Forensic Biology Richard Li, with additions and edits by Ruth Ballard

1. Lecture 3: Serology Tools Forensic BiologyRichard Li, with additions and edits by Ruth Ballard

2. Outline • Why is serology important? • Alternative Light Sources • Enzyme assays • Immunological assays • Antigens • Antibodies • Agglutination • Microscopy

3. Why is Serology Important? • Used to: • Locate stains • Especially those not visible to the human eye • E.g. Semen stain on a bed sheet • Identify the cellular source of biological evidence. • Support or refute victim’s or suspect’s version of events • People v Carrillo • Exploits presence of fluorescent molecules, enzymes or antigens specific to a body fluid

4. Alternative Light Sources • ALS excites fluorescent molecules in the stain • Many biological fluids contain such molecules • Especially useful for detecting semen stains • Fluorescence = emission of photons at low temp • Absorption of UV or visible radiation kicks electrons in the molecule up to a higher orbitial (higher energy state) • When electrons drop down to lower orbital, emit photons at a longer wavelength (lower energy) • We will use a Crime Lite in lab to detect semen

6. Emits (fluoresces) light at a longer wavelength Absorbs light at short wavelength

8. Enzyme Assays • Enzymes = molecules (usually proteins) that speed upchemical reactions in the cell • Tissue-specific expression • Examples: • Blood: catalase • Semen: acid phosphatase • Saliva: amylase • Detected by enzyme assays that detect a product of the reaction • “Feed” the stain an enzyme’s substrate and look for the appearance of the product

9. Why is Serology Important?

10. Immunological Assays • Animals are constantly under attack by pathogens (viruses, bacteria, protists, and fungi) • Animals have evolved ways to defend themselves • Innate immunity (all animals) • Acquired immunity (vertebrates only) • Antigens and antibodiesare exploited in forensics • Assays are often both tissue and species-specific

11. no memory • memory

12. Immunological Assays • Innate Immunity: • Barrier defense: • In mammals, epithelial cells including skin, mucous membranes • Epithelial cells = important source of DNA in forensics • Saliva and mucous • Tears • Acidic stomach juices • Acidic sweat • Coughing

13. Immunological Assays • Acquired Immunity: • Involves antigen recognition by lymphocytes • Antigens: Substances produced by invading organisms/cells that are recognized by the body as foreign • Usually a protein or polysaccharide • Presented on the surface of the invading cell (e.g. receptors, channel proteins, glycolipids)

14. Immunological Assays • B and T lymphocytes have specific antigen receptors embedded in plasma membrane • 100,000 per cell • Each cell expresses only one type of receptor • Receptors recognize a small accessible portion of their target antigen called an epitope

15. Immunological Assays: Antibodies • Receptors on B cells are Y-shaped • 2 light chains and 2 heavy chains • Disulfide bridges hold the chains together • Transmembrane region near one end of each heavy chain anchors receptor in cell membrane • A short tail region at the end of the heavy chain extends into the cytoplasm

17. Immunological Assays • Sometimes B cells give rise to plasma cells that secrete receptor (release it from the cell membrane) • The secreted receptor is called an antibody or immunoglobulin (Ig) • Found in serum portion of the blood • Can be “raised” in animals against human forms of proteins specific to various body fluids • Then used as a tool for identifying that body fluid in an evidence stain

18. Immunological Assays • Polyclonal Antibodies: • Generated by introducing an antigen (e.g. human hemoglobin) into a host animal (e.g. rabbit) • The animal’s B cells express receptors that bind various epitopes on the antigen • Some of these B cells become plasma cells and the receptors are released into the blood serum, forming a group of antibodies • Blood is removed from the host animal and allowed to clot; serum contains polyclonal antibodies • “Polyclonal” = Group of many different antibodies that recognize epitopes on the same antigen

19. Polyclonal antibodies

20. Immunological Assays • Monoclonal Antibodies • Preparation of antibodies from a single plasma cell; all antibodies are identical and recognize the same epitope • Plasma cells harvested from innoculated host animal’s spleen • Plasma cells are immortalized by fusing them with myeloma cells to form hybridoma cells • Pools of hybridoma cells are diluted into single clones and allowed to proliferate • Clones are screened for antibody of interest

22. Immunological Assays • Antiglobulins: Antibodies that recognize antibodies as antigens! • Antibodies from a specific animal (e.g. human) injected into host animal (e.g. mouse) • Mouse raises antiglobulins to the human antibodies • Antigens, antibodies, and antiglobulins used together in clever serological assays used in medicine and forensics • Detect human proteins specific to various body fluids • “Immunochromatographic test strip” assays • We will use in lab to detect human hemoglobin in blood stains

23. ABO Blood Typing • Agglutination reaction • Red blood cells present glycolipids on cell surface • Sugar groups: A, B, both, or none • Problem for blood transfusions • E.g. Mary presents B sugar but not A sugar (Type B) • Mary receives a blood transfusion from Todd, who presents only A group (A blood type) • Mary raises antibodies against A group • Mary’s antibodies bind A groups on Todd’s incoming blood cells

24. Red blood cell expressing A antigen Antibody to A antigen in blood serum of person with Type B blood = Agglutination (life threatening clots)

26. Microscopy • In forensic biology, especially important for: • Detecting sperm in sperm/epithelial cell mixtures • E.g. Vaginal swab from rape victim • Motile versus non-motile • Examining hair for morphological characteristics • Uses series of lenses to magnify image • Magnification dims image • Light source adds light back in so object can be seen

27. Microscopy