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Chapter 4: Serology Concepts
What is an antigen?
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An antigen is any substance that elicits an
immune response and is then capable of
binding to the subsequently produced
antibodies.
Antigens are generally proteins or
polysaccharides, but other substances such as
nucleic acids can also be antigens.
What is an antibody?
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An immunoglobulin
that is capable of
combining with
specificity to the
antigen that elicited its
production.
How, Why and Where are Antibodies
Produced?
(Massively condensed version)
Produced in Vertebrate Cells in response to a ‘nonself’ substance termed an antigen.
 The process begins with macrophages roaming the
bloodstream. Macrophages engulf ‘non-self’
molecules and then display portions of the
engulfed molecules on their outer plasma
membranes.
 After many recognition steps B-cells are stimulated
to produce specific antibodies.
 The antibodies then go off and bind to the foreign
substance thereby marking the substance for
destruction.
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What is an Epitope
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An epitope is the small site on the antigen
which is recognized by the antibody.
Usually between one and six sugars or
amino acids on the surface of the antigen.
Antibody-Antigen Interactions
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Binding of antibody to antigen is dependent on
hydrogen bonds, electrostatic attractions and Van
der Waals attractions.
These bonds are weak compared to covalent
bonds but the large number of weak bonds result
in a stable complex.
Antibody-antigen binding is reversible.
Binding site differences are due to amino acid
sequence differences.
The variable region is made up of 110 –130 amino
acids.
Polyclonal Antibodies
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Produced by immunizing an animal with the
appropriate antigen.
The immunized animal’s serum is collected.
Antibodies can then be purified from the serum.
Since one antigen induces the production of
many antibodies the result is a ‘polyclonal’
mixture of antibodies.
Polyclonal antibodies are much less expensive
than monoclonal antibodies.
Monoclonal Antibodies
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Much more complicated to produce than polyclonal
antibodies.
Process begins by immunizing an animal (most
commonly a mouse) with an antigen.
The animal’s spleen is removed.
B-cells are fused with myeloma cells resulting in
hybridomas.
Hybridomas are screened to find those producing
antibodies to the antigen with which they were
immunized.
Each hybridoma cell is derived from one B-cell so the
antibodies that a clonal population of hybridoma cells
produce are monoclonal antibodies.
Monoclonal antibodies recognize one epitope only.
Producing Monoclonal Antibodies
In the creation of a monoclonal antibody, a normal B cell (a type of lymphocyte, or white blood cell) is united with a myeloma cell (a type of cancer). This union results in the
formation of cloned hybridomas (hybrid cells) that have the cancer cell’s trait of dividing endlessly and the B cell’s ability to produce a specific type of antibody. Monoclonal
antibodies are designed to target specific molecules in the body. They have a variety of uses from pregnancy testing to diagnosing and treating certain diseases.
Producing Monoclonal Antibodies
In the creation of a monoclonal antibody, a normal B cell
(a type of lymphocyte, or white blood cell) is united with a
myeloma cell (a type of cancer). This union results in the
formation of cloned hybridomas (hybrid cells) that have the
cancer cell’s trait of dividing endlessly and the B cell’s ability
to produce a specific type of antibody. Monoclonal antibodies
are designed to target specific molecules in the body.
They have a variety of uses from pregnancy testing to
diagnosing and treating certain diseases.
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Antibodies- capable of binding to an antigen
 Also known as Immunoglobulins
▪ Found in serum portion of blood
▪ Five classes
▪
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▪
IgG
IgA
1gM
IgD
IgE
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Polyclonal Antibody
 Produced by different B lymphocyte clones in
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response to the different epitopes of the
immunogen
Avian- B Type
Rabbit- R Type
Horse- H Type
Polyclonal antiserum
Serum
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Monoclonal Antibody
 Spleen cells are utilized
 Fused cells are called Hybridoma Cells
 Limitations for use in serology assays
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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 energy of the interaction
between a single epitope on the antigen and
a single binding site on the corresponding
antibody
 Cross-reaction
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Avidity- the overall strength of binding
between an antibody and an antigen.
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Three types:
 Primary
▪ Most sensitive techniques
 Secondary
▪ Less sensitive but easier to perform
 Tertiary
▪ Not commonly employed in a forensic setting
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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
 Precipitin curve
▪ Prozone
▪ Zone of Equivalence
▪ Postzone
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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
▪ Initial Binding
▪ Lattice Formation
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