PowerPoint PresentationSlide 2Antigens & Antibodies IIPolyclonal antibodies vs Monoclonal antibodies Polyclonal antibodies: antibody preparations from immunized animals. Consist of complex mixtures of different antibodies produced by many different B cell clones Monoclonal Antibody: homogeneous antibody preparations produced in the laboratory. Consist of a single type of antigen binding site, produced by a single B cell clone (later we’ll talk about how these are made).Slide 5Affinity between two macromolecules can measured using a biosensor Technique: Surface Plasmon Resonance Instrument: BiocoreAffinity refers to strength of binding of single epitope to single antigen binding site. But antibodies have 2 or more identical binding sites. Most antigens are multivalent. What is impact of valence on strength of binding?Avidity (strength of binding) is influenced by both Affinity (Ka of single binding site) x Valence of interaction (number of interacting binding sites)low affinity interactions can have high avidity if valence is high. IgM tend to bind tightly, but have less specificity.Slide 10Cross-reactive AntigensSlide 12Slide 13Slide 14ABO blood types are due to presence of antibodies that cross-react with polysaccharide antigens on red blood cellsSlide 16Slide 17Why do A individuals have antibodies against B type RBC carbohydrates?Slide 19Rheumatic FeverTaking advantage of cross-reactivity in vaccine designCross-reactivity and self-toleranceSlide 23Slide 24Precipitation Reactions: Antibody and Antigen interactions in solution can lead to the formation of a lattice and precipitation of immune complexes. Antibody and antigen must be multivalent. Occurs most efficiently when antigen and antibody are at similar concentration.Can be generated by repeated immunization of animal (rabbit) with antigen (with adjuvant). polyclonal antibodies are a complex mixture of antibodies directed against different epitopes and that differ in their affinity for the antigen.Slide 27Polyclonal antibodies can form lattices with homogeneous, monomeric protein antigens because each antibody can interact with a different epitope on the antigen. Monoclonal antibodies do not form lattices with homogeneous, monomeric proteins, because only they can bind to only one epitope on the antigen.When do antibody-antigen lattices form?Slide 30Double-diffusion assay (Ouchterlony method)Slide 32Slide 33Slide 34Agglutination reaction based on antibody binding to the hormone human chorionic gondatrophic hormone (HCG). But first, a brief reminder about haptens and carriers. . .Hapten: ANY small molecule that cannot by itself induce an immune response, but can be an antigen.Slide 37Slide 38Slide 39Antibodies that bind to other antibodies (secondary antibodies)Slide 41Slide 42Slide 43Slide 44Slide 45Immunological Techniques Monoclonal Antibodies Radioimmune Assay (RIA) Enyzme Linked Immune Sorbant Assay (ELISA) Western blot Immunoprecipitation Flow cytometry Expression cloningPolyclonal antibodies are a complex mixture of antibodies directed against different epitopes and that differ in their affinity for the antigen. Each antisera preparation differs in specificity, averge affinity, cross-reactive specificies, etc. Supply is limited.Monoclonal antibodies Kohler and Milstein, 1975 a technique to generate inexhaustible supply of homogeneous antibody with useful specificities. Basic strategy: fuse 2 cell types to generate a “hybridoma”. Takes advantage of the properties of myeloma cell (unlimited growth capacity and cellular machinery to produce antibodies) and the antigen specificity of primary B cells.What is a clone? A population of cells derived from a single progenitor cell.Slide 50Slide 51HAT selection is used to select for growth of hybrids and against the growth of the parental myeloma.Cloning hybridomas from fusion Plate at limiting dilution (<1 cell/well) in 96 well plates. Allow clones to expand. Expand positive well and test for production of antibody of desired specificity in culture supernatantSlide 54Advantages of Monoclonal AbsThe basic antibody is a dimer of dimer (2 heavy chain-light chain pairs) composed of repeats of a single structural unit known as the “immunoglobulin domain”A Brief Review of Antibody StructureA Brief Review of Antibody StructurePrimary structureSecondary structureQuaternary structureTertiary structureAntigens & Antibodies IIDefinitionsA comparison of antigen recognition by B and T cellsFactors that influence immunogenicityQuantitating the strength of antibody-antigen interactionsEquilibrium constantsequilibrium dialysisimpact of multivalencyCross-reactivity of antibodiesMeasuring antibody-antigen bindingPolyclonal antibodies vs Monoclonal antibodiesPolyclonal antibodies: antibody preparations from immunized animals. Consist of complex mixtures of different antibodies produced by many different B cell clonesMonoclonal Antibody: homogeneous antibody preparations produced in the laboratory. Consist of a single type of antigen binding site, produced by a single B cell clone (later we’ll talk about how these are made).Affinity between two macromolecules can measured using a biosensor Technique: Surface Plasmon Resonance Instrument: Biocore-Resonance units are proportional to the degree of binding of soluble ligand to the immobilized receptor. (or soluble antibody to immobilized antigen, as shown here) - Determining the amount of binding at equilibrium with different known concentrations of receptor (antibody) and ligand (protein antigen) allows you to calculate equilibrium constants (Ka, Kd). -Rate of dissociation and association (koff, kon) can also be calculated.Affinity refers to strength of binding of single epitope to single antigen binding site. But antibodies have 2 or more identical binding sites.Most antigens are multivalent.What is impact of valence on strength of binding?Avidity (strength of binding) is influenced by both Affinity (Ka of single binding site) x Valence of interaction (number of interacting binding sites)Antibody-antigen interactions are multivalent when both the antibody and the antigen have multiple binding sites.Decavalent IgM Bivalent IgGlow affinity interactions can have high avidity if valence is high.IgM tend to bind tightly, but have less specificity.Avid binding due to high affinity.Binding of IgG tends to be more specific. (more perfect “fit” between antigen binding site and antigen)Antigens & Antibodies IIDefinitions and