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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Microscopy

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