Immunoglobulin Structure Function Assigned Reading l Performance Ojectives
Immunoglobulin Structure & Function Assigned Reading l Performance Ojectives l – Key terms – Key Concepts Content Outline l Short Answer Questions l 9/16/2020 1
Angels & Devils 9/16/2020 2
Assigned Reading Chapter: 5 pp 107 -142 l Janis Kuby’s Immunology 3 rd Ed l 9/16/2020 3
Content Outline l l l Basic Structure B Cell Receptor Antigenic Determinants of Igs Immunoglobulin Superfamily Ig Classes 9/16/2020 l l l Monoclonal Antibodies (MABS) Immunoglobulin Sequencing Studies Immunoglobulin Fine Structure 4
Background 9/16/2020 5
Tiselius 9/16/2020 6
Electrophoresis 9/16/2020 7
General Structure of Immunoglobulins Electron microscopy l Crystallography l Hinge region l light & heavy chains l binding site l domains l fragments l 9/16/2020 8
Ig. G Crystal 9/16/2020 9
X - Ray Crystallography l Crystallized Antigen or Antigen - Antibody Complex l Electron Density Map of the Molecule l Three Dimensional Images 9/16/2020 10
Antibodies contain a flexible hinge region EM evidence l Location l Chemical Structure l Advantages l 9/16/2020 11
Antibody Flexibility EMs of same conc of antibody mixed with different conc of antigen l Ag-Ab complexes form different shapes l Ag is inflexible l AB MUST BE FLEXIBLE l 9/16/2020 12
Ag-Ab Complexes 9/16/2020 13
Immunoglobulin flexible hinge 9/16/2020 14
Hinge Prolines 9/16/2020 15
Advantages of hinge Rotation around central point l Bind antigens at various distances l Bind various ratios of antigens l Blocks or binds to antigens over a wide range of concentrations 9/16/2020 16
Structure of Ig. G 9/16/2020 17
Ig. G: Domains 9/16/2020 18
Ig. G: Enzyme hydrolysis 9/16/2020 19
Ag-Ab complexes after Pepsin 9/16/2020 20
B Cell Receptor l BEFORE IMMUNIZATION – Ig genes are organized & Igs assembled – Anchored on membrane as specific receptors for immunogen l AFTER IMMUNIZATION – different expression of Ig genes – lose anchor>> secreted – different class of Ig secreted 9/16/2020 21
Antigenic Determinants of Igs l Immunoglobulins are immunogenic – Inject human antibodies into a mouse – Mouse develops anti-Human antibodies – Use these antibodies to probe structure of immunoglobulins l Three classes of anti-antibodies – Isotypes – Allotypes – Idiotypes 9/16/2020 22
Antibody Classes: Outline 5 classes of antibodies l Concentration of classes l Structure and function of l – Ig. G – Ig. M – Ig. A – Ig. D – Ig. E 9/16/2020 23
Immunoglobulin Classes Immunoglobulin A Ig. A, m. A l Immunoglobulin D Ig. D, m. D l Immunoglobulin E Ig. E, m. E l Immunoglobulin G Ig. G, m. G l Immunoglobulin M Ig. M, m. M l 9/16/2020 24
Immunoglobuin concentrations 9/16/2020 25
Ig Superfamily 9/16/2020 26
Ig. G: Physicochemical Properties l Molecular weight – Sedimentation constant 7 S – Total 146, 000 – Heavy chain 51, 000 Isotypes 4 l Heavy chain domains 4 l Carbohydrate (%) 2 -3 l half life (days) 21 l 9/16/2020 27
Structure of Ig. G 9/16/2020 28
Ig. G: Domains 9/16/2020 29
Ig. G subclasses 9/16/2020 30
Ig. G: Functions Bind antigen in tissue fluids l Promote destruction of antigen l – activates complement l Promote phagocytosis – binds to Fc receptors on macrophages l Passive protection of fetus – binds to receptors on placenta – transferred to fetal circulation 9/16/2020 31
Functions of Ig. G Domains VH + VL Antigen binding l CH 1 Complement binding c 4 b l CH 2 complement binding C 1 q l CH 3 Macrophage binding, Fc receptor l CH 2 +CH 3 neutrophil & K cell binding, bind to placental syncytiotrophoblast l 9/16/2020 32
Primary vs Secondary Response 9/16/2020 33
Ig. M: Physicochemical Properties l Molecular weight – Sedimentation constant 19 S – Total 900, 000 – Heavy chain 65, 000 Isotypes 1 l Heavy chain domains 5 l Carbohydrate (%) 12 l half life (days) 5 l 9/16/2020 34
Ig. M: Structure 9/16/2020 35
Ig. M: Heavy chains 9/16/2020 36
Ig. M: Functions Antigen receptor for B cell l Short term protection l – binds tightly to antigen – (high avidity) 9/16/2020 37
Ig. A: Physicochemical Properties Serum l Secretory Molecular weight – Sedimentation constant 7 S 11 S – Total 160, 000 385, 000 – Heavy chain 52 -56, 000 52, 56, 000 Isotypes l Heavy chain domains l Carbohydrate (%) l Half life (days) l 9/16/2020 2 2 4 7 -11 6 38
Structure of Ig. A 9/16/2020 39
Ig. A dimers 9/16/2020 40
EM of Ig. A 9/16/2020 41
Secretion of Ig. A 9/16/2020 42
Ig. A: Functions Protects body surfaces against invading microbes l Secretory Ig. A l – transported to epithelium – secreted in mucus, milk & tears l Blood Ig. A – transported to liver – binds to hepatocytes – secreted into bile 9/16/2020 43
J chain Small polypeptide chain l found in polymeric classes of antibodies l Acts as a clasp to join prototype subunits l produced by same cell that makes antibodies l 9/16/2020 44
J chain structure 9/16/2020 45
J chain: Beta sheets 9/16/2020 46
J Chain: Barrel 9/16/2020 47
Ig. E: Physicochemical Properties l Molecular weight 188, 000 – Sedimentation constant 8 S – Total 188, 000 – Heavy chain 72, 500 Isotypes 1 l Heavy chain domains 5 l Carbohydrate (%) 12 l half life (days) 2 l 9/16/2020 48
Structure of Ig. E 9/16/2020 49
Molecular model of Ig. E 9/16/2020 50
Ig. E: Functions l Mediates inflammatory response – Immunity to parasitic worms – Mediates many allergies (type I) 9/16/2020 51
Ig. D Physicochemical Properties l Molecular weight – Sedimentation constant 7 S – Total 184, 000 – Heavy chain 70, 000 Isotypes 1 l Heavy chain domains 4 l Carbohydrate (%) 9 -14 l half life (days) 3 l 9/16/2020 52
Ig. D Structure 9/16/2020 53
Ig. D Hinge region 9/16/2020 54
Substructure of Ig. D 9/16/2020 55
Mouse Ig. D 9/16/2020 56
Mouse Ig. D 9/16/2020 57
Ig. D Function l Activation of B cells by antigen – Ig. D : membrane antigen receptor l Indicator of Mature B cells – low levels of Ig. D & Ig. M in Bone marrow – high levels of Ig. D & Ig. M in peripheral lymphoid organs 9/16/2020 58
DONE!!! 9/16/2020 59
Performance Objectives 9/16/2020 60
Key Terms allotypes, allotypic determinants, Am determinants, Gm determinants, l Km determinants, Bence Jones proteins, Antibodies, immunoglobulin classes, l antibody subclasses, constant region (C), Fab fragment, Fab' fragment, l F(ab')2 fragment, Fc fragment, Framework residues (FR), Heavy chain, l 9/16/2020 61
hinge regions, chimeric antibodies, hybridoma, hypervariable regions (HVRs), l complematrarity determining regions(CDRs) l Hypoxantinine-Aminoopterin-Thymidine medium (HAT), idiotypes, idiotypic determinants, l immunoglobulin domains immunoglobulins, isotype, isotypic determinants, l 9/16/2020 62
joining chain (J), Light chain, monoclonal antibodies, multiple myeloma, l Secretory Iga, Segmental flexibility, variable regions (V) l 9/16/2020 63
Key Concepts Distinguish between the overall structure and the fine structure of immunoglobulins l Describe the variable and constant regions of immunoglobulins light and heavy chains l Explain the structural organization of the variable regions of an immunoglobulins light and heavy chains l 9/16/2020 64
Differentiate between Hypervariable regions and complementarity determining regions l Contrast monoclonal antibody production vs polyclonal antibody production l Discuss the differences in the biological effector functions of the different classes of immunoglobulins l 9/16/2020 65
Diagram the procedure for monoclonal antibody production l Construct a table comparing the biological characteristics of the five classes of immunoglobulins l Construct a table comparing the biochemical and biophysical characteristics of the five classes of immunoglobulins l 9/16/2020 66
Short Answer Questions 9/16/2020 67
The analysis of Ig. G molecules after hydrolysis by pepsin and papain led to similar yet different results. Explain. l What is the difference between and immunoglobulin and a myeloma protein? l Why were myeloma proteins and Bence Jones proteins critical to the early studies on antibody structure? What has largely replaced them? l 9/16/2020 68
Differentiate among, complementarity determining regions, hypervariable regions and framework regions. l Explain the statements: "Antibodies can be antigens" and "Antibodies can be used to characterize antibodies". l Discuss the terms immunoglobulin isotypes, immunoglobulin allotypes and immunoglobulin idiotypes and give examples of each. l 9/16/2020 69
Compare conventional antibody production with monocloanl antibody production and recombinant antibody production. l What are antibody domains? l Why can't light chains be used to classify antibodies? l The function of an immunoglobulin molecule is related to its structure. Explain. l 9/16/2020 70
l Different cellular fusion products can potentially occur in a hybridoma fusion. – – – l What are the potential products? Why are they a problem? How can you obtain the appropriate combination? Distinguish between idiotypes and antiidiotypes. 9/16/2020 71
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