The document discusses the structure and function of antibodies, which are gammaglobulin proteins essential for humoral immunity. It details the five classes of antibodies (IgG, IgM, IgA, IgD, IgE), their specific roles, and the mechanisms by which they neutralize pathogens. Additionally, it covers antibody structure, including variable and constant regions, and the process of creating monoclonal antibodies in the laboratory.

1. ANTIBODIES
DR. ROMA GOYAL
ASSISTANT PROFESSOR
DEPARTMENT OF MICROBIOLOGY

2. Antibody
⚫ Gammaglobulin proteins that react
specificallywith antigen thatstimulated their
production
⚫ 20% of plasma protein
⚫Fiveclassesof antibodies:
⚫IgG, IgM, IgA, IgD, and IgE
(based on differences in heavychains)
2

3. Antibodies
3
⚫Afterstimulation, B cells
differentiate into plasma cells, &
secreteantibodies,
(immunoglobulins), mediators
of humoral immunity

4. Antibodies Characteristics
4
⚫Diversity Respond todifferentantigens
⚫Long memory Respond manyyearsafter initial
exposuredue to memory T cellsand B cells

5. Antibodies Characteristics
5
⚫Specificity Actions specificallydirected 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)

6. Functions of antibodies
6
⚫Neutralizetoxinsand viruses
⚫Opsonize microbes to beeasilyphagocytosed
⚫Activate Complement, and preventattachmentof
microbes to mucosal surfaces
⚫Catalytic :Antibody can act as an enzyme to catalyze
synthesisof ozone (O3) that has microbicidal activity.

7. Structure of immunoglobulin
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⚫Simplestantibody molecule has Y
shape
⚫Consistsof fourpolypeptidechains:
⚫ Two H chains and two L chains
⚫Fourchains linked by disulfide
bonds
⚫Antibody moleculealwaysconsists
of identical H chainsand
identical L chains

8. Structure of immunoglobulin
8
⚫L and H chains subdivided into variable
and constant
regions.
⚫Regions composed of three-
dimensionally folded,
repeating segments called domains
⚫Each domain isabout 110 aminoacids
long
⚫
⚫ globular in shape
⚫ stabilized by intrachain disulphide bonds
⚫ Antigen binding sites located in variable domains
Amino terminus
Variable region
Carboxy terminus
Constant region
Complement fixing

9. Structure of immunoglobulin
9
⚫Variableregions of lightand heavy chain
responsible for antigen-binding
⚫Constantregionof heavy chain responsible for
biologic functions (e.g., complementactivation
and binding tocell surface receptors)

10. Structure of immunoglobulin
10
⚫ Lightchain : Onevariable region and one
constant region
⚫ L chain attached to H chain by disulphide &
non- covalent bonds

11. Structure of immunoglobulin
11
⚫L chains belong tooneof two types, κ (kappa) orλ
(lambda), due to amino acid differences in their
constantregions
⚫ Both typesoccur in all classesof immunoglobulins
(IgG, IgM, etc.), but any one immunoglobulin
moleculecontainsonlyone typeof L chain

12. Structure of immunoglobulin
12
⚫Heavy chain consists of a variable region and a
constantregion divided into three domains: CH1, CH2,
and CH3
⚫Each domain 110 aminoacids long
⚫ CH2 domain contains complement-binding site
⚫CH3 domain is siteof attachment of IgG to receptors on
neutrophilsand macrophages
⚫ H chains structurally & antigenicallydistinct
foreach class
H chains are distinct foreach of five immunoglobulin
classesand aredesignated γ, α, μ, ε, and δ

13. Structure of immunoglobulin
13
⚫Variable regions of L and H chains have three
extremely variable(hypervariable) amino acid
sequencesat amino-terminal end that form antigen-
binding site
⚫Specificityof antibodies is due to hypervariable
regions

14. Structure of immunoglobulin
14
⚫Amino-terminal portionof each L & H chain
participates in antigen-binding site
⚫Carboxy terminal forms Fc fragment, which has
biologicactivities

15. Heavy chain
15
H chain designated by Greek letter.
Ig class H chain
Ig G 
Ig M 
Ig A 
Ig D 
Ig E 

16. Structure of immunoglobulin
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17. Structure of immunoglobulin
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Antibody molecule treated with a proteolyticenzyme 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 forvariouscells,& other biologicactivities

18. Enzymatic digestion – generates
various fragments
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⚫Papain digestion
⚫Cleave Ig above disulfide bridge of hinge region
⚫Results in 3 fragments each
⚫Two Fab fragments – soluble fragments which bind
to Ag
⚫One Fc fragment – insoluble, crystallised in cold

19. PAPAIN CLEAVAGE

20. Enzymatic digestion – generates
various fragments
⚫Pepsin digestion
⚫Cleaves Ig molecule at point below disulfide
bridge of hinge region
⚫One F(ab’)2 fragment; 2 Fab subunits bound
together
⚫Many smaller fragments

21. PEPSIN DIGESTION

22. 3D ANTIBODY STRUCTURE

23. Classification of antibodies
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⚫ImmunoglobulinA (IgA)
⚫Immunoglobulin G (IgG)
⚫Immunoglobulin M (IgM)
⚫Immunoglobulin D (IgD)
⚫Immunoglobulin E (IgE)
⚫ Based on structural differences in constant regionsof
heavychains
⚫ Classes havespecialized effector functions

24. Ig M
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⚫ 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.

25. 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
25
Promotes neutraliza-
tion and cross-
linking of antigens;
very effective in
complement system
activation
Function

26. ⚫ 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
Immunoglobulin G
(IgG)
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27. Distribution
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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

28. Ig A
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⚫ 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.

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

30. Ig E
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⚫ 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

31. Distribution
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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

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

33. 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)
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34. Primary & Secondary antibody response
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⚫Primary Response
⚫Following exposure toan antigen, there is a slowrise in IgM
followed bya slow rise in IgG
⚫Secondary Response
⚫Following exposure to previously encountered antigen,
there is a rapid rise in IgG and slowor no rise in IgM
⚫ Memoryoranamnestic response

36. Abnormal Immunoglobulins
⚫Bence Jones proteins
⚫Produced in neoplastic condition of plasma cells called
• Multiple Myeloma
⚫Also called light chain disease
⚫Cancerous plasma cells produce excess light chain
(BJP) accumulated in pt serum and urine
⚫Waldenstrom’s Macroglobulinemia
⚫B cell lymphoma, produce excess IgM
⚫Heavy chain disease
⚫Cryoglobulinemia
⚫Blood contains cryoglobulin
⚫Associated with Multiple Myeloma and Hepatitis C
infection

37. 37
⚫Antibodies produced in response to antigens are
heterogeneous, formed bydifferentclonesof plasma
cells ( polyclonal)
⚫Antibodies thatarise from asinglecloneof cells
(e.g., in a plasma cell tumor [myeloma])are
homogeneous (monoclonal)

38. Hybridoma
Monoclonal antibodies made in laboratory by fusing a
myelomacell with an antibody-producing cell are
called hybridoma
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39. Hybridoma
39
Hybridomacells madeas following
(1) A mouse immunized with antigen of interest
(2)Spleen cells from this mousegrown in a culturedish
in the presence of mouse myelomacells
Myelomacells grow indefinitely in culture, & do not
produce immunoglobulins

40. Hybridoma
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3)Fusion of cells byadding certain chemicals
(e.g., polyethyleneglycol)
(4)Cells grown in a special culture medium(HAT
medium) thatsupportsgrowthof fused, hybrid cells
but notof “parental” cells
(5)Resulting clonesof cells screened forproductionof
antibody to antigen of interest

41. Production of monoclonal antibodies
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