Structure and Function of Antibodies Folder Title: antibodyNoTP Updated: October 08, 2015 See parts of Chapter 3, Kuby Edition 7; B and T-Cell Receptors & Signaling Antibody Structure: pages 80 – 94 This is anonymous. No name is needed or wanted. Have you done any research, or shadowing, or volunteering at Upstate Medical University? Response Yes or No This is anonymous. No name is needed or wanted. Have you taken any classroom courses (not laboratory research courses) at Upstate Medical University? Response Yes or No Following is a Turning Point short answer question. Please put all notes on the floor. Do not have any electronic devices other than your NXT transmitter. No consulting with other students. If you have a problem with your device, I can provide you with a loaner NXT device. If you have a problem using your NXT device, please ask Yifan for help. It is imperative that the integrity of these in-class Turning Point quizzes be maintained at the same level as we will do with the three written exams. Only one NXT device per person. We will count heads. Targets in the Specific Adaptive Immune Response: Antigenic Determinants (Epitopes on Antigens) Weapons of the Specific Adaptive Immune Response: Responds With: Lymphocyte Receptor Primary "Weapon" B-Cell Membrane-bound Antibody Extra-cellular Antibody T-Cell T-Cell Receptor Extra-cellular Cytokines Antibody Protein as a Passer or Runner in Football: Hands on the End of Arms = Recognize and Grasp the Ball Foot = Determines where the ball goes and what to do with it Fab = Bivalent antigen recognition (Two “hands” needed) Fc = Effector function or business end Need both! Fc Region Fab Region “Fc” = Fragment Crystallizable” “Fab” = Fragment Antibody Binding (not crystallizable) Protein Structure of Antibodies A dimeric protein Heavy and Light Chains 2 Heavy – 2 Light (For IgG, 150K MW Antibody) or Multiple Sets (e.g.: 4H + 4L) for IgA or 10H + 10L for IgM (Macroglobulin; 900K MW) Different antibody isotypes and antibody roles IgG, IgA, IgM, IgE, IgD Antibodies as Serum Proteins: Which serum proteins are they? What do they look like? See Box 3-1, Figure 1 Kuby 7th Edition Adding Soluble Antigen to Blood Serum Which protein fraction decreased? Where did it go? Untreated Blood Serum Antigen Added Tetrameric Structure of IgG See Figure 4-7 Kuby, 6th Edition p. 86 Figure 5-2, Kuby 3rd Ed. IgG4mer Papain Digestion Complementarity of Antibody – Antigen Binding Antigen - Antibody Binding Antibody Light Chain Variable Region Fig. 4-6a Kuby 3rd Ed AgAb Kiss Antibody Heavy Chain Influenza Virus Antigen Variable Region Antibodies as Proteins How Do Antibodies Bind to Virtually an Infinite Number of Different Possible Antigens? Proteins are not amorphous polymers. Proteins are not promiscuous in what they bind to. Representation of Sequence Comparisons Among Light Chains from Antibodies with Three Different Antigen Specificities H3N-Ser-Val-Ile-Thr-Gly-Gly-Tyr-Ala... Thr-Glu-Ala-Val-Tyr-Ser-Met-COO- H3N-Ser-Ile-Met-Thr-Arg-Leu-Tyr-Gly..Thr-Glu-Ala-Val-Tyr-Ser-Met-COOH3N-Thr-Gly-Gly-Thr-Lys-Leu-Tyr-Ile..Thr-Glu-Ala-Val-Tyr-Ser-Met-COOVariable Amino Terminal Half Conserved Carboxyl Terminal Half (Positions 1 to 107) (Positions 108 - 214) This shows the first 8 Amino acids from the amino terminal V-region This shows the last 7 amino acids from the carboxyl terminal conserved end. The Following Two Slides Show the Variable Amino Acid Positions in the Amino Terminal One-half of the light Chains (107 Residues) And in the Amino Terminal One-fourth of the Heavy Chains (about 110 residues) The Heavy chain of antibodies is twice as long as the light chains: 440 Amino acids in each of the two heavy chains 220 Amino acids in each of the two light chains Complementarity-determining Regions (CDR’s) from Light and Heavy Chains Come together in 3 dimensions to give the antigen-recognizing site. Ag Binding Site (Fab) Ag Binding Site (Fab) Confers Biological Activity (Kicks the Football) (Fc) Following are Turning Point short answer questions. Please put all notes on the floor. Do not have any electronic devices other than your NXT transmitter. No consulting with other students. If you have a problem with your device, I can provide you with a loaner NXT device. If you have a problem using your NXT device, please ask Elisabeth for help. It is imperative that the integrity of these in-class Turning Point quizzes be maintained at the same level as we will do with the three written exams. What Produces Antibody – Antigen Specificity? Why Do We get Specificity and Very Tight Affinity? Blue shows topology of pocket in MDM2 protein that binds to p53 Figure 9.13 The Biology of Cancer (© Garland Science 2007) How proteins recognize each other topologically (3-dimensional surfaces) Yellow is p53 protein showing peptide domain sequence that binds to MDM2 Control Protein Large surface protein epitope recognized Smaller low molecular size epitopes recognized Small peptide antigen binding to an Fab (Fragment-antibodybinding) fragment of a complementary antibody Light and heavy chain CDR regions move to better complement the antigen Peptide antigen from an HIV protein antigen Conformational change in antibody upon binding antigen (“induced fit”) Movement of Peptide-binding Pocket Accompanying Antigen Binding: Fab Fragment of Antibody to Hemagglutinin Peptide Figure 5-11 Kuby, 3rd Ed AgAbMove How does the antibody protein recognize its complementary antigen so precisely? Precise topological, spatial, and directional arrangement of stabilizing bonding. Minimization of repulsive interactions. Immunoglobulin Isotypes Structures and Functions: Effector Functions of Antibodies Controlled by Fc Regions of Isotypes Start Here: Thursday, October 1st, 2015 See Figure 4-6, Kuby 6th Edition p. 85 Variable Regions Including hypervariable CDR’s (Complementarity determining regions) κ or λ forms See Figure 4-6, Kuby 6th Edition p. 85 Heavy Chain Iso-forms The Heavy and light chains are labeled incorrectly in the Kuby Immunology Powerpoint slides. The figure is labeled correctly in the book. Chain Structures of the five immunoglobulin classes in humans (adapted from Kuby, 2nd edition) Class Heavy Light Sub-Classes Subunit Formula Chain Chain IgA γ α κ or λ γ1, γ2, γ3, γ4 γ2κ2 γ2λ2 κ or λ α1, α2 (α2κ2)n (α2λ2)n n =1,2,3,4 IgM μ κ or λ None IgD δ κ or λ None δ2κ2 δ2λ2 IgE ε κ or λ None ε2κ2 ε2λ2 IgG (μ2κ2)n (μ2λ2)n n = 1 or 5 Figure 5-17(a) IgAModl Kuby, 3rd Ed. Structures of Four Sub-types of IgG See Figure 4-18 Kuby, 6th Edition p. 98 IgGSubs Figure 5-16, Kuby, 3rd Ed. Properties & Activities of Human Serum Immunoglobulins (from Table 4-2, Kuby Immunology, 4th Ed. p. 96) Property or Activity IgG* Mol Wt (KD) 150 H Chain gamma Serum Conc (mg/ml) 0.5 – 9 Serum Half-life (dys) 8 - 23 Activate Complement Yes Cross Placenta Yes Membrane (mIg) Form No Fc Binds Macrophages Yes Mucosal Presence No Induces Mast Cell No IgA** IgM IgE 150 – 600 900 190 alpha mu epsilon 0.5 - 3 1.5 0.0003 6 5 2.5 No Strong No No No No No Yes*** No No ? No Strong Yes No No No Yes * 4 Sub-classes IgG1, IgG2, IgG3, IgG4 **2 Sub-classes IgA1, IgA2 (exists as mono-, di-, tri, tetramer) *** mIgM is monomer. Serum IgM is pentamer # Has no known effector function. Is a membrane-bound antigen receptor IgD# 150 delta 0.03 3 No No Yes No No No Effector Functions of Antibodies Functions of Fab Binding Neutralization or Blocking of Target Molecule or Particle Cross-linking and Agglutination of Target (Bivalent Fab Binding) Functions of Fc Region Complement Fixation and Lysis of Target Opsonization (Coating by Ab) and Phagocytosis of Target Targeting by Antibody for Cell-mediated Destruction (Antibody-dependent Cell-mediated Cytotoxicity: ADCC) Mast Cell Attraction and Activation by Bound IgE Immediate Type I Hypersensitivity, Type I Allergic Response, Localized and Systemic Anaphylaxsis Effector Following are Turning Point short answer questions. Please put all notes on the floor. Do not have any electronic devices other than your NXT transmitter. No consulting with other students. If you have a problem with your device, I can provide you with a loaner NXT device. If you have a problem using your NXT device, please ask Elisabeth for help. It is imperative that the integrity of these in-class Turning Point quizzes be maintained at the same level as we will do with the three written exams. Antibodies as Antigens Why does this matters? If we want to use antibodies as therapeutic agents in patients, we have to understand and control the immunogenicity of the antibodies, or they will generate damaging and dangerous allergic responses, and be cleared from the patient and would be ineffective at best. Antibodies are not cells, so they don’t have transplantation antigens and they don’t have to be histocompatibility matched, but they have to go unrecognized as foreign proteins and they cannot be allowed to generate allergic reactions in the recipient. Start Here Tuesday October 6, 2015 Antibodies as Antigens 1. Different Heavy Chain Isotypes (gamma, alpha, mu, epsilon, delta): Anti-isotype Antibodies (Anti-gamma, Anti-Alpha, Anti-Mu, etc) (Also differences in constant regions of kappa and lambda light chains) 2. Different individual mouse strains (or different people): Anti-allotype Antibodies (Antibodies from one person would raise anti-antibodies in a non-identical twin recipient) (1 and 2: Like any other proteins with multiple molecular forms) 3. Different antigen-recognition abilities: Anti-idiotype Antibodies. Anti-CDR’s for different antibodies Other proteins except for T-cell Receptors do not show these kinds of variations and are not immunogenic in this way Transplantation Concepts and Nomenclature: For All Proteins From self, from identical twin, or from and to inbred animals of the same strain: Syngeneic, Isogeneic, Isologous From same species but not self or identical twin: Allogeneic (“allo” = other) From different species (e.g mouse to human) Xenogeneic (“Xeno” = foreign) Transplantation Concepts and Nomenclature: Specifically for Antibodies Different Isotypes (IgG, IgM, etc.) in the Same Individual Isotypic Determinants The Same Isotype in Two Different Unrelated Individuals in the Same Species (CH1, CH2, CH3, and CL constant regions) And “Framework” Parts of VH and VL Allotypic Determinants (“allo” = other) The Same Isotype in Identical Twins or in Mice of Same Inbred Strain: Specific for Different Antigenic Determinants 6 Different CDR Regions or Idiotopes Idiotypic Determinants Isoforms of the same antibody specificity in the same individual. Recognizes same antigenic determinant with a different Ig Isoform Allotypic determinants (different individuals) are in the Constant Regions of Heavy and Light Chains Idiotypic determinants are in the V-regions of Heavy and Light Chains , especially at the CDR’s) Therapeutic Monoclonal antibody would use antibody matched as isotype and matched in the framework allotype in V-regions (i.e matched as close as possible to the patient). Then graft in the CDR’s from mouse to get the antigenic specificity that is needed. Foreign idiotopes could go unrecognized and be non-immunogenic. Matched human isotype and human allotype. Graft in mouse CDR’s Humanized monoclonal Ab Making Monoclonal Antibodies Now Immortal! Following are three Turning Point short answer questions. Please put all notes on the floor. Do not have any electronic devices other than your NXT transmitter. No consulting with other students. If you have a problem with your device, I can provide you with a loaner NXT device. If you have a problem using your NXT device, please ask Elisabeth for help. It is imperative that the integrity of these in-class Turning Point quizzes be maintained at the same level as we will do with the three written exams. View from OnLine at Textbook Web-site: Molecular Animation of Immunoglobulin Structure Molecular Animation of Cells of the Immune Response Molecular Visualization of Immunoglobulin Structure Molecular Visualization of Antigen-Epitope Interactions For OnLine Access to Immunology Edition 7 Information: http://bcs.whfreeman.com/immunology7e This link no longer works. Contact Freeman to see how to access the textbook resources that are part of the (expensive) textbook End Here: Tuesday October 6, 2015 Exam 1: Tuesday October 13th goes up to this point. This question and the next one are anonymous. They will not be graded and your name will not be recorded by the system. What language to you understand the best? 17% Rank 1 2 17% 17% 17% 3 4 17% 17% Responses 3 4 5 6 Other 1 2 5 6 This is also anonymous. It will not be graded and your name will not be recorded by the system. We are trying to determine the best way to present Immunology in class and the best way to give and to grade the quizzes and exams. With respect to English as spoken in class , as shown on the screen, and as used in the quizzes 33% 33% 33% lty td iff ic u Ih av ea lo ed es om Ih av fo l wi iff ic u lt y th E wi ble fo rt a m co m Ia ... .. n. .. A. I am comfortable with English and I can follow and respond with no language problem. B. I have some difficulty with English, but I can follow in class and respond to the quizzes most of the time. C. I have a lot difficulty following what you are saying in class and in sending in responses to the quiz questions.