1. ANTIBODY STRUCTURE AND THE GENERATION OF B-CELL DIVERSITY

2. WHAT ARE ANTIBODIES?
Antigen specific proteins produced by plasma cells
Belong to immunoglobulin superfamily
Located in blood and extravascular tissues, secretions and excretions
Bind pathogenic microorganism and their toxins in extracellular compartments
Secreted form of immunoglobulins

3. WHAT ARE IMMUNOGLOBULINS?
Antigen specific proteins produced by B lymphocytes
Belong to immunoglobulin superfamily
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

5. WHAT IS THE IMMUNOGLOBULIN SUPERFAMILY
Proteins with structural feature first defined in immunoglobulins
Characteristic structural feature
Sequence of Domains providing stable conformation
Domain
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

6. 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

7. 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)

10. 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

12. CLASSES (ISOTYPES) OF IMMUNOGLOBULINS
Additional classification based on light chains
Kappa
Lambda
Each IG has either kappa or lambda, not both
IgG kappa
IgG lambda
No functional differences between light chains

13. IT’S GREEK TO ME
Heavy chains, light chains and other molecules of the immune system identified using letters of the Greek alphabet

16. THREE DIMENSIONAL STRUCTURE OF ANTIBODIES
Antibodies function in setting of infectious process
Proteolytic enzymes, salt and pH differences
Antibodies remain stable based on
Sequence of domains
Single domain consists of
100 – 110 amino acids folded into compact and stable conformation
Domains
Variable (V)
Single V domain in H and L chains
Constant (C)
Single C domain in L chains
Three to four (C) domains in H chains

18. ANTIGEN BINDING SITES OF IMMUNOGLOBULINS
Antigen binding sites formed from hypervariable regions
Heavy chain V domain
Light chain V domain
Hypervariable regions of V domains
Amino acid sequence differences concentrated
Flanked by less variable framework regions
Three hypervariable regions in each V domain
Hypervariable regions also called
Complementarity-determining regions (CDR)

20. 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

22. ANTIBODIES AS DIAGNOSTIC AND THERAPEUTICS AGENTS
Based on specificity and affinity of antibodies
Both applications require large quantities of identical antibodies
Monoclonal antibodies
Monoclonal antibodies are produced using hybridoma cell line
Hybridoma cell line
Derived from single antibody producing cell fused with myeloma cell (neoplastic plasma cell)

24. IMMUNOGLOBULIN DIVERSITY IN B-CELLS BEFORE ENCOUNTER WITH ANTIGEN
Immune system capable of producing a limitless number of different immunoglobulins/antibodies
Mechanism
Genes for IG organized differently
Genes exist as nonfunctional segments
Variable (V), Joining (J), Diversity (D), Constant (C)
Genes are inherited in this form
Germline form (germline configuration)

25. IMMUNOGLOBULIN DIVERSITY IN B-CELLS BEFORE ENCOUNTER WITH ANTIGEN
Expression
Gene segments must be rearranged into functional gene
Gene Rearrangement
Takes place during development of B-cells
Mechanism of somatic recombination
Genes for IG located at 3 chromosomal locations
Heavy chain locus on chromosome 14
Kappa light chain locus on chromosome 2
Lambda light chain locus on chromosome 22

26. GERMLINE ORGANIZATION OF HUMAN IG HEAVY CHAIN AND LIGHT CHAIN LOCI
Lambda light chain locus
Gene segments
30 (V), 4 (J) and 4 (C)
Kappa light chain locus
40 (V), 5 (J) and 1 (C)
Heavy chain locus
65 (V), 27 (D), 6 (J) and 9 (C)

28. 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

30. SOMATIC RECOMBINATION
Performed by enzymes with cut and rejoin DNA
Directed by
Recombination signal sequences (RSS)
Recombination signal sequences
Recognition sites for enzymes
Recombination occurs between different types
9 / 12 / 7
9 / 23 / 7
Mechanism follows the 12/23 rule
Ensures segments joined in correct order

33. MECHANISMS OF GENETIC DIVERSITY IN V-REGION OF IMMUNOGLOBULINS
Random combination of
V and J segments in light chain genes
V, D and J segments in heavy chain genes
Addition of P (palindromic) and N (non-templated) nucleotides at junctions of gene segments during recombination
Junctional diversity
Association of H and L chains in different combinations

35. CONSTRUCTION OF B-CELL SURFACE IMMUNOGLOBULINS
Following rearrangement of VH gene segments, two CH loci are transcribed
IgM
IgD
M and D constant segments
Located nearest variable segments
M and D transcript processed by
Cleavage, polyadenylation and splicing
IgM and IgD enter endoplasmic reticulum

38. 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

40. DIVERSIFICATION OF ANTIBODIES AFTER B-CELLS ENCOUNTER ANTIGEN
Mature, naïve B cell has membrane bound IgM and IgD antigen receptors
Binding of antigen initiates proliferation and differentiation of B-cells into plasma cells
During differentiation, B cells switch from making immunoglobulin to antibody M and D isotypes
IgM
Produced in large amounts
Provides protective immunity
IgD
Produced in small amounts
No known function

41. MECHANISM OF SWITCHING FROM IMMUNOGLOBULIN TO ANTIBODY
Surface and secreted forms derived from same heavy chain gene by alternative RNA processing
Each heavy chain C gene has
Membrane coding (MC) region
Secretion coding (SC) region
Mechanism involves a switch in cleavage, polyadenylation and splicing
From pAm region to pAs region

43. 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)

44. 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

45. 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

47. 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)

49. CLASSES, SUBCLASSES AND PHYSICAL PROPERTIES OF IMMUNOGLOBULINS
Subclasses are numbered according to plasma concentration

51. 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

53. 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

54. 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

55. 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

57. IgE AND IgD ANTIBODIES OF THE IMMUNE RESPONSE
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