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Lecture 3: Serology Tools. Forensic Biology Richard Li, with additions and edits by Ruth Ballard. Outline. Why is serology important? Alternative Light Sources Enzyme assays Immunological assays Antigens Antibodies Agglutination Microscopy. Why is Serology Important?. Used to: - PowerPoint PPT Presentation
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Forensic BiologyRichard Li, with additions and edits by Ruth Ballard
Lecture 3: Serology Tools
Outline
Why is serology important?Alternative Light SourcesEnzyme assays Immunological assays
Antigens Antibodies Agglutination
Microscopy
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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 fluid3
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
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Absorbs light at short wavelength
Emits (fluoresces) light at a longer wavelength
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Enzyme Assays
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
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Why is Serology Important?
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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 antibodies are exploited in
forensics▪ Assays are often both tissue and species-specific
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• no memory
• memory
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
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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)
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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
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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
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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 17
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 antigen18
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Polyclonal antibodies
Immunological AssaysMonoclonal 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
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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
ABO Blood TypingAgglutination 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
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= Agglutination (life threatening clots)
Red blood cell expressing A antigen
Antibody to A antigen in blood serum of person
with Type B blood
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Blood Group Antigens Antibodies Can give blood
to Can receive blood from
AB A and B None AB AB, A, B, 0
A A B A and AB A and 0
B B A B and AB B and 0
0 None A and B AB, A, B, 0 0
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
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