1. Lecture 3: Serology Tools

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

3.  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 3

4.  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 4

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

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8.  Enzymes = molecules (usually proteins) that speed up chemical 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 8

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10.  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 antibodies are exploited in forensics ▪ Assays are often both tissue and species-specific 10

11. 11 no memory memory

12.  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 12

13.  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) 13

14.  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 14

15.  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 15

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17.  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 17

18.  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 18

19. 19 Polyclonal antibodies

20.  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 20

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22.  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.  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 23

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

25. 25 Blood GroupAntigensAntibodiesCan give blood to Can receive blood from ABA and BNoneABAB, A, B, 0 AABA and ABA and 0 BBAB and ABB and 0 0NoneA and BAB, A, B, 0 0

26.  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 26

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