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Antigens and Antibodies

Antigens and Antibodies. Antibodies. Antigens. Antigen : A substance that can be recognized by the adaptive arm of the immune system. Immunogen : Those antigens that induce a specific adaptive immune response in a host. Factors that influence the immunogenicity of an antigen. Size

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Antigens and Antibodies

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  1. Antigens andAntibodies

  2. Antibodies Antigens Antigen: A substance that can be recognized by the adaptive arm of the immune system. Immunogen: Those antigens that induce a specific adaptive immune response in a host.

  3. Factors that influence the immunogenicity of an antigen • Size • the larger the better • Chemical composition and heterogeneity • the more complex the better • Uptake and degradability • the less soluble the better • Foreignness • self molecules do not elicit immune responses

  4. Other Factors • Host Genotype (responder v. non-responder) • Route of Antigen Exposure • Dose of Antigen

  5. Adjuvant • A substance that when mixed with an antigen can enhance the immune response to that antigen. • An adjuvant can • Render the antigen insoluble • Prolong persistence of antigen at the site of entry • Activate inflammatory cells (source of PAMP) Examples: Bacterial components from Mycobacteria (Freund’s complete adjuvant), Cholera toxin, Bordetella, or insoluble aluminum salts.

  6. Linear and Conformational Epitopes (or AntigenicDeterminants) formed by 1o-4o Protein structure Model Protein with several potential epitopes

  7. crosslinking B cell B cell B-cells produce antibodies following contact with antigen. The antigen ‘cross-links’ antibody molecules on the surface of B-cells and sends signals to the B-cell to start secreting antibody.

  8. Molecules that are too small to cross-link surface immunoglobulin, are not immunogenic carrier Hapten: An antigen that can be bound and recognized by the immune system but cannot induce an immune response in the host Carrier: A substance that can help induce an immune response against a chemically linked hapten haptens

  9. L H VH CH2 CH3 CH1 Hyper variable Hinge CL VL Model Antibody (Immunoglobulin) Structure Constant Variable

  10. Immunoglobulin: quaternary structure

  11. F(ab') 2 Proteolytic Fragments of Immunoglobulin Pepsin treatment pepsin papain Papain treatment Fc + Fab

  12. Immunoglobulin heavy chain constant regions in humans fall into five major classes. These are: IgM (m), IgA (a), IgG(g), IgD (d) and IgE (e) IgG is further subdivided into four subclasses: IgG1, IgG2, IgG3, IgG4 (human) IgG1, IgG2a, IgG2b, IgG3 (mouse) IgA is subdivided into two subclasses IgA1 and IgA2 Immunoglobulin light chain constant regions are: Igk and Igl

  13. dimer J chain IgA Structural differences among Immunoglobulin isotypes monomer pentamer J chain IgM IgG, IgD, IgE Valence: number of antigen binding sites Avidity: Strength of binding based on valence

  14. Antibodies bind to antigens based on the ‘complementarity’ of the epitope on the antigen and the antigen-binding site of the antibody. Complementarity can be thought of as ‘goodness of fit’. The better the fit, the stronger the binding. The strength of the reaction is referred to as the affinity of the antibody. Generally speaking, a high affinity antibody is more protective than a low affinity antibody because it will bind antigens at lower concentrations. Ag + Ab AgAb

  15. Antibodies bind antigens through reversible non-covalent interactions These include: Electrostatic forces: Attraction between opposite charges. Hydrogen bonds: hydrogen ion shared between different groups create partial opposite charges. Van der Waals forces: Fluctuations in electron clouds around Molecules oppositely polarize neighboring atoms. Hydrophobic forces: hydrophobic amino acids attract .

  16. dimer J chain IgA Structural differences among Immunoglobulin isotypes monomer pentamer J chain IgM IgG, IgD , IgE Valence: number of antigen binding sites Avidity: Strength of binding based on valence

  17. IgM: the first isotype to be expressed in every B cell occurs in serum as a pentamer (valence = 10) can inhibit attachment of microbes can activate complement cascade IgG: the most abundant Ig subclass found in human serum occurs as a monomer (valence=2) can opsonize antigen for uptake by phagocytic cells can activate complement cascade cross placenta and protect offspring IgA: Major subclass of Ig in external secretions and mucosa occurs in serum as a monomer; in secretions as a dimer IgE: occurs at very low concentrations in serum can bind to mast cells and basophils and mediates allergy IgD: occurs primarily as surface Ig, co-expressed with IgM function unknown

  18. DO NOT MEMORIZE!!!!!

  19. 2o anti-A Mainly IgG Immune Response Antigen A Antigen A + Antigen B 1o anti-B IgM = 0 14 0 6 14 Time (Days) Hallmarks of the adaptive immune response: Specificity for antigen Induction of memory 1o anti-A Mainly IgM

  20. L H CH2 CH3 Variable COOH CH1 hv Hinge CL Idiotypic Allotypic VL NH2

  21. III. The Genetic Basis for Antibody Diversity It is estimated that a mammal can generate antibodies with as many as 108 different antibody specificities. Since antibodies are proteins and proteins are encoded by genes, it stands to reason that the diversity of antibodies must arise from diversity in the genome. The problem is that the genome does not contain enough genes to encode all of those different Ig specificities. In addition, each B-cell produces Ig of only 1 specificity so some mechanism must exist to limit the production of Ig by B-cells to Ig with only 1 specificity.  

  22. 1. Heavy chain and light chain combination Essentially any light chain can associate with any heavy chain. If there were 104 different light chains and 104 different heavy chains, we could derive 108 different specificities (104 104 = 108). Light chains Heavy chains

  23. 2. Multiple V genes The variable portion of light chains and heavy chains are encoded by gene segments called variable gene segments or V gene segments that are found in the respective gene complexes of the Ig light chain and Ig heavy chain. In different species, there are different numbers of V gene segments. The DNA between the V and C regions is not deleted, but the RNA transcript from the gene complex is processed so the final mRNA contains only the complete light chain transcript without the intervening sequences. VL1---VL2----VLn--------CL VH1---VH2----VHn--------CH

  24. Gene Sequence Ig Protein Liver Cell B Cell During B-cell development (in the bone marrow), genes that encode antibody heavy and light chains undergo breakage deletion, and repair to form ‘new’ genes. This process is called SOMATIC RECOMBINATION

  25. Somatic Recombination Transcription mRNA Splicing (Processing) Translation Germline DNA-------------- V--J Joined DNA----------- Primary RNA Transcript- Mature RNA Transcript-- Polypeptide chain----------

  26. Genomic Structure Heavy Chain

  27. 3. VJ and VDJ Recombination See animation of rearrangement 4. Recombination Inaccuracies See Animation 5. Somatic mutations Mutations occur within the gene complexes during the lifetime of the cell, resulting in the formation of antibodies that are slightly different that the original. Sometimes the resulting antibodies will bind to an antigen stronger than the original antibody. The new antibody molecule is said to have a higher affinity for the antigen. When antigen is present, these cells will preferentially bind antigen, proliferate, and secrete antibody of a higher affinity. This process is referred to as affinity maturation.

  28. Antigen-independent B-cell development pre B pro B stem cell immature B IgM IgM Exit bone marrow mature B IgD D-Jh Vh-DJh Vl-Jl

  29. Specificity of Antibodies Make them Ideal Reagents for Many Applications Therapy – Treatment against infection or intoxication Diagnostics – Appearance of pathogen-specific antibodies in serum is indicative of exposure/infection Research Reagents – Molecular probes for (protein) expression

  30. Therapy http://www.talecrisusa.com/products.asp

  31. Diagnostics A 20 year old diabetic college student returning from Spring Break was concerned that he became infected with HIV. How does his clinician help him? Examine his serum by ELISA for the resence of HIV-specific antibodies as a marker for infection

  32. Research: Immunohistochemistry Specific staining of pancreatic cells in murine pancreas using low-power antigen-retrieval (LAR) protocol. Insulin+ ß cells are shown in green and pancytokeratin (CK+) pancreatic duct epithelial cells in red (A1). Immunohistochemical (IHC) staining of insulin was exclusively specific for ß cells but not for duct epithelial cells (A2). Insulin+ ß cells are the dominant cell population in the islet (green), and cells are scattered around the periphery of the islet (red) (B1). Low magnification showing the highly specific staining in the large area of tissue (B2); pancreatic ß cells are stained with insulin in cytoplasm (green) and PDX-1 in nuclei (red) (C1). There was no unspecific binding in exocrine area (C2) and specific staining of pancreatic duct with CK (red) and vascular endothelial cells with PECAM (CD31) (green) (D1). Ab against PECAM stained only vessel but not duct (D2). Bars: A–C,D2 = 50 µm; D1 = 25 µm. From: Ge et al. Journal of Histochemistry and Cytochemistry 54: 843-847, 2006

  33. Antibody responses are generally polyclonal: Many individual ‘clones’ make antibodies of varying affinities against multiple epitopes found on the immunogen. A polyclonal response is the sum of all of the clonal responses (or monoclonal responses)

  34. Monoclonal Antibody Production The selection of hybridoma cells is accomplished by culturing the fused cell mixture in hypoxanthine-aminopterin-thymidine (HAT) medium. Aminopterin blocks the De Novo biosynthesis of thymine, purines and pyrimidines. Myeloma cells do not survive because they are incapable of growing when the De Novo nucleotide synthesis is blocked with HAT because they lack functional hypoxanthine-guanine phosphoribosyl-transferase (HGPRT-). B cells die in vitro within two weeks. The resulting hybridoma cells survive because they have received hypoxanthine-guanine phosphoribosyl transferase (HGPRT+)from the B cells and immortality from the myeloma cells.

  35. Monoclonal v. Polyclonal Antibodies Monoclonal: Limitless supply, defined specificity and subclass, typically less ‘background reactivity’ and less expensive, but sometimes application is limited because only 1 epitope is recognized. Polyclonal: Initially easier to produce, greater reactivity with antigen (usually more applications), but not a limitless supply. Specificity is less well-defined and background reactivity is more of problem

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