1. Chapter 4: Serology Concepts

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.

12. 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.

13. 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. Producing Monoclonal Antibodies

14.  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 Li14

15.  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 Li15

16.  Monoclonal Antibody  Spleen cells are utilized  Fused cells are called Hybridoma Cells  Limitations for use in serology assays Forensic Biology by Richard Li16

17.  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 Li17

18.  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 Li18

19.  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 Li19

20.  Secondary Reactions  Precipitation  Agglutination  Complement Fixation Forensic Biology by Richard Li20 Form the basis for many serologic assays in forensic labs Used less frequently in forensic serology

21.  Precipitation  Precipitin curve ▪ Prozone ▪ Zone of Equivalence ▪ Postzone Forensic Biology by Richard Li21

22.  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 Li22