1. ANTIBODY STRUCTURE AND FUNCTION

2. WHAT ARE IMMUNOGLOBULINS?
Antigen specific proteins produced by B lymphocytes
Belong to immunoglobulin super family (BCR, TCR, MHC)
Bound to surface of B lymphocytes
Function as binding (receptor) sites for specific antigens
Antigen receptor sites on mature B lymphocytes
IgM
IgD
Membrane-bound form of immunoglobulins

3. WHAT IS THE IMMUNOGLOBULIN SUPERFAMILY
Proteins with structural feature first defined in immunoglobulins
Characteristic structural feature
Sequence of Domains providing stable conformation
Domain (loop 60 – 70 a.a.)
Polypeptide (100 to 110 amino acids) chain folded into sandwich (2 slices of bread) held together by disulfide bond
IG superfamily members
Antibodies, B cell receptors, T cell receptors, MHC molecules and others

4. The Making of an Agent How Antibodies are Generated
Activation of a B cell and Clonal Expansion
1) Antigen binds the
BCR on a B-cell,
activating it
2) B-cell begins to
divide (Clonal
Expansion), forming
either plasma cells
(antigen factories)
or memory B-cells.
Abs has diversity
Activated B-cells undergo clonal expansion, where they either become antibody factories called Plasma cells, or they differentiate into memory B-cells. Memory B-cells survive for years and are essential for long term immunity. This allows the immune system to remember an antigen and respond faster the next time it is faced with this antigen. 4

6. IgD
IgG, IgA
IgM

7. STRUCTURE OF ANTIBODIES
Antibodies are glycoproteins composed of
Polypeptide chains and carbohydrate
Monomeric structure
Polypeptide chains
2 identical heavy chains
2 identical light chains
Polypeptide chains joined by disulfide bonds
Carbohydrate

9. Switch region

10. STRUCTURE OF ANTIBODIES
Polypeptide chains have variable and constant regions
Variable
N (amino)-terminal of polypeptide chain
Antigen binding site
Constant
C (carboxyl)-terminal of polypeptide chain
Binding sites for cell surface receptors and complement
Structure represented by the letter “Y”
Y shaped molecule cleaved by protease papain
Fragment antigen binding (Fab)
Fragment crystallizable (Fc)

11. Cys, Prolin rich

13. Variable domain of AntibodiesVH
FR1
FR2
FR3
FR4
CDR1
CDR2
CDR3 VL

16. CLASSES (ISOTYPES) OF IMMUNOGLOBULINS
Classes based on constant region of heavy chains
Immunoglobulin A (IgA)
Immunoglobulin D (IgD)
Immunoglobulin E (IgE)
Immunoglobulin G (IgG)
Immunoglobulin M (IgM)
Differentiation of heavy chains
Length of C region, location of disulfide bonds, hinge region, distribution of carbohydrate
Classes have different effector functions

18. No hing
No hing

19. CLASSES (ISOTYPES) OF IMMUNOGLOBULINS
Additional classification based on light chains
Kappa 60% in human 90% in mice
Lambda 40 % in human and 10% in mice
Each IG has either kappa or lambda, not both
IgG kappa
IgG lambda
No functional differences between light chains

20. J chain

22. The Assignment Antibody Isotype
Mammals express 5 different isotypes of antibodies (IgA, IgD, IgE, IgG and IgM) with different functions and locations
Class of antibody is defined by the heavy chain
The Assignment: Isotypes
Mammals express five different isotypes of antibodies, known as: IgA, IgD, IgE,IgG and IgM. The "Ig" prefix stands for immunoglobulin and the letter denotes the class of antibody. Each isotype has different functions and locations. The heavy chain defines the class of antibody, i.e. if the antibody is an IgA, IgD, IgE, IgG, or an IgM. 22

23. The Assignment Antibody Isotype
Basic antibody is composed of 1 Ig unit, i.e. is a monomer
Some are dimeric (IgA) or pentameric (IgM)
Isotype changes during development of B-cell
The Assignment: Isotypes
The basic antibody is composed of 1 Ig unit, that is, it is a monomer. However, secreted antibodies can also be dimeric with two Ig units as in the case of IgA, or pentameric with five Ig units, like IgM.
Antibody isotype changes during development and with activation of the B-cells. Immature B-cells that have never been exposed to antigen are known as naïve B-cells and express only cell surface bound IgM. As the B-cell begins to reach maturity, it expresses both IgM and IgD. After reaching maturity, the B-cell is ready to interact with circulating antigen. Once an antigen is bound, the B-cell becomes activated and begins to divide and differentiate into the antibody factory, a plasma cell. In this activated form, the B-cell can now secrete antibody, allowing it to circulate in your blood and serum.
Some daughter cells of the activated B-cells undergo isotype switching, whereby we get production of IgE, IgA or, most commonly, IgG. 23

24. ANTIGEN BINDING SITES OF IMMUNOGLOBULINS
Antigen binding sites vary with size and shape of antigen
Part of antigen to which antibody binds
Antigenic determinant (Epitope)
Antigen-Antibody binding based on non-covalent forces
Hydrogen bonds
Affinity
Strength of binding of one molecule to another by a single binding site
Avidity
Overall strength of binding between two molecules

25. CONSTRUCTION OF LIGHT CHAIN AND HEAVY CHAIN VARIABLE REGIONS
Constructed from 2 segments
1 (V) segment
1 (J) segment
Heavy chain
Constructed from 3 segments
1 (D) segment

26. SURFACE IMMUNOGLOBULINS ASSOCIATED WITH PROTEINS TO COMPLETE ANTIGEN RECEPTOR
In ER, IgM and IgD associated with transmembrane proteins
Ig-alpha
Ig-beta
Transmembrane proteins
Transport M and D to B cell surface
Communication of antigen binding to inside of B cell
Tails interact with intracellular signaling molecules
Complex of IgM and IgD with Ig-alpha and Ig-beta forms
B-cell receptor

27. DIVERSIFICATION OF ANTIBODIES AFTER B-CELLS ENCOUNTER ANTIGEN
Following antigen activation of B-cells, additional diversification occurs in V domain by
Somatic hypermutation
Introduction of random single nucleotide substitutions (point mutations) throughout V regions of H and L chains
Mechanism poorly understood
More common in hypervariable regions (CDRs)

28. OUTCOME OF SOMATIC HYPERMUTATION
Gives rise to some antibodies with higher
Affinity for antigen
Affinity
Strength of binding of one molecule to another by a single binding site
Higher affinity antibodies are produced as immune response proceeds
Affinity maturation

29. THE PRIMARY HUMORAL IMMUNE RESPONSE
Immune response initially produces IgM antibodies then switches to IgG antibodies
Question
Why switch from IgM to IgG?
Answer
Limited effector mechanisms for IgM
Range of effector mechanisms for IgG
Mechanism
Isotype or class switching

30. ISOTYPE OR CLASS SWITCHING
Process by which B cell changes class of IG produced while preserving antigenic specificity
Involves somatic recombination which attaches different heavy chain constant region to variable region
Occurs only during active immune response
Mechanisms involves recombination between
Switch sequences (regions)

32. CLASSES, SUBCLASSES AND PHYSICAL PROPERTIES OF IMMUNOGLOBULINS
IgGA2m1, has no disulfid bond between L,H but has L,L. IgA2 has more sucrose than IgA1 , but the type of sucrose in IgA1 and IgD is N.Acetylgalactosamine
S-IgA is responsible for immune exclusion.

35. S IgA production in lamina properia milieu
The role of SC component in SIgA2m1 is protection of Ab because Ab dose not contain disulfide bond between H,L.
“Protease A” from N. gonorrhoeae, Strep. Sanguis, H.influenza, Strep. Pneumoniae digest hing region of IgA1. Hing is not in IgA2.

36. FUNCTIONS AND PROPERTIES OF ANTIBODY
Neutralization
Direct inactivation of pathogen or toxin thereby preventing its interaction with human cells
Opsonization
Coating of pathogens for more efficient phagocytosis
Activation of complement
More efficient phagocytosis
Direct killing
Gate Keeper
Increase the concentration of Ab and complement proteins out of the arteries.
Mucosal immunity
SIgA
Destructive roles
Immune complex disease, Auto immunity and Allergy

37. The Mission Antibody Function
Antibodies are the main component of the Humoral Immune System
They bind antigen and flag them for elimination via 1 of 3 ways:
Function
Antibodies constitute the main component of the humoral immune system. They circulate freely in the bloodstream and search for harmful foreign agents. Antibodies protect the immune system from invading antigens by binding to them and flagging them for elimination via one of three ways: 37

38. Viruses and intracellular bacteria require a
Neutralization:
Viruses and intracellular bacteria require a
host cell in order to replicate
Antibodies prevent their entry into the cell
by binding the antigen, making it harder
for it to pass through the cell membrane.
Antibodies cannot attack pathogens hidden
within cells
Neutralization: Viruses and intracellular bacteria require a host cell in order to replicate and pass on disease. These antigens generally enter the cell either by fusing with receptors on the cell. Antibodies prevent this action by binding to the antigen, making it harder for it to pass through the cell membrane. Antibodies cannot attack pathogens within cells.

39. antigen, antibodies flag the foreign agent for destruction
Opsonization:
Upon binding to an
antigen, antibodies flag the
foreign agent for destruction
or elimination by other
immune cells, such as natural
killer cells or macrophages
Opsonization: Upon binding to an antigen, antibodies flag the foreign agent for destruction or elimination by other immune cells, such as natural killer cells or macrophages.

40. Activation of Complement: Similar to opsonisation, antibody will
flag the antigen for elimination.
However, elimination is initiated by a
cascade of proteins which collect on
the cell membrane and form a hole,
leading to cell lysis
Activation of Complement: Similar to opsonisation, antibody will flag the antigen for elimination. However, elimination is initiated by a cascade of proteins which collect on the cell membrane and form a hole, leading to cell lysis.

41. IgM ANTIBODY OF THE IMMUNE RESPONSE
First isotype produced in primary response
May or may not be produced in secondary response
Produced before B cells undergo somatic hypermutation
Occurs as pentamer with J chain
Found primarily in blood and lymph
Multiple binding sites confers high avidity and compensates for low affinity of monomers
Highly effective in complement activation
Functions as rheumatoid factor

42. IgG ANTIBODY OF THE IMMUNE RESPONSE
Second isotype produced in primary response
Primary isotype of
Secondary immune response
Memory immune response
Represents approximately 75% of total serum IG
Four subclassses (1-4)
Different effector functions
Transported across placenta
Functions as rheumatoid factor

43. IgA ANTIBODY OF THE IMMUNE RESPONSE
Two subclasses (IgA1 and IgA2) and two forms (monomeric and dimeric)
Monomeric
Located in blood and extracellular spaces
Predominately IgA1
Ratio of IgA1 to IgA2 is 10:1
Functions as rheumatoid factor
Dimeric
Located in mucous membranes and secretions
Predominately IgA2
Ratio of IgA2 to IgA1 is 3:2
J chain like IgM

45. IgE and IgD ANTIBODIES OF THE IMMUNE RESPONSE
IgE (Gate keeper)
Binds with high affinity to receptors on mast cells, basophils and activated eosinophils
Longer half-life when cell bound
Initiates a strong inflammatory reaction to parasites
Involved in allergic reactions
IgD
Antigen receptor on mature B-cells
No other known function

46. Antigenic marker on Immunoglobulin

47. Idiotypic:,VH,VL
Allotypic:CH, CL
Isotypic: CH, CL

48. Network theory Idiotype determinants are immunogen for hosts.
Niels K.Jerne express that after increase in Ab level, the Ab stimulate anti- idiotype Ab that suppress Ab response against Antigen.
Anti Idiotype is produced in the high level of specific Ab.

49. Antibody Production
Antibodies are generated by immunizing (injecting) animals with purified antigen. The animal responds by producing antibodies that specifically recognize and bind to the antigen.
Antibody reagents may be polyclonal or monoclonal. 19

50. Polyclonal Antibodies
Polyclonal antibody reagents are produced as different classes of immunoglobulins by many B-cells clones and react with various epitopes on an antigen. (Figure 2)
They are more tolerant of small changes in the nature of the antigen since they often recognize multiple epitopes.
They may be generated in a variety of animals like rabbit, goat, sheep, horse, etc. The rabbit is the most commonly used animal for generating polyclonal antibodies. 21

51. Polyclonal Antibodies
Figure 2
Polyclonal antibodies reacting with various epitopes
Each antibody is made by a different B-cell 22

52. Polyclonal Antibody Production
antigen
A rabbit is injected (intradermally or subcutaneously) with a purified dose of antigen. 24

53. Monoclonal Antibodies
Monoclonal antibodies are derived from a single B-cell and are produced as a single class of immunoglobulin.
They are raised by fusion of the specific B-cells with immortal myeloma (B-cell) cancer cells to form a hybridoma.
A hybridoma can multiply indefinitely and continuously produce a specific monoclonal antibody.
They react with a specific epitope on a given antigen (Figure 3), giving less background staining. 30

54. Monoclonal Antibodies
Figure 3
Monoclonal antibodies reacting with similar epitopes 32

55. Monoclonal Antibody Production
spleen
B-lymphocytes
The antibody-producing B-cells are harvested from the spleen or lymph nodes. 36

56. Monoclonal Antibody Production
myeloma cells
B-lymphocytes
The B-cells are fused with mouse myeloma cells forming immortal hybrid cells or hybridomas. 37

58. Humanization of Antibodies
Mouse
Chimaeric
Humanized
Human

59. Bispecific antibodies Affibody Plastic antibody

60. Thanks