HM

1. Antibody
Yanjun Lei
Ph.D & Associate Prof.

2. Antigen
Antibodies are major effector molecules and
representative for specificity in humoral immunity

3.  Membrane antibody: BCR
Membrane-bound receptors on the surface of B cell
Bind foreign antigens encountered by the host
 Secreted antibody/Soluble circulating antibody
Secreted by plasma cells.
Present in serum and tissue fluids
Mediate effector functions to neutralize or
eliminate antigen.

4. Content of Antibody Chapter
 The concept of Antibody/immunoglobulin,
monoclonal/polyclonal antibody;
 The structure of immunoglobulin ;
 The domain of immunoglobulin and the function of
each domain;
 The biological function of immunoglobulin;
 The characteristic and function of each Ig class

5. Kabat and Tiselius - 1938
immunized
rabbit with
ovalbumin
Before
After
γ-globulin fraction was identified as containing serum
antibodies.
Harvest serum
No
treatment
Mix with Ag
to remove
precipitation
Electrophoresis

6. Immunoglobulin (Ig)
Globulins( a family of glycoproteins) that have antibody
function
Immunoglobulin superfamily (IgSF)
Protein molecules that shared similar structural features
with Immunoglobulin. TCR, CK,CKR
1. Concept of antibody and Immunoglobulin
Antibodies (Ab)
Antibodies are globulins that are produced by plasma
cells in response to an antigen and react specifically with
the antigen that stimulated their production.
5 class: IgG, IgM, IgA, IgD and IgE

7. 2. Antibody Structure
2. “Y” shape: linked by
disulfide bonds
1. Consist of 4 polypeptide
chains
Two heavy (H) chains
Two light (L) chains
3. Two terminal
amino-terminal end (N)
carboxyl-terminal end (C)
Disulfide
bond
Fab
Fc

8. 4. Two regions
Constant (C) region: The C terminal
sequence (~ 110 residues of L chain
and remaining of H chain) was
constant for all antibodies
L chain 1/2-VL
H chain 1/4-VH
N-end
L chain 1/2-CL
H chain 3/4-CH
C-end
Variable (V) region : the sequence of the ~110 N terminal
residues of L chain and H chain was seen to be unique for
each antibody with differernt binding specificity.

9.  Hypervariable regions/
complementarity-
determining regions
(CDRs) form the antigen-
binding site
 The amino acids of V
region varies greatly among
antibodies with different
antigen specificity.
Variable (V) region

10. Hypervariable region (HVR)
complementarity-determining region(CDR)
 Within the variable regions of both heavy and light
chains, some polypeptide segments show exceptional
variability and are termed Hypervariable regions or
complementarity-determining regions(CDRs)
 There are 3 complementarity-determining regions(CDRs)
on both L and H chains.

11. Most of the differences among Abs with different
specificity fall within CDRs
CDRs on both L and H chains form the antigen-
binding site of the antibody molecules

13. Constant (C) region
 The regions of relatively
constant sequence beyond the
V regions
The Ig constant region domains take
part in various biological functions that
are determined by the amino acid
sequence of each domain.

14. In human, There are five distinct classes of antibody
/immunoglobulin molecule according to the sequence
of constant region of H chain
The class and subclass of immunoglobulinThe class and subclass of immunoglobulin
Ig IgM IgG IgA IgD IgE
H chain µ γ α δ ε
The H chain of a given antibody molecule determine
the class of that antibody.

15. General structure of the five major classes of secreted antibody

16.  Size: The length of the C region:
γ,α,δ: ~330 aa; µ, ε: ~440 aa
 Charge
 Amino acid sequence
 Carbohydrate content
IgG, IgM, IgA, IgE and IgD differ in:

17.  Minor differences in the amino acid sequences and
differences in position and numbers of disulfide
bond of the α and γ heavy chains led to further
classification of the heavy chains that determine the
subclass of antibody molecule they constitute.
α heavy chains: α 1 — IgA1
α 2 — IgA2
γ heavy chains: γ 1 — IgG1
γ 2 — IgG2
γ 3 — IgG3
γ 4 — IgG4

18.  In humans, 60% of light chains are kappa and 40% are
lambda; in mice 95% are kappa and 5% are lambda.
 Comparison of λλ chain sequences revealed
minor differences that could be used to classify
λλ chains into subtypes - 3 in mice and 4 in humans
 Each antibody has two identical heavy and light chains,
A normal antibody molecule contains only one light-
chain type, either κ or λ, never both.
2 light chain types:2 light chain types: κκ oror λλ

20. 5. Hinge region
IgG IgD and IgA
Is rich in proline residues
Give IgG IgD IgA segmental flexibility
The γ, δ and α heavy chain
contain an extended peptide
sequence between the CH1 and
CH2 domains that has no
homology with the other
domains.

21. 6. Accessorial structure
Joining (J) chain: facilitate the
polymerization of IgA and IgM
secretory piece (SP or SC):
is responsible for IgA
transporting through mucous
membrane epithelial cells

22. Basolateral
surface
luminal
surface
1. Dimeric IgA bind with Poly-
Ig receptor
2. Be internalized by receptor
mediated endocytosis.
3. Transport to luminal
surface
4. Poly-Ig is cleaved,
Releasing the SP bound to
the dimeric IgA
5. SP masks sites susceptible
to protease cleavage,
allowing the polymeric
molecule to exist longer in
the protease-rich mucosal
environment.

23. 3. The domains of antibody
Heavy and light chains are folded into domains,
each containing about 110 amino acid residues and
an intrachain disulfide bond that forms a loop of 60
amino acid

24. Constant Domain: CH1, CH2, CH3, CH4 ( IgM and IgE).
Variable Domain: VH, VL
CH1, CH2, CH3 ( IgA, IgD and IgG).

25. VL VH: antigen binding site
CL CH1: Facilitate interaction
with antigen and
increasing the maximum
rotation of the Fab arms.
Function of domains
Hinge region:
flexibility, enabling the 2 arms to move relatively
freely

26. CH2 of IgG/ CH3 of IgM:
activate the complement cascade.
cross the placental barrier. (IgG)
IgA, IgD, IgG IgE, IgM
CH1
Hinge
CH2
CH3
CH1
CH2
CH3
CH4

27. Secreted immunoglobulin:
 binding to cells by Fc receptors.
Membrane-bound
immunoglobulin （ BCR ） :
 An extracellular hydrophilic
“spacer”
sequence composed of 26 amino
acid residues.
 A hydrophobic transmembrane
sequence
 A short cytoplasmic tail
CH3 of IgG/ CH4 of IgE and IgM: bind with the cells

28. 4. Enzymatic Digestion Fragments of lgG4. Enzymatic Digestion Fragments of lgG
Fc: Fragment, crystallizable
Fab: Fragment, antigen binding
Proteolytic
enzyme

30. General structure of the five major classes of secreted antibody

32. 3 major categories:
Isotypic determinants
Allotypic determinants
Idiotypic determinants
Antigenic Determinants on
lmmunoglobulins

33. Definition - Isotypes are antigenic determinants that
characterize classes and subclasses of antibody.
Location - isotypes are found in the constant region of the
heavy chain and light
Isotype

34.  Different species inherit different constant-region
genes and therefore express different isotypes
 When an antibody from one species is injected into
another species, the isotypic determinants will be
recognized as foreign , inducing an antibody
response to the isotypic determinants on the foreign
antibody.
 Antibodies to isotypes are used for quantitation of Ig
classes and subclasses in research or clinic.
Occurrence - Isotypes are found in ALL NORMAL
individuals in the species.

35. Definition- are additional antigenic features of Ig that vary
among individuals of the same species and are under genetic
control.
Location - the allotypic differences are localized to the
constant region of the heavy and light chains

36.  Antibody to allotypic determinants can be produced by
injecting antibodies from one member of a species into
another member of the same species who carries different
allotypic determinants.
 Allotypes represent slight differences in the amino acid
sequences in the heavy or light chains of different
individuals
Occurrence - Individual allotypes are found in individual
members of a species. Any individual Ig
molecule will only have one allotype.

37. Definition - Unique antigenic determinants present on
individual antibody molecules or on
molecules of identical specificity.
Location - Idiotypes are localized on the variable region
of the Ig molecules

38. idiotypic determinants of the antibody can recognized by
self immune system to generate anti-idiotype Abs.
 The unique amino acid sequence of the VH and VL
domains of a given antibody can function not only as
an antigen binding site but also as a set of antigenic
determinants.
 Each individual antigenic determinant of the variable
region is refers to as an idiotype 。 Each antibody will
present multiple idiotypes.

39. 1. Neutralization
Antibodies bind specifically with the exotoxin or
virus to neutralize their toxicity or infectivity.
Function of Antibody

40. Neutralize the infectivity of Virus

41. 2. Activate the complement
3. Across epithelial layers
and across placenta
IgA (major)
IgM
Mucosal surface
of the respiratory,
gastrointestinal
and urogenital
tracts; breast milk
IgM and most of IgG
IgG fetus

42. 4. Antibodies bind to cell surface receptors on
macrophage, NK cells, mast cells and basophils
① IgG Fc —Mφ opsonization
② IgG Fc —NK ADCC
③ IgE Fc —mast cell Type I hypesensitivity

43. opsonization
Antibody can promote phagocytosis of antigens by
macrophage and neutrophils, is important factors in
antibacterial defenses.

44. ADCC ( Antibody-dependent cell-mediated
cytotoxicity)
The linking of Ab bound to target cells with the FcRs of a number of
cell types,particularly NK cells, can direct the cytotoxic activities of
the effector cell against the target cell. This process called ADCC
Perforin
granzyme

45. Mediate Type I hypesensitivity
Unique to IgE
Inappropriate and damaging
immune response that
result in serious disease,
tissue injury, or even death
Hypesensitivity:

46. The characteristics and function ofThe characteristics and function of
each Ab classeseach Ab classes

47. Is the most major Ig in serum and in extra vascular
space. constitutes about 75~80% of the total serum
Ig.
There are 4 human IgG subclasses.

48.  IgG is the only class of Ig that crosses the placenta
and play an important role in protecting the developing
fetus. Not all subclasses cross equally; IgG2 does not
cross well, IgG4 does not fix complement
 Not all subclasses activate complement equally well,
IgG3 is the most effective complement activator,
followed by IgG1, IgG2 is less efficient, and IgG4 dose
not activate complement.
 IgG1 and IgG3 bind with high affinity to Fc receptors
on the phagocytic cells and thus mediate opsonization.
IgG4 has an intermediate affinity for Fc receptors and
IgG2 has an extremely low affinity.

49.  IgM accounts for 5~10% of the total
serum Ig and is the 3rd most common
serum Ig.
 Monomeric IgM is expressed on
the surface of B cell, act as BCR.
 IgM secreted by plasma cells is
pentamer .
IgM is the first Ig to be made by the
fetus and the first Ig to be made by a
virgin B cells when it is stimulated by
antigen
 IgM is more efficient than IgG at
activating complement.
 IgM is “natural” antibodies to RBC
group
IgM

50.  Constitute 10-15% of the total Ig
in Serum
 Predominant Ig class In secretions
Breast milk, tears , saliva, mucus of
digestive tract, bronchial.
 Most serum IgA is monomer
 Secretory IgA is dimer
 J Chain (Joining) and secretory
component
IgA
 Play important protective effect
in mucosal immunity

51. Transport of IgA into Gut Lumen

52. 0.03 mg/ml, 0.2% of the
total Ig in Serum
Together with IgM ,is the
major membrane-bound Ig
expressed by mature B cells
its role in serum is
uncertain.
Immunoglobulin D (IgD)

53.  IgE is the least common serum
Ig since it binds very tightly to Fc
receptors on basophils and mast
cells even before interacting with
antigen.
Only 0.0003 mg/ml in serum
 Mediate allergic reactions.
IgE is involved in allergic
reactions. Binding of the allergen to
the IgE on the basophils an mast
cells results in the release of various
pharmacological mediators that
result in allergic symptoms.
Immunoglobulin E (IgE)

55. Polyclonal Antibody(pAb) /Antisera
Monoclonal Antibody(mAb)
Antibodies that are specific for one antigen
epitope and produced by one B cell clone, and is
produced by a B cell hybridoma
1975 Georges Kohler and Cesar Milstein
Antibodies that are derived from different B cell
clones. They are a mixture of immunoglobulin
molecules secreted against a specific antigen, each
recognizing a different epitope.
10. The preparation of Antibody