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Principles of Immunology Antigen-Antibody Interactions 4/25/06. Word/Terms List. Agglutinin EIA Equivalence zone FIA Immunodiffusion Immunoelectrophoresis RIA Titer. Low Affinity. High Affinity. Ab. Ab. Ag. Ag. Affinity. - PowerPoint PPT Presentation
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Principles of ImmunologyAntigen-Antibody Interactions
4/25/06
Word/Terms List Agglutinin EIA Equivalence zone FIA Immunodiffusion Immunoelectrophoresis RIA Titer
Affinity = attractive and repulsive forces
Ab
Ag
High Affinity
Ab
Ag
Low Affinity
Affinity
• Strength of the reaction between a single antigenic determinant and a single Ab combining site
Specificity The ability of an individual antibody
combining site to react with only one antigenic determinant.
The ability of a population of antibody molecules to react with only one antigen.
Cross Reactivity
• The ability of an individual Ab combining site to react with more than one antigenic determinant.
• The ability of a population of Ab molecules to react with more than one Ag
Anti-A Ab
Ag A
Anti-A Ab
Ag B
Shared epitope
Anti-A Ab
Ag C
Similar epitope
Cross reactions
Factors Affecting Measurement of Ag/Ab Reactions
• Affinity
• Avidity
• Ag:Ab ratio
• Physical form of Ag
Ab excess Ag excess
Equivalence – Lattice formation
Tests Based on Ag/Ab Reactions All tests based on Ag/Ab reactions will
have to depend on lattice formation or they will have to utilize ways to detect small immune complexes
All tests based on Ag/Ab reactions can be used to detect either Ag or Ab
Agglutination Tests
Lattice Formation
Agglutination/Hemagglutination
• Definition - tests that have as their endpoint the agglutination of a particulate antigen– Agglutinin/hemagglutinin
+
• Qualitative agglutination test– Ag or Ab
Agglutination/Hemagglutination
Quantitative agglutination test Titer Prozone
1/2
1/4
1/8
1/16
1/32
1/64
1/12
8
1/25
6
1/51
2
1/10
24
Pos.
Neg
.
Titer
648
512<232
128324
Patient
12345678
Agglutination/Hemagglutination
Definition Qualitative test Quantitative test• Applications
– Blood typing– Bacterial infections
–Fourfold rise in titer
• Practical considerations– Easy– Semi-quantitative
1/2
1/4
1/8
1/16
1/32
1/64
1/12
8
1/25
6
1/51
2
Passive Agglutination/Hemagglutination
Definition - agglutination test done with a soluble antigen coated onto a particle
+
• Applications– Measurement of antibodies to soluble antigens
Agglutination/Hemagglutination Inhibition
Definition - test based on the inhibition of agglutination due to competition with a soluble Ag
+
Prior to Test
+ +
Test
Patient’s sample
Agglutination/Hemagglutination Inhibition
Applications Measurement of soluble Ag
Practical considerations Same as agglutination test
• Definition
Precipitation Tests
Lattice Formation
Radial Immunodiffusion
Interpretation Diameter of ring
is proportional to the concentration
Quantitative Ig levels
• Method– Ab in gel– Ag in a well
Ag Concentration
Dia
met
er2
AgAgAgAg
Ab in gel
Immunoelectrophoresis
Method Ags are separated by electrophoresis
• Interpretation– Precipitin arc represent individual antigens
Ag-+
Ag
Ab
Ag
Ab
Immunoelectrophoresis
Method Interpretation Qualitative
Relative concentration
Radioimmuoassays (RIA)Enzyme-Linked Immunosorbent
Assays (EIA)
Lattice formation not required
Competitive RIA/ELISA for Ag
Method Determine
amount of Ab needed to bind to a known amount of labeled Ag
+
Prior to Test
Labeled Ag
+
Test
+Patient’ssample
LabeledAg
+– Use predetermined
amounts of labeled Ag and Ab and add a sample containing unlabeled Ag as a competitor
Solid Phase Non-Competitive RIA/ELISA
Ab detection Immobilize Ag Incubate with
sample Add labeled anti-Ig Amount of labeled
Ab bound is proportional to amount of Ab in the sample
SolidPhase
AgImmobilized
Ab in Patient’s
sample
LabeledAnti-Ig
Solid Phase Non-Competitive RIA/ELISA
Ag detection Immobilize Ab Incubate with sample Add labeled antibody Amount of labeled Ab
bound is proportional to the amount of Ag in the sample
SolidPhase
AgImmobilized
Ag in Patient’s
sample
LabeledAb
Tests for Cell Associated Antigens
Lattice formation not required
Immunofluorescence
• Direct– Ab to tissue Ag is labeled with fluorochrome
Ag
FluorochromeLabeled Ab
Tissue Section
Immunofluorescence
Indirect Ab to tissue Ag is
unlabeled Fluorochrome-labeled
anti-Ig is used to detect binding of the first Ab.
Ag
FluorochromeLabeled Anti-Ig
Tissue Section
UnlabeledAb
• Qualitative to Semi-Quantitative
Assays Based on Complement
Lattice formation not required
Complement Fixation
Ag mixed with test serum to be assayed for Ab– Standard amount of complement is added– Erythrocytes coated with Abs is added– Amount of erythrocyte lysis is determined
Ag
Patient’sserum
Ag No Ag
Ag