Immunoglobuins & their functions

Immunoglobuins & their functions

1. IMMUNOGLOBULINS AND THEIR FUNCTIONS PRESENTED BY : roshni maurya

2. WHAT ARE ANTIBODIES? (Ab) Proteins that recognize and bind to a particular antigen with very high specificity . Made in response to exposure to the antigen. One virus or microbe may have several antigenic determinant sites , to which different antibodies may bind. Each antibody has at least two identical sites that bind antigen: Antigen binding sites . Valence of an antibody: Number of antigen binding sites. Most are bivalent. Belong to a group of serum proteins called immunoglobulins (Igs).

3. WHAT ARE IMMUNOGLOBULINS ?(Ig) In 1964,endorsed by WHO, the generic term ‘ immunoglobulin’ was internationally accepted for ‘ proteins of animal origin endowed with known antibody activity and for certain proteins related to them by chemical structure.’ Ig are synthesized by plasma cells, to some extent by lymphocyte. Ig provide a structural and chemical concept, while the term ‘antibody’ is a biological and functional concept. All Ab are igs, but all igs may not be antibodies. Constitute 20-25% of total serum proteins.

4. 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

5. 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

6. 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 F ragment a ntigen b inding (Fab) F ragment c rystallizable (Fc)

7. H chains are distinct for each of the five Ig classes or isotypes and are designated γ α μ δ ε for the respective classes of Ig , namely IgG, IgA ,IgM , IgD IgE. L chains are one of two types designated κ and λ and only one type is found in Ig. Human Immunoglobulin Light Chain Types Kappa Lambda

8. L and H chains are subdivided into variable and constant regions. The regions are composed of three-dimensionally folded, repeating segments called domains. An L chain consists of one variable (VL) and one constant (CL) domain. Most H chains consist of one variable (VH) and three constant(CH) domains.(IgG and IgA have three CH domains,whereas IgM and IgE have four.)

9. The variable regions are responsible for antigenbinding ,whereas the constant regions are responsible for various biologic functions eg, complement activation and binding to cell surface receptors . The variable regions of L have three extremely variable (“ hypervariable / hot spots ”) amino acid sequence at the amino-terminal end that form the antigen-binding site .H chain have 4 such region.

10. Immunoglobulin Fragments: Structure/Function Relationships Ag Binding Complement Binding Site Placental Transfer Binding to Fc Receptors

11. Structure of the Variable Region Hypervariable (HVR) or complementarity determining regions (CDR) FR1 FR2 FR3 FR4 HVR1 HVR2 HVR3 Variability Index 25 75 50 100 Amino acid residue 150 100 50 Framework regions

12. A. Immunoglobulin classes - The immunoglobulins can be divided into 5 different classes based on differences in the amino acid sequences in the constant region of the heavy chains. All immunoglobulins within a given class will have very similar heavy chain constant regions. These differences can be detected by sequence studies or more commonly by serological means (i.e. by the use of antibodies directed to these differences). 1. IgG - Gamma   heavy chains 2. IgM - Mu   heavy chains 3. IgA - Alpha heavy chains 4. IgD - Delta   heavy chains 5. IgE - Epsilon   heavy chains Immunoglobulin Classes, Subclasses, Types and Subtypes

13. . Immunoglobulin Subclasses - The classes of immunoglobulins can de divided into subclasses based on small differences in the amino acid sequences in the constant region of the heavy chains. All immunoglobulins within a subclass will have very similar heavy chain constant region amino acid sequences. Again these differences are most commonly detected by serological means. 1. IgG Subclasses a) IgG1 - Gamma 1   heavy chains b) IgG2 - Gamma 2   heavy chains c) IgG3 - Gamma 3   heavy chains d) IgG4 - Gamma 4   heavy chains 2 . IgA Subclasses a) IgA1 - Alpha 1   heavy chains b) IgA2 - Alpha 2   heavy chains  

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

15. C . Immunoglobulin Types - Immunoglobulins can also be classified by the type of light chain that they have. Light chain types are based on differences in the amino acid sequence in the constant region of the light chain. These differences are detected by serological means. 1. Kappa light chains   2. Lambda light chains   D. Immunoglobulin Subtypes - The light chains can also be divided into subtypes based on differences in the amino acid sequences in the constant region of the light chain. 1. Lambda subtypes a) Lambda 1   b) Lambda 2   c) Lambda 3   d) Lambda 4   E. Nomenclature - Immunoglobulins are named based on the class, or subclass of the heavy chain and type or subtype of light chain. Unless it is stated precisely you are to assume that all subclass, types and subtypes are present. IgG means that all subclasses and types are present. F. Heterogeneity - Immunoglobulins considered as a population of molecules are normally very heterogeneous because they are composed of different classes and subclasses each of which has different types and subtypes of light chains. In addition, different immunoglobulin molecules can have different antigen binding properties because of different V H and V L regions.  

16. 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

17. IgG Structure Monomer (7S) IgG1, IgG2 and IgG4 IgG3

18. IgG Properties Major serum Ig Major Ig in extravascular spaces The only antibody to cross the placenta Fixes complement Binds to Fc receptors Phagocytes - opsonization NK cells – ADCC

19. drmsaiem BIOLOGICAL & CHEMICAL PROPERTIES OF Igs IgG. Is the major immunoglobulin in human serum, accounting for approximately 80%. Concentration of approximately 12 mg/ml IgG is a monomer consisting of identical pairs of H and L chains linked by disulfide bridges . Four subclasses of IgG have been identified, based on H chain differences: IgG1, IgG2, IgG3, and IgG4. IgG is the only immunoglobulin that can cross the placenta in humans and protect the infant during the first months of life. IgG molecules are capable of binding complement by the classical pathway (except for the IgG4, which activate by the alternative pathway).

20. drmsaiem BIOLOGICAL AND CHEMICAL PROPERTIES OF IMMUNOGLOBULINS IgG is the major antibody produced in the secondary immune response IgG has a half-life of approximately 23 days Effective antitoxic immunity is exclusively IgG. IgG is the major opsonizing immunoglobulin in phagocytosis; neutrophils have receptors for the Fc fragments of IgG I and IgG 3 . IgG appears late but persists for longer period. It appears after initial response which is IgM in nature.

21. IgM Structure Pentamer (19S) composed 5 H2L2 units plus one molecule of J chain Extra domain (C H4 ) J chain C 4 J Chain

22. IgM Properties 3rd highest serum Ig First Ig made by fetus and B cells Produced early in the primary response The most efficient Ig Fixes complement Tail Piece Agglutinating Ig Binds to Fc receptors B cell surface Ig

23. 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 . Represents about 8% to 10% of the total serum Igs . Concentration of ≈ I.2 mg/ml., half life is 5 days

24. Fixation of C1 by IgG and IgM Abs C1r C1s C1q C1r C1s C1q No activation Activation

25. IgA Structure Serum – monomer Secretions (sIgA) Dimer (11S), sIgA molecule consists of two H2L2 units plus one molecule each of J chain and secretory component(SC or SP) J Chain Secretory Piece

26. 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

27. Origin of sIgA: The SP is a polypeptide synthesized by epithelial cells that provides for IgA passage to the mucosal surface. It also protests IgA from being degraded in the intestinal tract. Y Y Y Y Y Y

28. IgA Properties 2nd highest serum Ig Major secretory Ig ( saliva, tears, respiratory, intestinal, and genital tract secretions.) Does not fix complement unless aggregated Binds to Fc receptors on some cells

29. IgD Structure Monomer Tail piece Tail Piece

30. IgD Properties 4th highest serum Ig B cell surface Ig Does not bind complement

31. IgE Structure Monomer Extra domain (C H4 ) C 4

32. IgE Structure Properties Least common serum Ig Allergic reactions Parasitic infections Does not fix complement

33. IgG IgA IgM IgD IgE Sedimentation coefficient (S) 7 7 19 7 8 Molecular weight 150,000 160,000 900,000 180,000 190,000 Serum concentration(mg/l) 12 2 1.2 0.03 0.00004 Half life (days) 23 6 5 2-8 1-5 Daily production (mg/kg) 34 24 3.3 0.4 0.0023 Intravascular distribution(%) 45 42 80 75 50 Carbohydrate (%) 3 8 12 13 12 Complement fixation Classical ++ - +++ - - Alternative - + - - - Placental transport + - - - - Present in milk + + - - - Selective secretion by seromucus gland - + - - - Heat stability (56 C) + + + + -

34. ABNORMAL IMMUNOGLOBULINS The earliest description of an abnormal immunoglobulins was the discovery Bence Jones (1847) of the protein that bears his name . Bence Jones protein is found typically in multiple myeloma. It can be identified in urine by its characteristics property of coagulation when heated to 50°C but redissolving at 70°C. Bence Jones are light chain immunoglobulins and so may occur as kappa or lambda forms. Multiple myeloma may affect plasma synthesising IgG , IgA, IgD or IgE. Similarly involvement of IgM producing cells is known as Waldenstrom’s macrolobulinemia. In this condition , there occurs excessive production of the respective myeloma protein (M protein) and of their chains (Bence Jones protein). Cryoglobulinemia is a condition in which there is the formation of a gel or a precipitate on cooling the serum, which redissolve on warming. Most cryoglobulins consists of either IgG , IgM or their mixed precipitates.

35. How Do B Cells Produce Antibodies? B cells develop from stem cells in the bone marrow of adults (liver of fetuses). After maturation B cells migrate to lymphoid organs (lymph node or spleen). Clonal Selection : When a B cell encounters an antigen it recognizes, it is stimulated and divides into many clones called plasma cells , which actively secrete antibodies. Each B cell produces antibodies that will recognize only one antigenic determinant.

36. Clonal Selection of B Cells is Caused by Antigenic Stimulation

37. Consequences of Antigen-Antibody Binding Antigen-Antibody Complex : Formed when an antibody binds to an antigen it recognizes. Affinity: A measure of binding strength. 1. Agglutination : Antibodies cause antigens (microbes) to clump together. IgM (decavalent) is more effective that IgG (bivalent). Hemagglutination: Agglutination of red blood cells. Used to determine ABO blood types and to detect influenza and measles viruses. 2. Opsonization: Antigen (microbe) is covered with antibodies that enhances its ingestion and lysis by phagocytic cells.

38. Consequences of Antibody Binding

39. Humoral Immunity (Continued) 3. Neutralization : IgG inactivates viruses by binding to their surface and neutralize toxins by blocking their active sites. 4. Antibody-dependent cell-mediated cytotoxicity : Used to destroy large organisms (e.g.: worms). Target organism is coated with antibodies and bombarded with chemicals from nonspecific immune cells. 5. Complement Activation : Both IgG and IgM trigger the complement system which results in cell lysis and inflammation.

40. Consequences of Antibody Binding

41. Immunological Memory Antibody Titer: The amount of antibody in the serum. Pattern of Antibody Levels During Infection Primary Response: After i nitial 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.

42. 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.

43. Antibody Response After Exposure to Antigen

44. drmsaiem Allo-antibodies & Auto-antibodies Antigens that initiate the immune cascades results in the formation of either allo -abs or auto -abs. Allo-antibodies are produced after exposure to genetically different or non-self, antigens of the same species. Auto-antibodies are produced in response to self antigens.

45. drmsaiem Laboratory Examination of Reaction between Ags and Abs In vitro testing for the detection of Ags or Abs may be accomplished by a variety of immunologic techniques. Such techniques as agglutination , precipitation , agglutination inhibition, and hemolysis are the most commonly used methods to detect the presence of blood group Ags or Abs . In blood bank testing, agglutination reactions are the major manifestation of the blood group Ag-Ab response. Typing for ABO, Rh, and other blood group Ags is accomplished by agglutination reaction.

46. drmsaiem Laboratory Examination of Reaction between Ags and Abs There are two stages for agglutination to develop; Stage 1 : known as sensitization , Ab binding occurs. Antigenic determinants on the red cell membrane combine with the antigen combining site ( Fab region ) on the variable regions of the immunoglobulin heavy and light chains. Stage 2 : a lattice structure composed of multiple Ag-Ab bridges between Abs and red cell Ags is formed visible agglutination is present during this stage.

47. CONCLUSION In general ,IgG protects the body fluids, IgA protects body surfaces and IgM the blood stream, while IgE mediates reaginic hypersensitivity. IgD is a recognition molecule on the surface of B lymphocytes.

48. thank u!