Slide 1Slide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Serological ReagentsSerological ReagentsSerological ReagentsThe Strength of Antigen-Antibody BindingThe Strength of Antigen-Antibody BindingAntigen-Antibody Binding ReactionsAntigen-Antibody Binding ReactionsAntigen-Antibody Binding ReactionsAntigen-Antibody Binding ReactionsForensic Biologyby Richard LiChapter 4: Serology ConceptsWhat is an antigen?An antigen is any substance that elicits an immune response and is then capable of binding to the subsequently produced antibodies.Antigens are generally proteins or polysaccharides, but other substances such as nucleic acids can also be antigens.What is an antibody?An immunoglobulin that is capable of combining with specificity to the antigen that elicited its production.How, Why and Where are Antibodies Produced?(Massively condensed version)Produced in Vertebrate Cells in response to a ‘non-self’ substance termed an antigen.The process begins with macrophages roaming the bloodstream. Macrophages engulf ‘non-self’ molecules and then display portions of the engulfed molecules on their outer plasma membranes.After many recognition steps B-cells are stimulated to produce specific antibodies.The antibodies then go off and bind to the foreign substance thereby marking the substance for destruction.What is an EpitopeAn epitope is the small site on the antigen which is recognized by the antibody.Usually between one and six sugars or amino acids on the surface of the antigen.Antibody-Antigen InteractionsBinding of antibody to antigen is dependent on hydrogen bonds, electrostatic attractions and Van der Waals attractions.These bonds are weak compared to covalent bonds but the large number of weak bonds result in a stable complex.Antibody-antigen binding is reversible.Binding site differences are due to amino acid sequence differences.The variable region is made up of 110 –130 amino acids.Polyclonal AntibodiesProduced by immunizing an animal with the appropriate antigen.The immunized animal’s serum is collected.Antibodies can then be purified from the serum.Since one antigen induces the production of many antibodies the result is a ‘polyclonal’ mixture of antibodies.Polyclonal antibodies are much less expensive than monoclonal antibodies.Monoclonal AntibodiesMuch more complicated to produce than polyclonal antibodies.Process begins by immunizing an animal (most commonly a mouse) with an antigen.The animal’s spleen is removed. B-cells are fused with myeloma cells resulting in hybridomas.Hybridomas are screened to find those producing antibodies to the antigen with which they were immunized.Each hybridoma cell is derived from one B-cell so the antibodies that a clonal population of hybridoma cells produce are monoclonal antibodies.Monoclonal antibodies recognize one epitope only.Producing Monoclonal Antibodies In the creation of a monoclonal antibody, a normal B cell (a type of lymphocyte, or white blood cell) is united with a myeloma cell (a type of cancer). This union results in the formation of cloned hybridomas (hybrid cells) that have the cancer cell’s trait of dividing endlessly and the B cell’s ability to produce a specific type of antibody. Monoclonal antibodies are designed to target specific molecules in the body. They have a variety of uses from pregnancy testing to diagnosing and treating certain diseases.In the creation of a monoclonal antibody, a normal B cell (a type of lymphocyte, or white blood cell) is united with a myeloma cell (a type of cancer). This union results in the formation of cloned hybridomas (hybrid cells) that have the cancer cell’s trait of dividing endlessly and the B cell’s abilityto produce a specific type of antibody. Monoclonal antibodies are designed to target specific molecules in the body. They have a variety of uses from pregnancy testing to diagnosing and treating certain diseases. Producing Monoclonal AntibodiesSerological ReagentsAntibodies- capable of binding to an antigenAlso known as Immunoglobulins▪Found in serum portion of blood▪Five classes▪IgG▪IgA▪1gM▪IgD▪IgEForensic Biology by Richard Li 14Serological ReagentsPolyclonal AntibodyProduced by different B lymphocyte clones in response to the different epitopes of the immunogenAvian- B TypeRabbit- R TypeHorse- H TypePolyclonal antiserumSerumForensic Biology by Richard Li 15Serological ReagentsMonoclonal AntibodySpleen cells are utilizedFused cells are called Hybridoma CellsLimitations for use in serology assaysForensic Biology by Richard Li 16The Strength of Antigen-Antibody BindingMediated by the interaction between epitope of the antigen and the binding site of the antibodyStrongest binding occurs only if the shape of the epitope fits the binding siteDepends on:▪Affinity▪AvidityForensic Biology by Richard Li 17The Strength of Antigen-Antibody BindingAffinity- the energy of the interaction between a single epitope on the antigen and a single binding site on the corresponding antibodyCross-reactionAvidity- the overall strength of binding between an antibody and an antigen.Forensic Biology by Richard Li 18Antigen-Antibody Binding ReactionsThree types:Primary ▪Most sensitive techniquesSecondary▪Less sensitive but easier to performTertiary▪Not commonly employed in a forensic settingForensic Biology by Richard Li 19Antigen-Antibody Binding ReactionsSecondary ReactionsPrecipitationAgglutinationComplement FixationForensic Biology by Richard Li 20Form the basis for many serologic assays in forensic labsUsed less frequently in forensic serologyAntigen-Antibody Binding ReactionsPrecipitationPrecipitin curve▪Prozone▪Zone of Equivalence▪PostzoneForensic Biology by Richard Li 21Antigen-Antibody Binding ReactionsAgglutinationIf antigens are located on the surface of cells or carriers, the interaction of antibodies and antigens will cause the cells or carriers to aggregate and form larger complexes▪Initial Binding▪Lattice FormationForensic Biology by Richard Li