This document provides an overview of antibodies (immunoglobulins). It discusses the basic structure of antibodies, including their Y-shape and polypeptide chains. There are five classes of antibodies (IgG, IgA, IgM, IgD, IgE) which have different properties and functions. The document also describes how antibodies carry out their effector functions, such as activating the complement system, opsonization to promote phagocytosis, and neutralization of toxins and pathogens.

1. ANTIBODY
Presented By- Sunita
PH.D (Ist year)
MBGE Deptt.

2. CONTENTS
 Introduction
 Properties of antibody
 Basic structure of antibody
 Types of antibody
 Class of antibody
 Enzymatic experiments with antibody
 Effectors function of antibody
 Conclusion

3. INTRODUCTION
 Antibodies are the antigens binding proteins present on B cell
membrane and secreted by plasma cells.
 Antibodies are active serum proteins formed in response to an
antigen and react specifically with that antigen.
 All antibodies are immunoglobulin's but all immunoglobulin's
may not be antibodies.

4. PROPERTY OF ANTIBODY
 Antibody is a glycoprotein.
 Antibody (Ab) also know as Immunoglobulin (Ig).
 Antibody is a “Y” shaped molecules.
 Antibody is made up of four polypeptide chain.
 Antibody is heterodimeric structure.
 Antibodies contain disulphide bonds.
 Each light chain is made of 214 Amino Acids.
 Each heavy chain is made up of 450 – 700 Amino acid.

5. STURCTURE OF ANTIBODY
 Antibodies are heavy (~150 kDa) globular plasma proteins.
 There are four polypeptide chains:-
 Two identical heavy chain and two light chains connected by
disulfide bonds.
 Light Chain (L) consists polypeptides of about 22,000 Da.
 Heavy Chain (H) consists larger polypeptides of around 50,000
Da or more.
 There are five types of Ig heavy chain (in mammal) denoted by
the Greek letters: α, δ, ε, γ, and μ.
 There are two types of Ig light chain (in mammal), which are
called lambda (λ) and kappa (κ).

6. STRUCTURE OF ANTIBODY

7.  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

8.  An antibody is made up of a variable region and constant.
 CONSTANT REGION:- The region that has a constant structure
is called the constant region.
 Constant region in the c-terminal end.
 VERIABLE REGION:- The region show the variability is called
the variable region, variable region is located at the
extremity in the N terminal end.
 COMPLEMENTARITY DETERMINING REGIONS: antigen
binding site is complimentarily to the structure of epitope these
area are called CDR.
 Framework :-the VL and VH domain exhibit,less variation
these stretches are called framework.

9. HEAVY AND LIGHT CHAIN
 All 4 Heavy and Light chains are bound to each other by
disulfide bonds and non covalent interactions such as salt
linkages.
 All chains have 2 ends– an amino terminal end (NH3) and a
carboxy terminal end (COOH).
 There are 5 classes of Heavy chains and 2 classes of L chains.

10. DIFFRENCE BETWEEN HEAVY AND
LIGHT CHAIN
 5 classes
 Each designated by Greek
letter
 5 classes of Ig (IgG,
IgA, IgM, IgD and IgE)
classified based on AA
sequence of heavy
chains
 2 types
 Kappa (κ) and lambda (λ)
 In humans, L chains, 60%
kappa and 40% lambda
 Both light chains of Ab
molecule should be same
type, either κ or λ, never
both
HEAVY CHAIN LIGHT CHAIN

11. TYPE OF ANTIBODIES
 Isotype :- It is distinct form of heavy and light chain which
Present in all member of species, encoded at distinct genetic
loci. (Kappa & lambda and µ,δ).
 Idiotype :- A single antigenic determinant in variable domains
of antibody or T-Cell receptor also called as idiotypic
determinant . (VH and VL )
 Allotype :- An antigenic determinant that varies among
member of a species. The constant region of antibodies possess
allotypic determinant.

13. ENZYMATIC DIGESTION-GENERATES
VARIOUS FRAGMENT
 Cleave Ig above
disulfide bridge of hinge
region.
 Results in 3 fragments
each.
 Two Fab fragments
soluble fragments which
bind to Antigen.
 One Fc fragment
insoluble, crystallized in
cold.
 Cleaves Ig molecule at
point below disulfide
bridge of hinge region.
 One F(ab’)2 fragment;
2 Fab subunits bound
together.
 Many smaller fragments.
Mercaptoethanol:- Reduction
(Eliminates Disulfide Bonds)
And Alkylation Showed.
Papain digestion Pepsin digestion

15. IMMUNOGLOBULIN CLASSES
 Based on 5 types of heavy
chains = 5 classes of Ig
 IgG, IgA, IgM, IgD and IgE

16. IMMUNOGLOBULIN IgG
IgG
 70-80% of total Ig in body.
 Maximum daily production, longest half life of 23 days.
 Highest serum concentration.
 Has 4 subclasses – IgG1, IgG2, IgG3 and IgG4, all different
Subclasses vary in biological function, length of hinge region
and No. of disulfide bridges.

17. TYPES OF IgG

18. FUNCTION
 Can cross placenta: provide immunity to fetus and newborn.
 Plays major role in neutralization of toxins as it can easily
diffuse into extra vascular space.

19. IgM
 5% to10% of total serum immunoglobulin
 IgM is pentameric structure
 IgM is first Ig class produce in a primary response
 Highest molecular weight(180,000 Da)
 Not in body fluids or secretions

20. IgM

21. FUNCTION
 Acute infection: 1st Ab to be produced following infection. Represents
acute or recent infection. Also called primary immune response Ab
 Present on B cell surface. Serves as B cell receptor for Ag binding
 Acts as Opsonin: binds as antigen which is then easily recognized
and removed
 Fetal immunity: 1st Ab to be synthesized in fetal life.

22. IgA
 Second most abundant Ig molecules
 Constitutes 10-15% of total serum Ig
 IgA are the dimeric structure
 2 subclasses: IgA1 and IgA2
 Serum IgA and Secretary IgA
 All J-chain containing IgA are called secretory IgA.

23. IgA FUNCTION
 Dimeric in nature
 Also secretory component present
 Location: found in milk, saliva, tears, etc
 Function: mediates local or mucosal immunity
 Effective against bacteria like Salmonella, Vibrio, etc
 Breast milk rich in Secretory IgA; protect to infant

24. IgE
 Lowest serum concentration,0.3%
 shortest half life and minimum daily production
 life span of IgE is 1 to 5 days

25. IgD
 First discovered Ig molecules
 Total serum Ig 0.2%
 Life span of IgD immunoglobulin is 2 to 8 days
 Membrane bound immunoglobulin express by
mature B cell

27. EFFECTOR FUNCTION OF ANTIBODY
 Antibody do not kill or remove pathogen solely by
binding to them then effectors function and antibody are
effect the pathogen that result the removal the pathogen
and death.
 COMPLEMENT SYSTEM
 OPSONIZATION
 NEUTRILIZATION
 ADCC (ANTIBODY-DEPENDENT-CELL-MEDIATED-CYTOIXICITY)

28. COMPLEMENT SYSTEM
 A collection of antiserum glycoprotein called the complement.
 The complement activation pathway is a protein fragment
called c3b
 Complement system represents a group of about 30 proteins.
 The activation of the complement cascade may be initiated by
several protein that circulate in our body.
 The complement works as a cascade system.

29.  Their activation via major three pathways.
 Classical pathway:-antibody dependent pathway and
triggered by formation of soluble antigen-antibody complex
or by binding of the antibody to the antigen present on the
target cell surface.
 Alternative pathway:- antibody independent pathway
stimulated by antigen directly e.g.. Bacterial cell surface
components.
 Mannose binding Lectin pathway:-Also antibody
independent but resembles classical pathway.

32. Classical pathway
 The formation of antigen and antibody complex(immune
complex).
 The initiation stage of activation involves components
C1,C2,C3,C4 which are present in plasma inactive form.
 Three subcomponent of C1 –C1q,C1r,C1s.
 C1q molecules are 18 polypeptide chain that associate to
form six collagen-triple helical arm.
 C1q binding site in CH2 domain in fc portion of antibody
molecule.
 Each C1r and C1s are monomer contain catalytic domain.

33. The building of a C3 activation
complex
 Once C1 is activated, it activates 2 other complement proteins,
C2 and C4 by cutting them in half.
 C2 is cleaved into C2a and C2b.
 C4 is cleaved into C4a and C4b.
 Both C2a and C4b bind together on the surface of the bacteria
C2b and C4a diffuse away.

34. C3 Activation complex
 C2a and C4b bind together on the surface to form a
C3 activation complex.
 The function of the C3 activation complex is to
activate C3 proteins.
 This is done by cleaving C3 into C3a and C3b.

35.  C3b is an opsonin
 Opsonins are molecules that bind both to bacteria
and phagocytes.
 Opsonization increases phagocytosis by 1,000
fold.
C3b

36. c3a
 C3a increases the inflammatory response by binding
to mast cells and causing them to release histamine.

37. Building the C5 activation complex
 Eventually enough C3b is cleaved that the surface
of the bacteria begins to become saturated with it.
 C2a and C4b which make up the C3 activation
complex .
 When C3b binds to C2a and C4b it forms a new
complex referred to as the C5 activation complex.

38. The C5 activation complex
 The C5 activation complex (C2a, C4b, C3b)
activates C5 proteins by cleaving them into C5a
and C5b
 Many C5b proteins are produced by the
C5activation complex. These C5b begin to coat the
surface of the bacteria.

39. The function of C5a
 C5a disperses away from the bacteria.
 Binds to mast cells and increases inflammation.
 Most powerful chemotactic factor known for
leukocytes

40. Building the Membrane Attack
complex
 C5b on the surface of bacteria binds to C6
 The binding of C6 to C5b activates C6 so that it
can bind to C7
 C7 binds to C8 which in turn binds to many C9’s
 Together these proteins form a circular complex
called the Membrane attack complex (MAC)

41. Membrane Attack complex
 The MAC causes Cytolysis.
 The circular membrane attack complex acts as a
channel in which cytoplasm can rush out of and
water rushes in.
 The cells inner integrity is compromised and it dies

42. Alternative pathway
 The alternative pathway is part of the non-specific
defense because it does not need antibodies to
initiate the pathway.
 The alternative pathway is slower than the Classical
pathway

43. Initiation of The Alternative pathway
 C3 contains in
unstable thioester
bond.
 This unstable bond
makes C3 subject to
slow spontaneous
hydrolysis to C3b and
C3a
 The C3b is able to
bind to foreign
surface antigens.

45. FACTOR B
 C3b on the surface
of a foreign cells
binds to another
plasma protein
called factor B

46. Factor D
 The binding of C3b
to factor B allows a
protein enzyme
called Factor D to
cleave Factor B to
Ba and Bb.
 Factor Bb remains
bound to C3b while
Ba and Factor D
disperse away.

47. The C3 activation complex
 Properdin, also called factor P, binds to the
C3bBb complex to stabilize it.
 C3bBbP make up the C3 activation complex
for the alternative pathway

48. C3 ACTIVATION COMPLEX
 The C3 activation
complex causes the
production of more
C3b.
 This allows the initial
steps of this pathway
to be repeated and
amplified
 2X106 molecules can
be generated in 5
minutes

49. C5 ACTIVATION
 When an additional
C3b binds to the C3
activation complex it
converts it into a C5
activation complex.
 The C5 activation
complex cleaves C5 into
C5a and C5b.
 C5b begins the
production of the MAC.

50. LECTIN MANNOSE PATHWAY
 First step is binding of Mannose
Binding Lectin (MBL) to mannose
residue on the surface of microbes.
 To the MBL bound to microbe, MBL
–Associated Serine Proteases
(MASP-1and MASP-2) will bind.
 MASP 1 and 2 is similar in structure
and function to C1s and C1r
 This complex will cleave C4 and
C2

51. Lectin Binding Pathway
 Independent of antibodies.
 Lectins are proteins that recognize and bind to
specific carbohydrate targets.
 Activated by Mannose binding lectin (MBL) – lectin
that binds to mannose residues on the microbes.
 MBL is similar to C1q in structure and function.

52.  The lectin pathway is activated by the binding of
mannose binding lectin (MBL) to mannose residue on
glycoprotein or carbohydrate on the surface of
microorganisms.
 MBL is a acute phase protein and its concentration
increase during inflammatory response.
 Its function in the complement pathway similar to
C1q.
 After MBL binds to cb residue on the surface of
pathogen.

53.  MBL associate serine protease,MASP-1and MASP-2
Bind to MBL.
 The active complex formed by this association cause
cleavage and activation of C4 and C2.
 MASP-1 and MASP-2 similar to C1r and C1s.
 This means C2 and C4 form a C5 convertase .

55. Opsonisation
Opsonization → enhance the activity of phagocytosis.
 Promotion of phagocytosis of antigen by
macrophage and neutrophils.
 Protein molecules called fc receptor which can bind
the constant region of Ig molecules that are present
on the surface of macrophage and neutrophils.
 The principal opsonins are IgG opsonin and C3b
opsonin.

57. phagocytosis
 Phagein = "to eat", cytos = "cell“,
 osis = “process”
 Phagocytosis is the process of engulfment and
destruction of foreign particle particles such as
bacteria.

58. ENGULFMENT STAGE

59. Neutralization
 The antibody bind to specific microbe or microbial
toxin.
 Block pathogen entry into the cell and neutralize their
infectivity.
 Microbes and microbial toxin bind to surface molecule
of host.
 Antibody bind to the pathogen.
 Neutralization encourage or prevent a pathogen form
initiating an infection.

60. single-chain variable fragment (scFv)
 An scfv fragment consist of the smallest functional antigen –
binding domain of an antibody ( 30 kDa).
 The variable heavy (VH)and variable light(VL) chains are
joined together by a flexible peptide linker (fusion protein).
 A short linker peptide of ten to about 25 amino acids.
 The linker is usually rich in glycine for flexibility,
 They are bind to the epitope of antigen.
 The two-hybrid system, phage display is used for the high-
throughput screening of protein interactions. ( determination of
interaction partners of a protein).

61. Antibody dependent cell mediated
cytotoxicity
 The linking of antibody bound to target cell with fc receptor.
 a number of cell type NK cell can direct the cytotoxic
activities of the effectors cell against the target cell this
process is called antibody dependent cell mediated
cytotoxicity.
 When macrophage, neutrophils bind to target cell way of fc
receptor and that result the level of lytic enzyme.
 Their cytoplamic lysosomes and release of these lytic enzyme.
At the site of the fc mediated contact may in damage to target
cell ,start the process of degranulation.
 Macrophages NK cell have been secrete tumor nacrosis factor.
(TNF)

63. THANK YOU.