LECTURE FOR MBBS, IMMUNOLOGY ANTIBODY React specifically & in some observable manner with Ag which stimulated their production  globulins produced in response to antigenic stimulation

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

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

5. Antibodies Characteristics
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⚫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
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⚫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
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⚫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
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⚫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
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⚫ Lightchain : Onevariable region and one
constant region
⚫ L chain attached to H chain by disulphide &
non- covalent bonds

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

15. Heavy chain
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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
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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
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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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