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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
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Basic Immunologic Procedures Part 1
November 11, 2014 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 Three Distinct Phases of Antigen/Antibody
Reactions
Primary Phenomenon Sensitization Secondary Phenomenon Lattice
formation Tertiary Phenomenon Detected by affect on tissues or
cells. Primary phenomenon Sensitization binding of single antibody
to single antigen site These tests are Difficult Complex Expensive
Require special equipment Time consuming Primary phenomenon
Sensitization binding of antibody to antigen not visible Primary
Phenomenon Tests
Techniques include: Immunofluorescence Radioimmunoassay Enzyme
immunoassay Secondary Phenomenon Sensitization taken a step further
called lattice formation Antibody molecule binds to two separate
antigens on adjacent antigens If antigen on large structures such
as RBCs causes agglutination. If both antibody and antigen are
soluble results in precipitation precipitation agglutination
Secondary Phenomenon These tests are: Downside is Easy to
perform
Less expensive Less time consuming Do not require special equipment
Downside is Less sensitive Less specific More interference
Secondary Phenomenon Examples of tests: Precipitation
Agglutination
Complement Fixation Tertiary Phenomenon Reaction not visible,
detected by affect of reaction on tissues or cells. Tests include:
Inflammation Phagocytosis Deposition of immune complexes Immune
adherence Chemotaxis Phagocytosis Secondary Phenomena Most
Frequently Utilized
Precipitation soluble antibody reacts with soluble antigen
Agglutination particulate antigens bound together by antibody
Complement Fixation antibody binding to antigen triggers activation
of complement Antigen-Antibody Binding
Affinity-The higher the affinity of the antibody for the antigen,
more stable will be the interaction Avidity-Reactions between
multivalent antigens and multivalent antibodies are more stable and
thus easier to detect Law of Mass Action Antigen-Antibody
Binding
Union of antigen and antibody requires Affinity Avidity Affinity
and avidity determined by Law of Mass Action Antibody Affinity
Describes the strength of a single Ag-Ab bond.
When 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. Affinity
Avidity 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. Law of Mass Action Governs
the reversibility of the antigen-antibody reaction. Reversible
reaction, visible reaction occurs when the rate of binding exceeds
the rate of dissociation. Precipitation Curve 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 Antibody Excess Antigen Excess
Precipitation Curve Measurement of Precipitation by Light
Antigen-antibody complexes, when formed at a high rate, will
precipitate out of a solution resulting in a turbid or cloudy
appearance. Turbidimetry measures the turbidity or cloudiness of a
solution by measuring amount of light directly passing through a
solution. Nephelometry indirect measurement, measures amount of
light scattered by the antigen-antibody complexes.
Precipitation/Flocculation
When soluble antibody binds to soluble antigen (sensitization)
there will come a point where lattice formation will occur
resulting in precipitation occurring resulting in a visible
reaction These immune complexes have fallen out of solution. The Ab
at the bottom in the illustration at right is still in the soluble
phase. Turbidimetry Measures turbidity or cloudiness of a solution
by measuring the amount of light PASSING THROUGH the solution.
Soluble antigen and antibody join and once they join in sufficient
amounts precipitate, results in cloudiness. The more cloudy the
solution, the less light can pass through. Nephelometry Measures
SCATTERED light bouncing off antigen-antibody complexes. Passive
Immunodiffusion
Reactions in gels Migrate towards each other and where they meet in
optimal proportions form a precipitate. Four Methodologies Single
diffusion, single dimension
Single diffusion, double dimension Double diffusion, single
dimension Double diffusion, double dimension Oudin Single
Diffusion, Single Dimension Oudin Precipitation Solution of
antibody is carefully layered on top of a solution of antigen, such
that there is no mixing between the two. With time at the interface
where the two layers meet, antigen-antibody complexes form a
visible precipitate. The other two tubes are negative controls,
containing only antibody or only antigen plus an irrelevant protein
in the second layer. Radial Immunodiffusion (The Mancini
method)
In radial immunodiffusion, an antigen sample is placed in a well
and allowed to diffuse into agar containing a suitable dilution of
an antiserum. The antigen diffuses in all directions from the well,
and accordingly the region of equivalence is established and a ring
of precipitation (precipitin ring) forms around the well. The area
of the precipitin ring is proportional to the concentration of
antigen. The diameter of the area of precipitation (including the
well diameter) is measured to determine the concentration of
antigen. Double immunodiffusion (The Ouchterlony method)
In double immunodiffusion , if antigen to be detected, a known
reagent antibody is placed in the center well and the unknown
samples are placed in the surrounding well. If antibody is to be
detected, unknown antigen is placed in the center. After each of
the samples and reagents have been added to the appropriate wells,
diffusion occurs and both antigen and antibody diffuse radially
from wells toward each other, thereby establishing a concentration
gradient. A line of precipitation forms at the zone of equivalence.
Ouchterlony Gel Diffusion
Holes punched in agar. Known antibody or antigen added to center
well. Known sample added to outer well. Unknown sample added to
outer well next to unknown sample. Wait for bands to form.
Ouchterlony Immunodiffusion Ouchterlony - Identity
The antibodies in the antiserum react with both the antigens
resulting in a smooth line of precipitate. The antibodies cannot
distinguish between the two antigens. i.e., the two antigens are
immunologically identical. Ouchterlony Partial Identity
In the pattern of partial identity, the antibodies in the antiserum
react more with one of the antigens than the other. The spur is
thought to result from the determinants present in one antigen but
lacking in the other antigen Ouchterlony Non-Identity
In the pattern of non-identity, none of the antibodies in the
antiserum react with antigenic determinants that may be present in
both the antigens, i.e., the two antigens are immunologically
unrelated as far as that antiserum is concerned. End of Part 1