ANTIBODIES

BALBASTRO, KENNETH
DE GUZMAN, GIAN
LUNA, NENUEL
PANGILINAN RAFAEL
• PART I: STRUCTURES
• PARTII: IMMUNOGLOBULIN CLASSES
• PART III: THEORIES AND GENES
ANTIBODY STRUCTURE
HOMOLOGY UNIT

– four-chain polypeptide unit linked by

noncovalent
and disulfide (S-S) bonds
 pairs of identical heavy(H) and light(L) chains
N-terminal
--antigen-binding
portion
V-Region
--portion that shows
heterogeneity in terms of
amino acid sequencing

--the V Regions of one light

and one heavy chain form
an antigen-binding site
C-Region
--Unlike V-region,
this region has a
constant amino
acid sequences
WITHIN are CDRs
or
Complementary-
determining
Regions
CDR
-HYPERvariable
loops

-present in both H
and L chains

(later, we will know

the purpose of
CDR)
IF THE INTERCHAIN
DISULFIDE BONDS ARE
TREATED TO BREAK—

4 polypeptide chains
are separated:
two identical L chains
and two identical H
chains
Heavy chains

μ γ

α ε
 Light chains
κ

L-CHAIN

λ
Light Chain
– small chains (25,000 D)

– 1 constant domain
• Types: kappa (κ) and
lambda (λ)
– 200-220 amino
acids
– no functional
difference
– found in all five
classes of
immunoglobulins
– one type
present in each molecule
κ : 65%
λ : 35%
 Heavy Chain
– larger chains (50,000-77,000 D)

– 1 variable domain: 110-120 acids

– 3 or more constant domains ( CH1, CH2, CH3)

GLYCOSYLATED
 allotypes
Variants due to idiotypes
isotype
intraspecies genetic Variants due to the
Types: (determines
differences large amount of
the class and
Each individual has a structural
subclass of a
particular variant at heterogeneity in the
molecule)
each Ig gene locus, Ig V regions
α – IgA γ – IgG
which will often This is related to the
δ – IgD μ –
differ from those in diversity required to
IgM
other indiviudals bind different
ε – IgE
Occur for gamma, antigens
alpha, epsilon
isotype allotype idiotype
Same
●
Variations
●
Variations
●

heavy in in
chains for constant variable
each class regions regions
Hinge Region
– high content of proline
and hydrophilic residues_

-SECTION of Fc and Fab Regions which contains the inter-heavy –

chain disulphide bonds

-confers the flexibility on the antibody molecule

Proline Residues

●
– “flexibility”
●
– lets the Ag-binding sites operate independently

Hydrophilic Residues

●
– open
●
– accessible to proteolytic cleavage with enzymes
●
 Pepsin, Papain
Papain
– splits approximately equal-size fragments:
 2 antigen-binding fragments
“Fab fragments”
 a crystallizable fragment
“Fc fragments”
Pepsin
– Fc fragment is completely destroyed
splitting it into tiny peptides
– Fab fragments combines  F(ab)’2
fragment

• Fd fragment = a L chain + half of a H

chain
 3D
structure
 blilize
ond
hecompact
forth
H andthe
L
ns
ubunits
f the regions

t
s
h
e
b
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e

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t

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ns
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e
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pt
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H
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 Antigen bi nd to a small number
of ami no acids at the
HPERVARIABLE REGIONS

There are three small

hypervariable regions

These regions are

called the CDRs

THE antigen-binding site i s

determined by the apposition
of the si x loops, three from
each chai n.
•– basic unit: DOMAIN (homology unit)
•  Variable (V) region
• – N-terminal end
• – antigen-binding portion
•  Constant (C) region
• IgG, IgD and IgE: monomers
• IgA: both monomeric and polymeric
• IgM: pentamer
 Antibody
Syntheses,
Monoclonal Bonds, Influence
Antibodies of Agglutination,
Prperties Genes Coding for
Immunoglobulin

Immunoglobu
lin classes

structures
 Functions
C H2 bind complement and control the rate of Ig catabolism (breakdown)
CH2 and CH3 bind phagocyte FcR (Fc Receptor) to stimulate antigen uptake;
mediate binding of Ig to Fc receptors expressed on the surfaces of
various cell types including B cells themselves
CH region which anchors the molecule to the lipid bilayer.

CH1 and CH2 In between is the hinge region

VH and VL fold together to form an antigen-binding site, so each Ig molecule has
two identical antigen-binding sites.
CDR3 Binds to peptide;
 properties
• Glycoproteins
• Can be isolated in the gamma globulin fraction
of protein by electrophoresis
• Can be found in many blood plasma and body
fluids
Ab
 Bifuncti Specifici Cross-
onal ty Reactivity
●
Antigen-
binding
●
Binds to Similar to
●

the
function specific determinan
●
Effector
functions antigen t of antigen
Affinity Avidity
“Tightn
●
Overall
●

ess of strengt
the Fit” h
 classifications

IgG IgM

IgA IgD
 IgG IgM IgA IgD IgE
MW 190,000 900,000 160,000- 180,000 190,000
Sedimenta 7S 19S 7S 7S 8S
tion Coef.
H-Chain 50,000- 70,000 55,000- 62,000 70,000-
MW 60,000 60,000 75,000
Percent in 70-75 10 10-15 Less than 0.002
Total Ig 1
Pool
Serum 800-1600 120-150 70-350 1-3 0.005
Conc.
Mg/dl
Half-life in 23 6 5 1-3 2-3
days
Complem YES YES
ent
Fixation
 IgG IgM IgA IgD IgE
Distribution Vascular Vascular Vascular Membran FcεR on
intra and Intra (intra)+ e of (naive mast cells,
extra Mucosa cells) basophils

Subclasses 4 2
Breast + +++
Milk/Colos
trum
Placental ++
Transfer
 IgG
• Predominant Immunoglobulin in humans
• 70-75% of total Ig pool
• High diffusion coefficient allowing it to enter
extravascular spaces more readily
• 7S Molecule
• 150,000 MW
Can cross the placenta

Complement can be activated

Longest half-life of any immunoglobulin class (23-25 days)

Providing immunity for the newborn

Major Functions
Fixation of complement

• Major Functions:
–
Opsonization

Neutralization of toxins and viruses

Participation in agglutination and precipitation reactions

 4 Major Subclasses
IgG1 – IgG2 –
66% 23%

IgG3 – 7% IgG4 – 4%

• Differ mainly in number and position of disulfide

bridges
• Also found in Cord Blood
and CSF
• Normal Values
Adult 800-1800 mg/dl (90-210 IU/ml)
Infants 350-400 mg/dl (40-45 IU/ml)
(3-4 mos.)
Children 700-800 mg/dl (80-90 IU/ml)
(1st year)
• Decreased levels of IgG:
Primary – genetic
Secondary – Acquired
Infectious diseases – Hepa, Rubella, IM
Collagen disorders – RA, SLE
Hematologic disorders – polyclonal and
monoclonal gammopathy, Hodgkin’s
disease, monocytic leukemia
 classifications

IgG IgM

IgA IgD
Immunoglobulin A

IgA
 Immunoglobulin A
• Monomer
• MW: 160,000 Daltons
• Sedimentation Coefficient: 7S
• H (alpha) chain: 55-60,000 Daltons with 472
AA’s
• Two Isotypes: IgA1 and IgA2
• Two forms: Serum and Secretory
• Capable of acting as Opsonin
 Serum IgA
• 15-20% of total normal serum Ig pool
• 200mg/dL
• 80% is in monomeric form
• Linked by disulfide bonds and joining J chains
• Fixes complement through alternative
pathway
 Secretory IgA
• • Found
Foundasasaadimer
dimerininbody
bodysecretions
secretions
• • Synthesized
Synthesizedininplasma
plasmacells
cells
• • Predominant
PredominantIgIgininsecretions
secretions
• • Protect mucosal surfaces
Protect mucosal surfaces
• • Low
LowIgA=
IgA=Increased
Increasedmucosal
mucosalinfections
infections
Secretory Component

●
Taken inside the cell

●
Released to opposite surface via Transcytosis

●
Vesicle fuses with membrane on opposite side

●
Small SC fragment cleaved

●
IgA dimer liberated
 classifications

IgG IgM

IgA IgD
Immunoglobulin D

IgD
 Immunoglobulin D
• Extremely scarce in the serum
• MW: 184,000 Daltons
• Appears to have an extended hinge region
 Function
• Signals b cell activation
• Bind to basophils and mast cells
• Activate cells that produce antimicrobial
factors
 classifications

IgG IgM

IgA IgD
 IgE
The least abundant immunoglobulin in the serum

Most heat-labile of all Immunoglobulins

Incapable of crossing the placenta

IgE does not participate in typical immunoglobulin reactions

it attaches to basophils and tissue mast cells by means of Fcε RI receptors

Plasma cells that produce IgE are located primarily in the lung and the skin
• When two adjacent IgE molecules on a mast
cell bind specific antigen, a cascade of cellular
events is initiated which will result to:
– Degranulation of the mast cells: release of
vasoactive amines such as:
• Histamines
• Heparin
– Induction of type 1 immediate hypersensitivity or
allergic reaction
 Anaphyla
ctic Shock
Asthm
a

Hives
Typical
Reactions

Vomiti
ng

Hay
FEVER
• Note: it has protective roles also—triggering
an acute inflammatory reaction
Neutorp that recruits
– Neutophils Eosinop
hils
hils
– eosinophils
Monoclonal Antibodies

Purified antibodies cloned from a

single cell

Exhibit exceptional purity and

specificity

Able to recognize and bind to specific

antigen
Antibody Synthesis
 Lag No antibody is detectable
●

The antibody titer increases

Log
●

logarithmically

Plateau The antibody titer stabilizes

●

Decline The antibody is catabolized

●
• IMMUNE COMPLEXES
– Noncovalent combination of antigen with its
respective, specific antibody
– Small(soluble) or large(precipitating)
– Antibody can react with antigen that is fixed or
localized in tissues or with antigen that is released
or present in the circulation
• Once in the circulation, the immune complex is usually
removed by phagocytic cells but normally does not lead
to pathologic conditions
• Factors determining immune complexes
1. Nature of the antigen
2. Nature of the antibody
3. Degree of lattice formation
4. Rate of formation
5. Rate of clearance
 Types of Bonding

Hydrogen
Van der
Hydrophobic Electrostatic
BondWaals
 Type of Bonding
1. Hydrophobic Bonding—major bonds
between antigens and antibodies
 Many nonpolar side chains of proteins are
hydrophobic
 The exclusion of water frees some of the
constraints imposed by the proteins
 Gain in energy and forms an energetically stable
complex
2. HYDROGEN BONDS—results from the formation
of hydrogen bridges between appropriate
atoms
 Major hydrogen bonds in ag-ab interactions
 O-H-O
 N-H-N
 O-H-N

3. VAN DER WAALS FORCES—nonspecific, attractive

forces generated by the interaction between
electron clouds and hydrophobic bonds
4. ELECTROSTATIC FORCES—result from the
attraction of oppositely charged amino acids
located on the side chains of two amino acid
residues
Theories of Antibody Diversity
 Ehrlich’s Side Chain Theory
• Paul Ehrlich in the 1900s
Cells had specific receptors for antigen present before contact with antigen occurred

Once antigen was introduced, it would select the cell with the proper receptors

Combination would take place

Receptors break off and enter the

Circulation as antibody molecules
New receptors are
born from those that
have broken off

Pr
o
c
es
s
m
a
y
b
e
re
p
e
at
e
d
 • Emergence of two key premises

1) Lock and key concept for antibody and

antigen
2) Antigen selected cells with a built in capacity
to respond to it
 The Template Theory
• Felix Haurowitz in the 1930s
Second Major theory
Also known as the instructive theory

Antibody producing cells synthesize a generalized type of

antibody

Antigen serves as a mold and alters protein synthesis so

that antibody of specific fit is made
• Contradictions:
1) Protein synthesis – 3D structure is
genetically predetermined making it not
subject to alteration
2) Self tolerance – cannot be explained, high
levels of self antigen should mean many
templates for antibody production
 Clonal Selection
Macfarlane Burnet

CLONAL SELECTION

Niels Jerne
 Preprogrammed
Preprogrammed
Genetically
Genetically

Specific Antigen finds

Specific
cells Antigen finds
capable of
cells capabletoofit
responding
responding to it

Key Premises

Activation
Lymphocytes
Activation
Lymphocytes
n
n
Proliferatio
Proliferatio

ofof
BB
Heavy Chain Rearrangement
• From a single region of chromosome 14
• Divided into three groups:
– Vh
–D
–J
• Constant region
• Disulfide Bond
REGIONS
 Constant Region

• Mu and Epsilon with 4 Ig

domains
• Alpha, Gamma and Delta
with 3 tandem domain
 Variable Region
• Differs between B cells
• Same for all Ig produced by same B cell
• Composed of a single Ig domain
 Synthesis
• Composed of:
– 1 Vh gene
– 1 D gene
– 1 J gene
– 1 Constant region
 DNA level
V geneD
one joined
and toJDJ
V gene joined to DJ
one D==and
complex V(D)J
complexjoined
J
gene
gene V(D)J
gene joined
gene

Cl
a
s
s
s
w
it
c
hi
n
g
 RNA level

Constant
region
Mu H spliced to
chains V(D)J
complex
synthesized
Looping out
and deletion
of other C
regions

Heavy Chain
Light Chain Rearrangement

light chains
Occurs after μ
lack a D
chains appear
segment
Chromo Coding for κ chains
●

some 2
Chromo Coding for λ chains
●

some 22
 2

1 Ck Region

5 J regions
32 Vk
regions
 J Ck
V

V/J Joining

Transcriptio

RNA Splicin
 Thank You

Balbastro
De Guzman
Luna
Pangilinan