Forensic Biology by Richard Li

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Forensic Biologyby Richard Li

• Chapter 4 Serology Concepts

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Serological Reagents

• 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)

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Serological Reagents

• Innate Immunity
• Barrier defense
• In mammals, epithelial cells including skin,
  mucous membranes
• Saliva, mucous, and tears
• Acidic stomach juices
• Acidic sweat

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Serological Reagents

• Innate Immunity
• Internal defenses
• Phagocytic white blood cells (leukocytes)
• Neutrophils and macrophages
• Receptor-mediated recognition of fragments of
  molecules characteristics of a set of pathogens
• E.g. flagellin, lipopolysaccharides, dsRNA
• Engulf invaders and degrade in lysosomes
• Antimicrobial peptides and proteins
• E.g. interferons, complement system

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Serological Reagents

• Innate Immunity
• Internal defenses
• Inflammatory response

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Serological Reagents

• Acquired Immunity
• Involves antigen recognition by lymphocytes
• Antigens foreign substances produced by invading
  organisms, usually proteins or polysaccharides
• Immunogen-natural antigen
• Usually macromolecules such as glycolipids (ABO
  system) and glycoproteins (Rh, Lewis systems)
• Haptens- small molecules that are not natural
  antigens
• Can conjugate with larger molecules to
  artificially elicit antibody formation (cocaine,
  amphetamines)

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Serological Reagents

• Acquired Immunity
• B and T lymphocytes have specific antigen
  receptors embedded in plasma membrane
• 100,000 per cell
• Each cell expresses only one type of receptor
• The receptors recognize a small accessible
  portion of the antigen called an epitope

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Serological Reagents

• Acquired Immunity
• Most antigens are multivalent
• Have more than one epitope

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Serological Reagents

• Acquired Immunity
• 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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Serological Reagents

• Acquired Immunity
• Sometimes B cells give rise to plasma cells that
  secrete a soluble form of the antigen receptor
• The secreted receptor is called an antibody or
  immunoglobulin (Ig)
• Found in serum portion of the blood
• Five classes IgG, IgA, IgM, IgD, and IgE

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Serological Reagents

• Antigens and antibodies are important tools in
  forensic serology
• Antibodies to specific proteins can be raised in
  animals and used in the identification of bodily
  fluids
• Hemoglobin in blood
• Prostate specific antigen (PSA) in semen
• Amylase in saliva

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Serological Reagents

• Polyclonal Antibodies
• Generated by introducing an antigen (e.g. human
  hemoglobin) into a host animal (e.g. rabbit)
• The animals 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 soluble antibodies
• Blood is removed from the host animal and allowed
  to clot serum contains antibodies

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Serological Reagents

• 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
  animals 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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Serology Reagents

• Antiglobulins Immunoglobins that are, themseves,
  immunogens
• Antiglobulins recognize a common epitope on the
  immunoglobulins produced by a particular animal
• E.g. Goat anti-mouse antiglobulins are raised in
  goat against mouse immunoglobulins
• Have important uses in forensic science
• Use in immunochromatographic assays we will
  perform in lab

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The Strength of Antigen-Antibody Binding

• 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

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The Strength of Antigen-Antibody Binding

• Affinity- the strength of the interaction between
  a single epitope on the antigen and a single
  binding site on the corresponding antibody
• Depends on specificity of antibody for antigen
• Cross-reactions can occur
• Avidity- the overall strength of binding between
  an antibody and an antigen
• Overall synergistic strength of all interactions

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Antigen-Antibody Binding Reactions

• Two types used in forensic serology
• Primary
• Initial binding of a single epitope of an antigen
  and single binding site of an antibody to form an
  antigen-antibody complex
• Most sensitive techniques for detecting amounts
  of antigen and antibody in a sample
• Secondary
• Less sensitive but easier to perform
• Three types precipitation, agglutination, and
  complement fixation

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Antigen-Antibody Binding Reactions

• Secondary Reactions
• Precipitation
• Agglutination
• Complement Fixation

Form the basis for many serologic assays in
forensic labs
Used less frequently in forensic serology
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Antigen-Antibody Binding Reactions

• Precipitation
• May occur when a soluble antigen is incubated
  with its antibody
• The antigen-antibody complex cross-link to one
  another to form an insoluble precipitate
• Whether or not a precipitation reaction occurs
  depends on the relative concentrations of antigen
  and antibody
• Precipitin Curve

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Antigen-Antibody Binding Reactions
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Antigen-Antibody Binding Reactions

• Prozone
• There is far more antibody than antigen
• Cross-linking does not occur because there is not
  enough antigen

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Antigen-Antibody Binding Reactions

• Zone of Equivalence
• Reached as more antigen is added
• Cross-linking occurs between neighboring
  complexes

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Antigen-Antibody Binding Reactions

• Post Zone
• Antigen is in excess
• There is not enough antibody for cross-links to
  form

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Antigen-Antibody Binding Reactions

• 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
• If the antigen is located on a red blood cell,
  reaction is called hemagglutination
• A visible clump is observed in the test tube if
  an agglutination reaction has occurred

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