Antibodies: Structure And

Function
 Antibody structure
Antibodies belong to a class of proteins called
immunoglobulins. Antibodies bind antigen with a
high degree of specificity and affinity.
Immunoglobulins are Y shaped proteins. The
arms of the Y bind antigens. The tail of the
Y is responsible for biological activity eg. C
activity or binding to cells.
Ability of immunoglobulins to bind antigen
determined by AA sequence in variable region.
Antibody molecules belong to one of five classes
i.e. IgG, IgM, IgA, IgD & IgE
 General Characteristics of
antibody
Antigen-specific products of B cells
First of the molecules participating in immune response
to be characterized, best understood
Basic building block, immunoglobulin domain, is
used in molecules of both immune system & other
biological recognition systems
Antibody molecule has 2 separable functions:
1) specific binding to antigen eliciting response
2) biological activity - recruit cells & components
designed to destroy agent to which ab developed
 Antibody Structure
Antibodies Are Made Up Of:
2 Light Chains (identical) ~25 KDa
2 Heavy Chains (identical) ~50 KDa
Each Light Chain Bound To Heavy Chain By Disulfide (H-
L)
Heavy Chain Bound to Heavy Chain (H-H)
First 100 a/a Of Amino Terminal Vary of Both H and L Chain
Are Variable.
Referred To As VL , VH, CH And CL
CDR (Complementarity Determining Regions) Are What
Bind Ag
Remaining Regions Are Very Similar Within Same Class
 Antibody Structure
Repeating Domains of ~110 a/a
Intrachain disulfide bonds within each domain
Heavy chains
1 VH and either 3 or 4 CH (CH1, CH2, CH3, CH4)
Light chains
1 VL and 1 CL
Hinge Region
Rich in proline residues (flexible)
Hinge found in IgG, IgA and IgD
Proline residues are target for proteolytic digestion (papain and
pepsin)
Rich in cysteine residues (disulfide bonds)
IgM and IgE lack hinge region between CH1 & CH2No
They instead have extra CH4 Domain
 Hinge Region
Consists of approx. 12 AA between CH1 & CH2
homology between AA sequence of hinge & heavy chains
AA sequence differs with different classes
Cysteine involved in formation of interchain disulfide bonds
Proline prevents folding in a globular structure, allowing
flexibility between two Fab arms of the Y-shaped antibody;
allows open & close to accommodate binding to two epitopes;
because it is open, it can be cleaved by proteases (e.g. papain)
to generate the Fab & Fc fragments
 AA sequence in hinge region
Hinge region
Light Chain
Cys
-Arg-Val-Glu-Pro-Lys-Ser- Cys-Asp-Lys-Thr-His-Thr-Cys-Pro-Pro-Cys -Pro-Ala-Pro-Glu-

-Arg-Val-Glu-Pro-Lys-Ser- Cys-Asp-Lys-Thr-His-Thr-Cys-Pro-Pro-Cys -Pro-Ala-Pro-Glu-

Cys

Light Chain
Papain

Fab region Fc region

Enzymatic Digestion Of Antibodies
Digestion With Papain Yields
3 Fragments
2 identical Fab and 1 Fc
Fab Because Fragment That is Antigen Binding
Fc Because Found To Crystallize In Cold Storage
Pepsin Digestion
F(ab`)2
No Fc Recovery, Digested Entirely
Mercaptoethanol Reduction (Eliminates Disulfide
Bonds) And Alkylation Showed
 Porter (England)
Treatment with proteolytic enzyme,
papain, resulted in three approximately
Fab Fab
equal sized fragments:
2 capable of ag rx (fragment antigen
SS S S binding) Fab specifically bind antigen,
univalent (cant ppt) one binding site each,
identical to each other.
S S

1 could be crystallized (fragment-

Fc crystallizable). Fc crystallizable (thus
homogenous) cant bind ag responsible for
biological activity of molecule after ag
bound to Fab portions.
 Eddleman (USA)
Treatment with mercaptoethanol = 4
chains: (mercaptoethanol breaks S-S
SH HS SH HS SH HS
bonds)
2 chains = 53,000 daltons
2 chains = 22,000 daltons
All immunoglobulins basic unit
consisting of 4 polypeptides i.e. 2 H,
2L

Pepsin digestion results in Fab2

cleavage @ C terminal
SS SS SS
portion, resulting in a divalent
fragment (Fab2) joins by S-S
& several Fc fragments
C-terminus
Cleavage of Ig
SH HS SH HS
SH HS
E 4 polypeptide chains
ce dM
du
Re
Fab Fab

Papain SS SS
SS 2 Fragment ag binding

1 Fragment crystallizable
Pe
p sin

1 Fab2
SS SS SS

Several small pieces

Fc
 Light chain sequences
N-terminus

N-terminus
Fab Fab

SS SS 214 AA in
SS two domains

C terminus Analysis from

C terminus several myelomas

Different
sequences Variable (VL)
in N-termin.
domain
Identical
sequences
Constant (CL)
in C-termin.
domain
 Heavy-chain sequence
N-terminal domain varies
Heavy chain consists of 445
(HV)
AA in 4 domains
Other 3 domains constant
(HC)
Flexible
Hinge
Region

N-terminus
Fab Fab

SS SS
SS

C terminus
 Sequencing Of Heavy Chains

Sequencing Of Several Immunoglobulins Revealed

100-110 Amino Terminus, Highly Variable (V)
Five Basic Sequence Patterns
,, , ,
IgA, IgG, IgD, IgE and IgM
The Above Classes Are Called Isotype
Each class can have either or light chains
Minor Differences Led To Sub-classes For IgA and IgG
IgA1, IgGA2 and IgG1, IgG2, IgG3, IgG4
 H chains
H chain confers unique biologic properties of molecule e.g.
1/2 life, receptor binding , enzyme-, C activation with ag
Immunoglobulin isotype Heavy chain
IgM
IgG
IgA
IgD
IgE
Individual of species produces all H chains, in proportion
characteristic for species, but ab molecule H chains are
identical ( i.e., 2 etc.)
 Structural features
Structural features of IgM
- IgM
5-10% of serum immunoglobulin
SS
IgM only exists as a monomer on
SS
SS the surface of B cells, has a very

SS
SS

low affinity for antigen

SS Pentameric version is secreted
SS

SS
First Ig produced following
SS

J chain
S S
S immunization.
SS Macroglobulin (M),900 kD, 19S
SS S

SS
SS Doesnt have hinge region has
additional H domain
Has a J chain (one of 2 Ig
isotypes)15 kD
 IgM facts and figures

Heavy chain: - Mu
Half-life: 5 to 10 days
% of Ig in serum: 10
Serum level (mgml-1): 0.25 - 3.1
Complement activation: ++++ by classical pathway
Interactions with cells: Phagocytes via C3b receptors
Epithelial cells via polymeric Ig receptor
Transplacental transfer: No
Affinity for antigen: Monomeric IgM - low affinity - valency of 2
Pentameric IgM - high avidity - valency of 10
 IgG
Two H chains, Two L chains (either or but not both)
Each H chain = 50 kD, Each L chain = 25 kD
150 kD, 7S, globulin
Most abundant immunoglobin 80% of serum Ig
~10mg/mL
IgG1,2,3,4 (decreasing serum concentration)
IgG1, IgG3 and IgG4 cross placenta
IgG3 Most effective complement activator
IgG1 and IgG3 High affinity for FcR on phagocytic cells,
good for opsonization
 Highest concentration in
serum
IgG
Plays major role in immune
defence.
MW approx. 150 kDa
Small size ppt in surfaces
(e.g. cross placental barrier)
Opsinize, aggl. & ppt ag
Only activates classical C Subclasses show close overall relation
pathway Heavy chains =
All normal indiv. have all
IgG1>IgG2>IgG3>IgG4
IgG3 has shortest 1/2 life,
highest catabolic rate,
highest # S-S
IgG1 IgG2 IgG4 = monoval. no aggl
ppt rx., autoab to clot fac
Autoab to DNA = IgG1,IgG3

IgG3 IgG4
 IgG facts and figures
Heavy chains: 123 4 - Gamma 1 - 4
Half-life: IgG1 21 - 24 days IgG2 21 - 24 days
IgG3 7 - 8 days IgG4 21 - 24 days
Serum level (mgml-1): IgG1 5 - 12 IgG2 2-6
IgG3 0.5 - 1 IgG4 0.2 - 1
% of Ig in serum: IgG1 45 - 53 IgG2 11 - 15
IgG3 3-6 IgG4 1-4
Complement activation: IgG1 +++ IgG2 +
IgG3 ++++ IgG4 No
Interactions with cells: All subclasses via IgG receptors on macrophages
and phagocytes
Transplacental transfer: IgG1 ++ IgG2 +
IgG3 ++ IgG4 ++
 IgA
10-15% of serum IgG
Predominant Ig in external secretions
Milk, saliva, tears, mucus
5-15 g of IgA released in secretions!!!!
165 kD, 7S, migrates as fast
Serum mainly monomeric, polymers
possible not common though
Secretions, as dimer or tetramer+J-chain
IgA. Two IgA
molecules joined
polyptetide+secretory component (Poly
by J chain. IgR)
IgA Antibody Transport Across Cell
(Transcytosis)

The J chain
facilitates transport
of IgA across
mucosal epithelia.
The J chain also
facilitates transfer
of IgA to newborns
to confer neonatal
passive immunity.
 IgA facts and figures
Heavy chains: 1 or 2 - Alpha 1 or 2
Half-life: IgA1 5 - 7 days
IgA2 4 - 6 days
Serum levels (mgml-1): IgA1 1.4 - 4.2
IgA2 0.2 - 0.5
% of Ig in serum: IgA1 11 - 14
IgA2 1 - 4
Complement activation: IgA1 - by alternative and lectin pathway
IgA2 - No
Interactions with cells: Epithelial cells by pIgR
Phagocytes by IgA receptor
Transplacental transfer: No
 IgE
Very low serum concentration, 0.3g/mL
Participate in immediate hypersensitivities reations.
Ex. Asthma, anaphylaxis, hives
Sometimes called reaginic ag
190 kDa, 8S, migrates as fast
Contains an extra domain (CH4) which binds to
mast cells & basophils
May remain attached for long time when ag
reappears, cross links IgE on mast cell surface,
release mast-cell granules (Histamine) & signs of
anaphylaxis
 IgE facts and figures
Heavy chain: - Epsilon
Half-life: 1 - 5 days
Serum level (mgml-1): 0.0001 - 0.0002
% of Ig in serum: 0.004
Complement activation: No
Interactions with cells: Via high affinity IgE receptors expressed by
mast cells, eosinophils, basophils and
Langerhans cells
Via low affinity IgE receptor on B cells
and monocytes
Transplacental transfer: No

IgE appears late in evolution in accordance with its role in protecting against parasite
infections
Most IgE is absorbed onto the high affinity IgE receptors of effector cells
IgE is also closely linked with allergic diseases
Cross-Linkage of Bound IgE Antibody
With Allergen Causes
 Structural IgD
features of IgD
Primarily a B cell antigen receptor
IgM and IgD, Expressed on B-cell Surface
Long exposed hinge region
170kD, 7S, migrates as fast
No interchain S-S bridges in H chains
Low serum concentrations, ~30g/mL
the function of IgD in host defence is unknown

Readily denatured
 IgD facts and figures
Heavy chain: - Delta
Half-life: 2 to 8 days
% of Ig in serum: 0.2
Serum level (mgml-1): 0.03 - 0.4
Complement activation: No
Interactions with cells: T cells via lectin like IgD receptor
Transplacental transfer: No

IgD is co-expressed with IgM on B cells due to differential RNA splicing

Level of expression exceeds IgM on nave B cells
IgD plasma cells are found in the nasal mucosa - however the function of IgD in
host defence is unknown - knockout mice inconclusive
Ligation of IgD with antigen can activate, delete or anergise B cells
Extended hinge region confers susceptibility to proteolytic degradation
 Antibodies Act As Immunogens
Antigenic Determinants on Abs Fall in 3
Categories
Isotypic
Allotypic
Idiotypic
Isotypic
Constant Region Of Ab
If you inject Ab in a different species Anti-
Isotype is generated
If within same species, No Anti-isotype
 Antibodies Act As Immunogens
Allotype
Even though same isotypes within one species
small differences (1-4 a/a) arise in different
individuals (form of polymorphism)
If injected with such Ab you generate anti-
allotype Ab
Ex. During pregnancy
Blood transfusion
 Antibodies Act As Immunogens
Idiotype
Unique VH AND VL binds antigen but can also behave
as antigenic determinant
If you inject a monoclonal antibody into a
genetically identical recipient then anti-idiotypic
antibodies are generated
No anti-isotypic and no anti-allotypic Abs will be
generated