forensic biology by richard li
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Chapter 4: Serology Concepts. Forensic Biology by Richard Li. 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). - PowerPoint PPT PresentationTRANSCRIPT
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Chapter 4: Serology Concepts
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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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•no memory
•memory
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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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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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Innate Immunity: Internal defenses:▪ Inflammatory response
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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)7
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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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Acquired Immunity: Most antigens are multivalent▪ Have more than one epitope
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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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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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Fragment
antigen binding
(Fragment crystallizable)Lower part of heavy chains = constant domains
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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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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 soluble antibodies
Blood is removed from the host animal and allowed to clot; serum contains antibodies
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Polyclonal antibodies
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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
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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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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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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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AFFINITY AVIDITY
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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 fixation23
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Primary antigen-antibody reaction
affinity constant
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Secondary Reactions
Precipitation Agglutination
Complement Fixation
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Form the basis for many serologic assays in forensic labs
Used less frequently in forensic serology
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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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Prozone: There is far more antibody than antigen Cross-linking does not occur because
there is not enough antigen
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Zone of Equivalence: Reached as more antigen is added Cross-linking occurs between neighboring
complexes
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Post Zone: Antigen is in excess There is not enough antibody for cross-
links to form
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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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Agglutination
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