alleles exist for
• Some of the genes

In same species
Allotype
These alleles
• Encode subtle amino acid differences
• Called allotypic determinants

Occur in some but not all Members of a species

Idiotype
Idiotypic Determinants
Arise from sequence of
• Heavy- and light-chain variable regions
Idiotype
Individual antigenic determinant of the variable
region
Quiz
Serum of individual from one species is inserted
in individual of another species, the antibodies
formed are
A. Anti-isotype
B. Anti-allotype
C. Anti-idiotype
Quiz
Serum of individual from one species is inserted
in individual of same species, the antibodies
formed are
A. Anti-isotype
B. Anti-allotype
C. Anti-idiotype
Quiz
Monoclonal antibodies are inserted to an
induvial, the antibodies formed are
A. Anti-isotype
B. Anti-allotype
C. Anti-idiotype
Antibody-Mediated Effector Functions
• Antibodies must not only recognize antigen, but
also invoke responses
Effector functions—that will result in
• Removal of the antigen and
• Death of the pathogen.
Antibody-Mediated Effector Functions
• Opsonization Is Promoted by Antibody
• Antibodies Activate Complement
• Antibody-Dependent Cell-Mediated Cytotoxicity
(ADCC) Kills Cells
• Some Antibodies Can Cross Epithelial Layers by
Transcytosis