Principles of Immunology
Antigen-Antibody Interactions
4/25/06
Word/Terms List
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Agglutinin
EIA
Equivalence zone
FIA
Immunodiffusion
Immunoelectrophoresis
RIA
Titer
Affinity
• Strength of the reaction between a single antigenic
determinant and a single Ab combining site
High Affinity
Low Affinity
Ab
Ab
Ag
Ag
Affinity =  attractive and repulsive forces
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
Cross reactions
Anti-A
Ab
Anti-A
Ab
Anti-A
Ab
Ag A
Ag B
Ag C
Shared epitope
Similar epitope
Factors Affecting Measurement of
Ag/Ab Reactions
• Affinity
• Avidity
Ab excess
Ag excess
• Ag:Ab ratio
• Physical form of Ag
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
Neg.
Pos.
1/1024
1/512
1/256
1/128
1/64
1/32
1/16
1/8
1/4
Patient
1/2
 Quantitative agglutination test
 Titer
 Prozone
Titer
1
2
3
64
8
512
4
5
<2
32
6
7
8
128
32
4
• Applications
– Blood typing
– Bacterial infections
–Fourfold rise in titer
• Practical considerations
– Easy
– Semi-quantitative
1/512
1/256
1/128
1/64
1/32
1/16
1/8
1/4
 Definition
 Qualitative test
 Quantitative test
1/2
Agglutination/Hemagglutination
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
• Definition
 Applications
 Measurement of soluble Ag
 Practical considerations
 Same as agglutination test
Precipitation Tests
Lattice Formation
Radial Immunodiffusion
• Method
Ab in gel
– Ab in gel
– Ag in a well
Ag
Ag
Ag
 Diameter of ring
is proportional to
the
concentration
 Quantitative
Diameter2
 Interpretation
 Ig levels
Ag Concentration
Ag
Immunoelectrophoresis
 Method
 Ags are separated by electrophoresis
+
Ag
Ag
Ab
Ag
Ab
•
Interpretation
– Precipitin arc represent individual antigens
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
– Use predetermined
amounts of labeled Ag
and Ab and add a
sample containing
unlabeled Ag as a
competitor
Prior to Test

+
Labeled
Ag
Test
+
Labeled
Ag
+

Patient’s
sample
+
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
Labeled
Anti-Ig
Ab in
Patient’s
sample
Immobilized
Ag
Solid
Phase
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
Labeled
Ab
Ag in
Patient’s
sample
Ag
Immobilized
Solid
Phase
Tests for Cell Associated
Antigens
Lattice formation not required
Immunofluorescence
• Direct
– Ab to tissue Ag is labeled with fluorochrome
Fluorochrome
Labeled Ab
Ag
Tissue Section
Immunofluorescence
 Indirect
 Ab to tissue Ag is
unlabeled
 Fluorochrome-labeled
anti-Ig is used to
detect binding of the
first Ab.
• Qualitative to SemiQuantitative
Unlabeled
Ab
Fluorochrome
Labeled Anti-Ig
Ag
Tissue Section
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
No Ag
Ag
Ag
Patient’s
serum
Ag