Forensic Biology by Richard Li

1. Chapter 4: Serology Concepts Forensic Biologyby Richard Li

2. What 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.

3. What is an antibody? • An immunoglobulin that is capable of combining with specificity to the antigen that elicited its production.

5. 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.

6. 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.

7. 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.

10. 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.

11. 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.

13. 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.

14. Serological Reagents • Antibodies- capable of binding to an antigen • Also known as Immunoglobulins • Found in serum portion of blood • Five classes • IgG • IgA • 1gM • IgD • IgE Forensic Biology by Richard Li

15. Serological 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 • Serum Forensic Biology by Richard Li

16. Serological Reagents • Monoclonal Antibody • Spleen cells are utilized • Fused cells are called Hybridoma Cells • Limitations for use in serology assays Forensic Biology by Richard Li

17. The 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 • Avidity Forensic Biology by Richard Li

18. The 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

19. Antigen-Antibody Binding Reactions • Three types: • Primary • Most sensitive techniques • Secondary • Less sensitive but easier to perform • Tertiary • Not commonly employed in a forensic setting Forensic Biology by Richard Li

20. Antigen-Antibody Binding Reactions • Secondary Reactions • Precipitation • Agglutination • Complement Fixation Form the basis for many serologic assays in forensic labs Used less frequently in forensic serology Forensic Biology by Richard Li

21. Antigen-Antibody Binding Reactions • Precipitation • Precipitin curve • Prozone • Zone of Equivalence • Postzone Forensic Biology by Richard Li

22. Antigen-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 Formation Forensic Biology by Richard Li