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1. By Dr Tabassum Mahmood
1

2. Antigen
Any substance which
when introduced into
body, is capable of
provoking an immune
response(antibodies
production )
2

3. Antigens Characteristics
 Foreignness: Molecules recognized as “self” are not
immunogenic
 Molecular Size: Small foreign molecule with
molecular weight below 10,000 (hapten ) weakly
immunogenic & must be coupled to carrier molecule
to be antigenic
 Once antibodies are formed they recognize hapten
3

4. Antigen Characteristics
 Chemical–Structural Complexity: Amino acid
homopolymers less immunogenic than
heteropolymers (containing two or three different
amino acids)
4

5. Antigens Characteristics
 Antigenic Determinants (Epitopes) :Small chemical
groups on antigen molecule that can elicit
immunological response & react with antibody
 Dosage , Route & Timing of Antigen
administration: These factors affect immunogenicity
5

6. Adjuvants
 Enhance immune response to antigen
 Chemically unrelated to antigen
 Differ from a carrier protein :
Not covalently bound to antigen like carrier protein
 Cause slow release of antigen, prolonging stimulus
6

7. Adjuvants
 Stimulate Toll-like receptors at surface of
macrophages, cytokine production enhances response
of T cells and B cells to antigen
 Some vaccines contain adjuvants i.e. aluminum
hydroxide or lipids
7

8. Epitope
 Small part of antigen that interacts with an antibody
 Any given antigen may have several epitopes
 Each epitope is recognized by a different antibody
8

9. Super antigen
 They are able to bind to MHC molecules outside the
peptide binding cleft
 They cause upto 10% of T cells to be nonspecifically
stimulated/ activated
9

10. Epitopes: Antigen Regions Interact with Antibodies
10

11. Antibody
 Gamma globulin proteins that react
specifically with antigen that stimulated their
production
 20% of plasma protein
 Five classes of antibodies:
 IgG, IgM, IgA, IgD, and IgE
(based on differences in heavy chains)
11

12. Functions of antibodies
 Neutralize toxins and viruses
 Opsonize microbes to be easily phagocytosed
 Activate Complement, and prevent attachment of
microbes to mucosal surfaces
 Catalytic :Antibody can act as an enzyme to catalyze
synthesis of ozone (O3) that has microbicidal activity.
12

13. Antigen-Antibody Reactions

14. Antibodies
 After stimulation, B cells differentiate into plasma
cells, & secrete antibodies,(immunoglobulins)
,mediators of humoral immunity
14

15. Antibodies Characteristics
 Diversity Respond to different antigens
 Long memory Respond many years after initial
exposure due to memory T cells and B cells
15

16. Antibodies Characteristics
 Specificity Actions specifically directed against
antigen that initiated response
 Inflammatory response : Combined effect of cells
(e.g., T cells, B cells, macrophages & neutrophils) &
proteins (e.g., interleukins, antibodies & complement)
16

17. Structure of immunoglobulin
 Simplest antibody molecule has Y shape
 Consists of four polypeptide chains:
 Two H chains and two L chains
 Four chains linked by disulfide bonds
 Antibody molecule always consists of identical H
chains and identical L chains
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18. 18

19. Structure of immunoglobulin
 L and H chains subdivided into variable and constant
regions.
 Regions composed of three-dimensionally folded,
repeating segments called domains
 Each domain is about 110 amino acids long
 globular in shape
 stabilized by intrachain disulphide bonds
 Antigen binding sites located in variable domains
19

20. Structure of immunoglobulin
20

21. Structure of immunoglobulin
 Variable regions of light and heavy chain
responsible for antigen-binding
 Constant region of heavy chain responsible for
biologic functions (e.g., complement activation
and binding to cell surface receptors)
21

22. Structure of immunoglobulin
 Light chain : One variable region and one
constant region
 L chain attached to H chain by disulphide &
non- covalent bonds
22

23. Structure of immunoglobulin
 L chains belong to one of two types, κ (kappa) or λ
(lambda), due to amino acid differences in their
constant regions
 Both types occur in all classes of immunoglobulins
(IgG, IgM, etc.), but any one immunoglobulin
molecule contains only one type of L chain
23

24. Structure of immunoglobulin
 Heavy chain consists of a variable region and a
constant region divided into three domains: CH1, CH2,
and CH3
 Each domain 110 amino acids long
 CH2 domain contains complement-binding site
 CH3 domain is site of attachment of IgG to receptors on
neutrophils and macrophages
 H chains structurally & antigenically distinct
for each class
H chains are distinct for each of five immunoglobulin
classes and are designated γ, α, μ, ε, and δ
24

25. Structure of immunoglobulin
 Variable regions of L and H chains have three
extremely variable(hypervariable) amino acid
sequences at amino-terminal end that form antigen-
binding site
 Specificity of antibodies is due to hypervariable
regions
25

26. Structure of immunoglobulin
 Amino-terminal portion of each L & H chain
participates in antigen-binding site
 Carboxy terminal forms Fc fragment, which has
biologic activities
26

27. Heavy chain
H chain designated by Greek letter.
Ig class H chain
Ig G 
Ig M 
Ig A 
Ig D 
Ig E 
27

28. Structure of immunoglobulin
Antibody molecule treated with a proteolytic enzyme s
papain,break peptide bonds in “hinge” region ,
producing two identical Fab fragments, which carry
antigen-binding sites, and one Fc fragment, involved
in placental transfer, complement fixation, attachment
site for various cells,& other biologic activities
28

29. 29

30. Antigen-Antibody Responses
30

31. Classification of antibodies
 Immunoglobulin A (IgA)
 Immunoglobulin G (IgG)
 Immunoglobulin M (IgM)
 Immunoglobulin D (IgD)
 Immunoglobulin E (IgE)
 Based on structural differences in constant regions of
heavy chains
 Classes have specialized effector functions
31

32. Ig M
 5-8 % of serum immunoglobulins
 Short lived
 Pentameric structure
 Predominant Ab in primary immune response.
 Earliest Ab synthesized by fetus
 Confined to intravascular pool due to large size
 Not transported across placenta
 Presence of IgM in newborn indicates intra uterine
infection
 Useful in the diagnosis of congenital infections like
syphilis, rubella, HIV, toxoplasmosis etc.
32

33. Distribution
Class of Immuno-
globulin (Antibody)
IgM
(pentamer)
J chain
First Ig class
produced after
initial exposure to
antigen; then its
concentration in
the blood declines
Promotes neutraliza-
tion and cross-
linking of antigens;
very effective in
complement system
activation
Function
33

34.  75% of total immunoglobulins
 4 subclasses – IgG1, IgG2, IgG3 & IgG4
Each having a distinct type of gamma chain
 Major Antibody of secondary response, found in
serum & body fluids
 Only maternal Ig to be transported across placenta –
natural passive immunity in newborn
 Participates in complement fixation, precipitation &
neutralization of viruses & toxins
IgG
34

35. Distribution Function
Class of Immuno-
globulin (Antibody)
IgG
(monomer)
Most abundant Ig
class in blood;
also present in
tissue fluids
Promotes opsoniza-
tion, neutralization,
and cross-linking of
antigens; less effec-
tive in activation of
complement system
than IgM
Only Ig class that
crosses placenta,
thus conferring
passive immunity
on fetus
35

36. Ig A
 2nd most abundant 10-13 %
 Major Ig in colostrum, saliva, tears & other body
fluids.
 Two forms : IgA1 & IgA2.
 Secretory IgA in dimeric form – composed of 2 basic
chain units, a J chain & the secretory component.
 Secretory component helps to transport dimer from
submucosa to mucosal cell surface.
 Secretory component protects IgA from proteolytic
digestion and denaturation.
36

37. Distribution Function
Class of Immuno-
globulin (Antibody)
IgA
(dimer)
J chain
Secretory
component
Present in
secretions such
as tears, saliva,
mucus, and
breast milk
Provides localized
defense of mucous
membranes by
cross-linking and
neutralization of
antigens
Presence in breast
milk confers
passive immunity
on nursing infant
37

38. Ig E
 Low levels in serum
 On surface of mast cells & basophils which have
specific receptors for Fc portion of IgE
 Produced in linings of respiratory & intestinal tracts
 Causes anaphylactic type of hypersensitivity
 Defense against parasitic infections
38

39. Distribution Function
Class of Immuno-
globulin (Antibody)
IgE
(monomer)
Present in blood
at low concen-
trations
Triggers release from
mast cells and
basophils of hista-
mine and other
chemicals that cause
allergic reactions
39

40. Ig D
 Resembles Ig G structurally
 Present with Ig M on B cell surface
 Susceptible to proteolytic attack
40

41. Distribution Function
Class of Immuno-
globulin (Antibody)
IgD
(monomer)
Trans-
membrane
region
Present primarily
on surface of
B cells that have
not been exposed
to antigens
Acts as antigen
receptor in the
antigen-stimulated
proliferation and
differentiation of
B cells (clonal
selection)
41

42. Isotypes
 Antigenic (amino acid) differences in constant
regions of heavy chains
 IgG and IgM are different isotypes; constant region of
their H chains (γ and μ) is different antigenically
42

43. Allotype
 Additional antigenic features of immunoglobulins
that vary among individuals
 Genes coding for L and H chains are polymorphic,
and individuals can have different alleles.
 e.g. γH chain contains an allotype called Gm, which is
due to a one– or two–amino acid difference that
provides a different antigenecity to the molecule
 Each individual inherits different allelic genes that code
for one or another amino acid at the Gm site
43

44. Idiotype
 Antigenic determinants formed by specific amino
acids in hypervariable region
 Each idiotype is unique for immunoglobulin
produced by a specific clone of antibody-producing
cells
 Anti-idiotype antibody reacts only with hypervariable
region of specific immunoglobulin molecule that
induced it
44

45. Immunoglobulin class switching
(isotype switching)
 Initially, all B cells carry IgM specific for an antigen
and produce IgM antibody in response to exposure to
that antigen
 Later, gene rearrangement permits elaboration of
antibodies of same antigenic specificity but of
different immunoglobulin classes
45

46. Immunoglobulin class switching
(isotype switching
 Antigenic specificity remains same for lifetime of
B cell and plasma cell because specificity is
determined by variable region genes (V, D, and J genes
on the heavy chain and V and J genes on the light
chain) no matter which heavy-chain constant region is
utilized
46

47. Immunoglobulin class switching
(isotype switching
 Same assembled VH gene can sequentially
associate with different CH genes
 Immunoglobulins produced (IgG, IgA, or IgE) are
specific for same antigen as original IgM but have
different biologic characteristics
47

48. Immunoglobulin class switching
(isotype switching
 Mature B cells can express both IgM and IgD
 Once a B cell has “class” switched past a certain H
chain gene, it can no longer make that class of H chain
because intervening DNA is excised and discarded.
.
48

49. Immunoglobulin class switching
(isotype switching
 Class switching occurs only with heavy chains;
light chains do not undergo class switching.
“Switch recombinase” enzyme catalyzes
rearrangement of VDJ genes during class switching.
 Control of class switching is dependent on
1.concentration of various interleukins
 (IL)-4 enhances production of IgE,IL-5 increases IgA
2.Interaction of CD40 protein on B cell with CD40
ligand protein on the helper T cell
49

50. Primary & Secondary antibody response
 Primary Response
 Following exposure to an antigen, there is a slow rise
in IgM followed by a slow rise in IgG
 Secondary Response
 Following exposure to previously encountered
antigen, there is a rapid rise in IgG and slow or no
rise in IgM
 Memory or anamnestic response
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53. Abnormal Immunoglobulins
 Bence Jones protein in multiple myeloma – light
chains of Immunoglobulins
 Cryoglobulinemia – formation of gel or
precipitate on cooling serum which redissolves
on warming – in myelomas, SLE
53

54. Monoclonal Antibodies
 Antibodies produced in response to antigens are
heterogeneous, formed by different clones of plasma
cells ( polyclonal)
 Antibodies that arise from a single clone of cells
(e.g., in a plasma cell tumor [myeloma])are
homogeneous (monoclonal)
54

55. Hybridoma
Monoclonal antibodies made in laboratory by fusing a
myeloma cell with an antibody-producing cell are
called hybridoma
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56. Hybridoma
Hybridoma cells made as following
(1) A mouse immunized with antigen of interest
(2) Spleen cells from this mouse grown in a culture dish
in the presence of mouse myeloma cells
Myeloma cells grow indefinitely in culture, & do not
produce immunoglobulins
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57. Hybridoma
3)Fusion of cells by adding certain chemicals
(e.g., polyethylene glycol)
(4) Cells grown in a special culture medium(HAT
medium) that supports growth of fused, hybrid cells
but not of “parental” cells
(5) Resulting clones of cells screened for production of
antibody to antigen of interest
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58. Production of monoclonal antibodies
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