Humoral Immunity Antibody Mediated

1. Humoral ImmunityAntibody Mediated Dr.T.V.Rao MD Dr.T.V.Rao MD

2. Humoral Immunity • Results in production of proteins called “immunoglobulin's” or “antibodies”. • Body exposed to “foreign” material termed “antigen” which may be harmful to body: virus, bacteria, etc. • Antigen has bypassed other protective mechanisms, ie, first and second line of defense. Dr.T.V.Rao MD

3. 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 immunoglobulin's Dr.T.V.Rao MD

4. 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 Dr.T.V.Rao MD

5. Different classes of Antibodies Dr.T.V.Rao MD

6. Structural configuration of Antibody Dr.T.V.Rao MD

7. 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 Dr.T.V.Rao MD

8. Antibodies are Produced by B Lymphocytes Dr.T.V.Rao MD

9. Immunoglobulin Classes I. IgG • Structure: Monomer • Percentage serum antibodies: 80% • Location: Blood, lymph, intestine • Half-life in serum: 23 days • Complement Fixation: Yes • Placental Transfer: Yes • Known Functions: Enhances phagocytosis, neutralizes toxins and viruses, protects fetus and newborn. Dr.T.V.Rao MD

10. Immunoglobulin Classes II. IgM • Structure: Pentamer • Percentage serum antibodies: 5-10% • Location: Blood, lymph, B cell surface (monomer) • Half-life in serum: 5 days • Complement Fixation: Yes • Placental Transfer: No • Known Functions: First antibodies produced during an infection. Effective against microbes and agglutinating antigens. Dr.T.V.Rao MD

11. Immunoglobulin Classes III. IgA • Structure: Dimer • Percentage serum antibodies: 10-15% • Location: Secretions (tears, saliva, intestine, milk), blood and lymph. • Half-life in serum: 6 days • Complement Fixation: No • Placental Transfer: No • Known Functions: Localized protection of mucosal surfaces. Provides immunity to infant digestive tract. Dr.T.V.Rao MD

12. Immunoglobulin Classes IV. IgD • Structure: Monomer • Percentage serum antibodies: 0.2% • Location: B-cell surface, blood, and lymph • Half-life in serum: 3 days • Complement Fixation: No • Placental Transfer: No • Known Functions: In serum function is unknown. On B cell surface, initiate immune response. Dr.T.V.Rao MD

13. Immunoglobulin Classes V. IgE • Structure: Monomer • Percentage serum antibodies: 0.002% • Location: Bound to mast cells and basophils throughout body. Blood. • Half-life in serum: 2 days • Complement Fixation: No • Placental Transfer: No • Known Functions: Allergic reactions. Possibly lysis of worms. Dr.T.V.Rao MD

14. 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 Dr.T.V.Rao MD

15. Antigen- binding site Antigen- binding site Disulfide bridge V V V V Variable regions Light chain C C Constant regions C C Transmembrane region Plasma membrane Heavy chains B cell Cytoplasm of B cell (a) A B cell receptor consists of two identical heavy chains and two identical light chains linked by several disulfide bridges. B Cell Receptors for Antigens • B cell receptors • Bind to specific, intact antigens • Are often called membrane antibodies or membrane immunoglobulin's Dr.T.V.Rao MD

16. Antibodies bond to antigenic determinants Antigenic determinants are portions of the antigen Dr.T.V.Rao MD

17. Antibodies are Proteins that Recognize Specific Antigens Dr.T.V.Rao MD

18. Epitopes: Antigen Regions that Interact with Antibodies Dr.T.V.Rao MD

19. Dynamics of Antibody Production • Primary immune response • Latent period • Gradual rise in antibody production taking days to weeks • Plateau reached • Antibody level declines Dr.T.V.Rao MD

20. Secondary Response • Second exposure to SAME antigen. • Memory cells are a beautiful thing. • Recognition of antigen is immediate. • Results in immediate production of protective antibody, mainly IgG but may see some IgM Dr.T.V.Rao MD

21. Dynamics of Antibody Production • Antibody production • Initial antibody produced in IgM • Lasts 10-12 days • Followed by production of IgG • Lasts 4-5 days • Without continued antigenic challenge antibody levels drop off, although IgG may continue to be produced. Dr.T.V.Rao MD

22. 1 Secondary response to anti- gen A produces antibodies to A; primary response to anti- gen B produces antibodies to B Day 1: First exposure to antigen A 4 Primary response to antigen A produces anti- bodies to A 2 Day 28: Second exposure to antigen A; first exposure to antigen B 3 104 103 Antibody concentration (arbitrary units) 102 Antibodies to A Antibodies to B 101 100 35 28 21 42 49 56 0 14 7 Time (days) • In the secondary immune response • Memory cells facilitate a faster, more efficient response Dr.T.V.Rao MD

23. 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 • Isotope or class switching Dr.T.V.Rao MD

24. Humoral (antibody-mediated) Immunity IL 1 IL 2 Autocrine stimulation Dr.T.V.Rao MD

25. 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) Dr.T.V.Rao MD

26. Antigen molecules bind to the antigen receptors of only one of the three B cells shown. Antigen molecules B cells that differ in antigen specificity Antigen receptor The selected B cell proliferates, forming a clone of identical cells bearing receptors for the selecting antigen. Some proliferating cells develop into short-lived plasma cells that secrete antibodies specific for the antigen. Some proliferating cells develop into long-lived memory cells that can respond rapidly upon subsequent exposure to the same antigen. Antibody molecules Clone of memory cells Clone of plasma cells • Clonal selection of B cells • Generates a clone of short-lived activated effector cells and a clone of long-lived memory cells Dr.T.V.Rao MD

27. Humoral (antibody-mediated) Immunity Memory Cells Dr.T.V.Rao MD

28. Benefits of Immunological Memory Dr.T.V.Rao MD

29. Clonal Selection Only one type of antibody—and one type of B cell—responds to the antigenic determinant That cell type then produces a large number of clones Dr.T.V.Rao MD

30. 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 Dr.T.V.Rao MD

31. 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 Dr.T.V.Rao MD

32. DIVERSIFICATION OF ANTIBODIES AFTER B-CELLS ENCOUNTER ANTIGEN • Following antigen activation of B-cells, additional diversification occurs in V domain by • Somatic hyper mutation • Somatic hyper mutation • Introduction of random single nucleotide substitutions (point mutations) throughout V regions of H and L chains • Mechanism poorly understood • More common in hyper variable regions (CDRs) Dr.T.V.Rao MD

33. 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 Dr.T.V.Rao MD

34. 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 Dr.T.V.Rao MD

35. 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 subclasses (1-4) • Different effector functions • Transported across placenta • Functions as rheumatoid factor Dr.T.V.Rao MD

36. 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 Dr.T.V.Rao MD

37. IgE AND IgD ANTIBODIES OF THE IMMUNE RESPONSE • IgE • 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 Dr.T.V.Rao MD

38. Immunological Memory Antibody Titer: The amount of antibody in the serum. Pattern of Antibody Levels During Infection Primary Response: • After initial exposure to antigen, no antibodies are found in serum for several days. • A gradual increase in titer, first of IgM and then of IgG is observed. • Most B cells become plasma cells, but some B cells become long living memory cells. • Gradual decline of antibodies follows. Dr.T.V.Rao MD

39. Immunological Memory (Continued) Secondary Response: • Subsequent exposure to the same antigen displays a faster and more intense antibody response. • Increased antibody response is due to the existence of memory cells, which rapidly produce plasma cells upon antigen stimulation. Dr.T.V.Rao MD

40. 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) Dr.T.V.Rao MD

41. Programme Created By Dr.T.V.Rao MD for basic learning for Medical and Paramedical Students in the Developing World • Email • doctortvrao@gmail.com Dr.T.V.Rao MD