2. Basic Immunologic
Procedures
Terry Kotrla, MS, MT(ASCP)BB
Introduction
Detection of antigen/antibody reactions difficult
 Can measure EITHER antigen or antibody.
 Sensitization is the binding of a specific antibody
to its’ specific antigen
 Sensitization cannot be visualized
 Multitude of laboratory methods have been
developed to make this reaction visible

Factors Which Affect Reactions
Concentrations of reactants
 Temperature
 Length of incubation
 pH of test system
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Three Distinct Phases of
Antigen/Antibody Reactions
Primary Phenomenon – Sensitization
 Secondary Phenomenon – Lattice formation
 Tertiary Phenomenon – Detected by affect on
tissues or cells.
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Primary phenomenon

Sensitization – binding of antibody to antigen – not
visible
Primary phenomenon
Sensitization – binding of single antibody to
single antigen site
 These tests are
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Difficult
Complex
Expensive
Require special equipment
Time consuming
Primary Phenomenon Tests

Techniques include:
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Immunofluorescence
Radioimmunoassay
Enzyme immunoassay
Secondary Phenomenon
Sensitization taken a step further to lattice
formation
 Fab of Antibody molecule binds to two separate
antigens on adjacent antigens
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If antigen on large structures such as RBCs causes
agglutination.
If both antibody and antigen are soluble results in
precipitation
Secondary Phenomenon Reactions
Secondary Phenomenon

These tests are:
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Easy to perform
Less expensive
Less time consuming
Do not require special equipment
Downside is
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Less sensitive
Less specific
More interference
Secondary Phenomenon
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Examples of tests:
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Precipitation
Agglutination
Complement Fixation
Tertiary Phenomenon
Reaction not visible, detected by affect of
reaction on tissues or cells.
 Tests include:
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Inflammation
Phagocytosis
Deposition of immune complexes
Immune adherence
Chemotaxis
http://www.cellsalive.com/mac.htm
Phagocytosis
Secondary Phenomena Most
Frequently Utilized
Precipitation – soluble antibody reacts with
soluble antigen
 Agglutination – particulate antigens bound
together by antibody to form visible complex
 Complement Fixation – antibody binding to
antigen triggers activation of complement,
results in cell lysis
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Antigen-Antibody Binding

Union of antigen and antibody requires
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Affinity
Avidity
Affinity and avidity determined by Law of Mass
Action
Antibody Affinity
Describes the strength of a single Ag-Ab bond.
 As Ag and Ab come close together a chemical
bond forms which is weak and can dissociate.
 How well the Ab fits the Ag will determine
stability of bond, “lock and key” fit has strongest
affinity.
 Ab may react with structurally similar Ags,
results in cross reactivity.
 Most antibodies have a high affinity for their
antigens.
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Affinity
Avidity
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Describes the combined strength of multiple Ag-Ab
bonds.
Initially bond is easily broken, but multiple bindings at the
same time the dissociation is overcome by the sheer
number of bonds remaining.
Avidity is influenced by both the valence of the antibody
and the valence of the antigen.
Avidity
Affinity versus Affinity

Affinity refers to the strength of binding between
a single antigenic determinant and an individual
antibody combining site whereas avidity refers to
the overall strength of binding between
multivalent antigens and antibodies.
IgM and IgG
Most frequently detected immunoglobulins.
 IgM has low affinity but high avidity due to 10
binding sites.
 IgG has 2 strong binding sites, high affinity and
avidity.
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Law of Mass Action
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Governs the reversibility of the antigen-antibody
reaction.
Reversible reaction, visible reaction occurs when the
rate of binding exceeds the rate of dissociation.
As affinity and avidity increases, strengthens reaction.
Precipitation Curve
Depends on concentration of Ag and Ab.
 Prozone – antibody excess, many antibodies
coat all antigen sites- results in false negative
 Postzone – antigen excess, antibody coats
antigen but cannot get lattice formation, results
in false negative
 Zone of Equivalence – antigen and antibody
present in optimal proportions to bind and give
visible reaction
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Precipitation Curve
Precipitation Curve