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![Affinity & Avidity Affinity is the strength of Ag-Ab bonds between a single epitope and an individual Ab’s binding site Avidity : The binding strength between a multivalent Ab (polyclonal Ab) and a multivalent Ag Ag + Ab Ag..Ab K = [Ag..Ab]/[Ag][Ab] The higher [Ag..Ab], the larger is K (the associated Ab and Ag), the higher is affinity of the Ab to the Ag. K = Equilibrium constant = Association constant = Ab affinity](https://image.slidesharecdn.com/pharm-immuno5-6servadaptiveimmuneresponseag-ab-101213225856-phpapp01/75/Pharm-immuno5-6serv-adaptive-immune-response-ag-ab-46-2048.jpg)
![Affinity maturity Ag + Ab Ag..Ab K D = [Ag][Ab] / [Ag..Ab] The lower the K D ( dissociation constant ) the higher the affinity Affinity maturity : after repeated exposure to the Ag the affinity increases](https://image.slidesharecdn.com/pharm-immuno5-6servadaptiveimmuneresponseag-ab-101213225856-phpapp01/75/Pharm-immuno5-6serv-adaptive-immune-response-ag-ab-47-2048.jpg)
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Pharm immuno5-6serv adaptive immune response ag-ab
- 1. LECOM-Pharmacy Immunology 5& 6 Adaptive (acquired) immune response Antigen & Antibody Dr. Saber Hussein
- 2. Objectives Define antigenicityand immunogenicity Four characteristics of immuogenic molecule ( requirements for immunogenicity) Foreignness High molecular weight Chemical complexity Degradability Define haptens and their functions Define antigenic determinants Capture of protein antigens by antigen presenting cells Antigen recognized by T lymphocytes Antigens recognized by B lymphocytes
- 3. Learning Objectives Basicstructure of Abs in relation to specificity & diversity Variable & constant regions of light & heavy chain Biological & chemical characteristics of the 5 classes of Ab Compare polyclonal & monoclonal Ab Three characteristics of primary Ag-Ab reaction The forces that foster the primary Ag-Ab reaction Affinity & avidity of Abs Secondary Ag-Ab reaction; lattice formation
- 4. Objectives 9. Knowthe role of receptors in the recognition of antigen in the adaptive immune system: Antibodies as receptors of B lymphocytes T cell receptor (TCR) for antigens 10. Development of immune repertoires Maturation of lymphocytes Production of diverse antigen receptors Maturation and selection of B lymphocytes Maturation and selection of T lymphocytes
- 5. Antigenicity & ImmunogenicityAntigenicity : The ability to bind an Ab or an activated T cell Every immunogen is an antigen, BUT not every antigen is an immunogen Immunogenicity : Ability to elicit immune response Only proteins can induce cellular immunity Humoral immunity can be induced by: Proteins Lipopolysaccharides Nucleic acids Other substances
- 6. Features of anImmunogen High molecular weight Chemical complexity Solubility or biodegradability Foreignness or nonself
- 7. Ab cross-reactions withdifferent Ags Abs react most strongly with homologous Ag Sometimes they cross-react with other Ags Cross-reaction The reaction between an Ag and an Ab that was generated against a different Ag but with some similarity with the cross-reacting Ag Cross-reactions are related to chemical structure of Ags: i. Chemical nature of hapten’s groups ii. Position of substitutions iii. Size of substituted groups iv. Charge v. Stereoisomerism
- 8. Haptens, Antigenic Determinants(Epitopes) Ag has 2 functional regions: Hapten Carrier Epitopes are immunologically active portions of Ag Epitopes on an Ag are recognized by B cells and T cells Antigenic determinants serve as fingerprint of macromolecules Size of an epitope is determined by the size of the Ab’s Ag-binding site Size of recognizable epitope by an Ab: 6 sugar residues 15-20 amino acids (Some linear epitopes are as small as 5 aas) Ab Carrier Hapten
- 9. Haptens &Antigenic DeterminantsHaptens Usually small molecules Not immunogenic by themselves Always antigenic with a specific Ab Immunogenic when combined with a carrier molecule (large) Simple hapten: only 1 antigenic determinant Complex hapten: > 2 antigenic determinants Antigenic Determinants Small part of the molecule Few amino acids A short carbohydrate moiety- few sugars Must be accessible to be functional Charge & polarity Conformation dependent
- 10. Ag recognized byT lymphocytes T lymphocytes recognize only protein antigens Proteins must be presented in the form of short peptides They must be presented by an APC with the appropriate MHC molecule: MHC I presents antigen to the cytotoxic T cell MHC II presents antigen to the helper T cell
- 11. Antigens recognized byB lymphocytes T-cell in dependent route of antigen recognition: B lymphocytes recognize certain antigens without the help of the T H cell These include: Lipopolysaccharides Nucleic acids: DNA & RNA No long term immunity results through this route Only IgM is produced No memory cells
- 12. Antigens recognized byB lymphocytes T-cell dependent route: Recognizes protein antigens Long term immunity IgG is produced by class switching Memory cells
- 13. Antigens recognized byB lymphocytes T-cell dependent Recognizes protein antigens Long term immunity IgG is produced by class switching Memory cells T-cell in dependent Recognizable Ags: Lipopolysaccharides Nucleic acids: DNA & RNA No long term immunity results through this route Only IgM is produced No memory cells
- 14. Antigen Capture andPresentation to Lymphocytes A model of how a T cell receptor (TCR) recognizes a complex of a peptide antigen displayed by a major histocompatibility (MHC) molecule MHC molecules are expressed on antigen-presenting cells and function to display peptides derived from protein antigens Peptides bind to the MHC molecules by anchor residues , which attach the peptides to pockets in the MHC molecules The TCR of every T cell recognizes some residues of the peptide and some (polymorphic) residues of the MHC molecule Fig. 3-1
- 15. The capture anddisplay of microbial antigens Microbes enter the body through an epithelium and are captured by antigen-presenting cells resident in the epithelium enter lymphatic vessels or blood vessels The microbes and their antigens are transported to peripheral lymphoid organs the lymph nodes, the spleen, where protein antigens are displayed for recognition by T lymphocytes Fig. 3-2
- 16. The capture andpresentation of protein antigens by dendritic cells Immature dendritic cells in the epithelium capture microbial antigens by surface receptors and leave the epithelium The dendritic cells migrate to draining lymph nodes, being attracted there by chemokines produced in the nodes During their migration, and in response to the microbe, the dendritic cells mature Mature DC express high level MHC & costimulators In the lymph nodes, the dendritic cells present antigens to naive T cells Fig 3-4 Langerhans cells
- 17. Properties of MHCmolecules and genes Some of the important features of MHC molecules are listed, with their significance for immune responses Fig 3-8
- 18. Role of MHCin Antigen Presentation to T Cells Ag processing The event whereby the Ag is prepared to be presented to lymphocytes in a form they can recognize It includes fragmentation of the protein Ag into small peptides in the macrophage and the presentation to T cells Ag-presenting cells (APCs) bind peptide Ags to their MHC II and present it to the CD4 + helper T cells APCs present peptides to CD8 + cytotoxic T cells with their MHC I Ag-presenting cells (APCs) include macrophages and other cells
- 19.
- 20. MHC Restriction ofT Cells The process by which the MHC controls interactions between immune cells It involves the recognition of foreign antigens in association with class I or II molecules The following reactions are MHC-restricted: Antigen presentation T- and B-cell cooperation Cytotoxic T-cell interaction with target cells Malignant cells Viral infected cell
- 21. What Regions of HLA Complex Encode MHC I & MHC II? Coding Regions: MHC-I coding region: HLA-A, HLA-B and HLA-C MHC-II coding region: HLA-D [ DN, DO, DP , DQ & DR ]
- 22.
- 23. What kind ofT cell do we see here? Antigen-MHC Class II Complex
- 24. Fig 4-1 :Properties of antibodies and T cell antigen receptors (TCRs) Antibodies may be expressed as membrane receptors or secreted proteins TCRs only function as membrane receptors When Ig or TCR molecules recognize antigens, signals are delivered to the lymphocytes by proteins associated with the antigen receptors The antigen receptors and attached signaling proteins form the B cell receptor (BCR) & TCR complexes Single antigen receptors are shown recognizing antigens, Signaling requires the cross-linking of two or more receptors by binding to adjacent antigen molecules
- 25. Fig 4-1 CD4,8Fig 4-1 T cell B cell
- 26. Fig 4-1 : zeta )
- 27. Fig 4-2: Thestructure of antibodies Schematic diagrams of a secreted IgG (A) and a membrane form of IgM (B) illustrate the domains of the heavy and light chains and the regions of the proteins that participate in antigen recognition and effector functions N and C refer to the amino-terminal and carboxy-terminal ends of the polypeptide chains, respectively
- 28.
- 29. Fig 4-3: Features of the major isotypes (classes) of antibodies The table summarizes some important features of the major antibody isotypes of humans. Isotypes are classified on the basis of their heavy chains Each isotype may contain either or light chain Each of the 5 classes differ in their locations in our body and how they stimulate the innate system to remove antigen The schematic diagrams illustrate the distinct shapes of the secreted forms of these antibodies IgA consists of two subclasses : IgA1 and IgA2 IgG consists of 4 subclasses : IgG1, IgG2, IgG3, & IgG4 The serum concentrations are average values in normal individuals
- 30. Fig 4-3: Features of the major isotypes of Abs Breast-fed neonates get it with the mother’s milk Antiparasitic activity with mother’s milk
- 31. Fig 4-3: Features of the major isotypes of Abs Diagnostic for acute infections
- 32.
- 33. Polyclonal & MonoclonalAbs Polyclonal Abs Heterogeneous mix of Abs With specificity against the same Ag Produced by variety of Ab-producing cells They are many clones of cells Polyclonal Abs recognize & react against different epitopes on the Ag Avidity Monoclonal Abs Produced by a single clone of cells Resultant Abs are identical in all aspects Same affinity Same binding specificity Recognize the same epitope They are produced in hybridoma between activated B cells and malignant plasma cells (fusion)
- 34. Three dimensional representationof the IgG molecule IgG molecule
- 35. IgG IgG digestionwith papain produces 3 fragments 2 identical Fab fragments Fab fragments , are capable of binding Ag because they contain the Ag-binding site Fc fragment : a fragment composed of H chain only . It crystallizes in the cold
- 36. Pentameric structure ofIgM The structure of IgM is similar to that of IgG except the IgM heavy chain has an extra domain . A small, cysteine-rich protein called J chain initiates cross linking of C3 and C4 of five IgM monomers to make the circulating, pentameric form of IgM
- 37. Dimeric structure ofIgA Dimeric IgA held together by J chain and secretory component J chain Secretory component
- 38. Secretory IgA IgArepresents 15-20% of serum immunoglobulin It constitutes the majority of Ab found in secretions Humans have 2 types of IgA: IgA1 and IgA2 IgA1 is the prominent subclass in serum and is found mainly as monomer IgA2 is the prominent Ig in secretions (saliva, gut, respiratory mucus) and occurs as a dimer with two Fc ends of the Abs bound together by a J chain Secretion across the mucosa is mediated by a specific secretory component which binds to a cell receptor
- 39. IgE IgE is similar to IgG except it has an extra constant region domain on the H-chain Functions: Type I hypersensitivity Anti-parasitic Degranulation of mast cells
- 40. IgD IgD is similar to the structure of IgG. Its only known function is as part of the signaling complex of B cells
- 41. Primary Ag-Ab reactionThe first interaction between Ag & Ab Key-lock principle Ag-Ab interaction is precise = specific Characteristics of Ag-Ab reaction: i. Rapid, in seconds ii. Independent of electrolytes, salt, buffer iii. Not visible
- 42. Fig 4-4: Binding of an Ag by an Ab This model of a protein antigen bound to an antibody molecule shows how the antigen-binding site can accommodate soluble macromolecules in their native (folded) conformation . The heavy chains (H) of the antibody are red L chains: yellow Ag is blue
- 43. Chemical forces fosterAb-Ag Four noncovalent interactions hold antigenic determinants w/in Ab-binding site: Coulombic (electrostatic, ionic) interactions Van der Waals forces Hydrogen bonds Hydrophobic interactions
- 44. Secondary Ag-Ab reaction& Secondary response Secondary Ag-Ab reaction : The conversion of the invisible primary reactions macroscopically visible ones as in the case of precipitation and agglutination Secondary response : The immune response which follows a second encounter with a particular Ag It is usually stronger ( affinity maturity )
- 45. Lattice formation Occurswhen Ag-Ab complexes aggregate in form of precipitation in liquid medium - Agglutination , including particulate components , other than Ag and Ab, such as cells Ag Ab
- 46. Affinity & AvidityAffinity is the strength of Ag-Ab bonds between a single epitope and an individual Ab’s binding site Avidity : The binding strength between a multivalent Ab (polyclonal Ab) and a multivalent Ag Ag + Ab Ag..Ab K = [Ag..Ab]/[Ag][Ab] The higher [Ag..Ab], the larger is K (the associated Ab and Ag), the higher is affinity of the Ab to the Ag. K = Equilibrium constant = Association constant = Ab affinity
- 47. Affinity maturity Ag+ Ab Ag..Ab K D = [Ag][Ab] / [Ag..Ab] The lower the K D ( dissociation constant ) the higher the affinity Affinity maturity : after repeated exposure to the Ag the affinity increases
- 48. Monoclonal Ab productionImmunize animals, rats or mice, with Ag When the animals start to make a good Ab response remove their spleens and prepare a cell suspension Fuse spleen cells with a myeloma cell line by the addition of polyethylene glycol (PEG), which promotes membrane fusion Only a small proportion of the cells fuse successfully The fusion mixture is then set up in culture with medium containing “HAT” HAT = Mixture of Hypoxanthine Aminopterin (powerful toxin that blocks a metabolic pathway) Thymidine (H & T intermediate metabolites help the cell bypass the pathway when added) Spleen cells can grow/survive in HAT Myeloma cells are sensitive to HAT because of metabolic defect that prevents them from using the bypass HAT culture contains: Spleen cells: die naturally in 1-2 weeks Myeloma cells: Killed by HAT Fused cells (hybridoma): Survive because of immortality of myeloma and HAT-resistance of the spleen cells Some produce antibody Any wells containing growing cells are tested for the production of the desired Ab (often by solid phase immunoassay) Positive ones are cloned by plating out so that there is only one cell in each well This produces a clone derived from a single progenitor, which is both: Immortal Producer of monoclonal Ab
- 49.
- 50. B cells produceAbs B cells are specialized white blood cells produced in the bone marrow. Each B cell contains multiple copies of one kind of antibody as a surface receptor for antigen. The entire population of B cells has the ability to specifically bind to millions of different antigens When the antibody on the surface of a B cell binds to an antigen , the cell can be stimulated to undergo proliferation and differentiation. This process is called clonal selection .
- 51. Clonal selection Thecells produced make the same Ab, but become memory cells and plasma cells Memory cells insure that subsequent infections by the pathogen receive a more rapid response. Plasma cells secrete large amounts of the Ag-specific Ab T helper cells are required for the clonal selection of B cells Ab secreted by plasma cells forms complexes with free pathogens and their toxic products The complexes can: inactivate pathogens & stimulate other innate systems including phagocytes and complement to eliminate the danger from our extracellular fluids
- 52. Abs and theirdiversity An Ab immunoglobulin is a "Y" shaped molecule made up of two identical "light" and "heavy" chains of amino acids. The variable region includes the N-terminal 110-130 amino acids of the light and heavy chains, and is responsible for binding to antigen. The constant region is the C-terminal end and contains similar amino acids for each class of Ab.
- 53.
- 54. Abs Diversity (con)When a stem cell changes to become a B cell, DNA segments for both heavy (VDJ) and light (VJ) chains are randomly combined. Each B cell ends up with functional genes for making one light and one heavy chain coding for an Ab as a membrane-bound receptor. Ab specificity depends on the gene fragments used. Abs are produced that can react with almost any chemical structure in nature.
- 55. The immune systemcreates billions of different Abs with a limited number of genes by rearranging DNA segments during B cell development , prior to Ag exposure. Mutation can also increase genetic variation in Abs Abs Diversity Heavy chain Light chain
- 56. Ab Class switchingAt first, B cells contain IgM molecules only. Class switching occurs after Ag binding , when plasma cells are produced. Class switching refers to a DNA rearrangement changing the heavy chain constant gene in memory cells. Loss of coding regions for the constant part of the heavy chain causes IgG, IgA, and IgE to be produced.
- 57. Ab Class switchingto produce IgA
Editor's Notes
- #17 Dendritic cells at different stages of their maturation may express different membrane proteins Immature dendritic cells express surface receptors that capture microbial antigens Mature dendritic cells express high levels of MHC molecules and costimulators, which function to stimulate T cells (skin, in the example shown, where the dendritic cells are called Langerhans cells)
- #30 (IgG subclasses are given different names in other species, for historical reasons; in mice, they are called IgG1, IgG2a, IgG2b, and IgG3.) Light chains may be lambda or kappa. The five major classes of heavy chain are IgM, IgG, IgA, IgD, and IgE. Each of these classes differ in their locations in our body and how they stimulate the innate system to remove antigen.
- #35 Three dimensional representation of the IgG molecule
- #36 Digestion of IgG with papain produces 2 identical fragments: Fab fragments, are capable of binding Ag because they contain the Ag-binding site, and a third fragment composed of H chain only. It crystallizes in the cold, hence the name: Fc fragment
- #46 lattice (l²t“¹s) n. 1.a. An open framework made of strips of metal, wood, or similar material overlapped or overlaid in a regular, usually crisscross pattern. b. A structure, such as a window, screen, or trellis, made of or containing such a framework. 2. Something, such as a decorative motif or heraldic bearing, that resembles an open, patterned framework. 3. Physics. a. A regular, periodic configuration of points, particles, or objects throughout an area or a space, especially the arrangement of ions or molecules in a crystalline solid. b. The spatial arrangement of fissionable and nonfissionable materials in a nuclear reactor. --lat·tice tr.v. lat·ticed , lat·tic·ing , lat·tic·es . To construct or furnish with a lattice or latticework. [Middle English latis , from Old French lattis , from latte , lath, of Germanic origin.] --lat“ticed adj. ag·glu·ti·na·tion (…-gl›t”n-³“sh…n) n. 1. The act or process of agglutinating; adhesion of distinct parts. 2. A clumped mass of material formed by agglutination. Also called agglutinate. 3. Linguistics. The formation of words from morphemes that retain their original forms and meanings with little change during the combination process. 4. The clumping together of red blood cells or bacteria, usually in response to a particular antibody.
- #47 K = Equilibrium constant = Association constant = Ab affinity The higher [Ag..Ab], the larger is K (the associated Ab and Ag), the higher is affinity of the Ab to the Ag.
- #48 K = Equilibrium constant = Association constant = Ab affinity The higher [Ag..Ab], the larger is K (the associated Ab and Ag), the higher is affinity of the Ab to the Ag.
- #49 Lymph node cells may be used for cell suspension in addition to spleen cells
- #54 So how much variation is possible through recombining gene fragments? Over 15,000,000 combinations of Variable, Diversity and Joining gene segments are possible. Imprecise recombination and mutation increase the variability into billions of possible combinations.