Principles of Immunology

Antigen-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
/
1
6

1
/
3
2

1
/
6
4

1
/
1
2
8

1
/
2
5
6

1
/
5
1
2

1
/
1
0
2
4

P
o
s
.

N
e
g
.

Titer
64
8
512
<2
32
128
32
4
Patient
1
2
3
4
5
6
7
8
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
/
1
6

1
/
3
2

1
/
6
4

1
/
1
2
8

1
/
2
5
6

1
/
5
1
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
Patients 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
D
i
a
m
e
t
e
r
2

Ag Ag Ag Ag
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
+
Patients
sample
Labeled
Ag
+

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
Solid
Phase
Ag
Immobilized
Ab in
Patients
sample
Labeled
Anti-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
Solid
Phase
Ag
Immobilized
Ag in
Patients
sample
Labeled
Ab
Tests for Cell Associated
Antigens
Lattice formation not required
Immunofluorescence
Direct
Ab to tissue Ag is labeled with fluorochrome
Ag
Fluorochrome
Labeled 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
Fluorochrome
Labeled Anti-Ig
Tissue Section
Unlabeled
Ab
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
Patients
serum

Ag No Ag
Ag