Chapter 4: Serology Concepts

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

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

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

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

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

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

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

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