Ag-Ab reactions

Tests for Ag-Ab reactions

Nature of Ag/Ab Reactions
Lock and Key Concept
Non-covalent Bonds
Hydrogen bonds
Electrostatic bonds
Van der Waal forces
Hydrophobic bonds
Reversible
Multiple Bonds
Source: Li, Y., Li, H., Smith-Gill, S. J.,
Mariuzza, R. A., Biochemistry 39, 6296, 2000
http://www.med.sc.edu:85/chime2/lyso-abfr.htm
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
Calculation of Affinity
Ag + Ab Ag-Ab
K
eq
=
[Ag-Ab]
[Ag] x [Ab]
Applying the Law of Mass Action:
Avidity
The overall strength of binding between an Ag
with many determinants and multivalent Abs
K
eq
=
10
4

Affinity
10
6

Avidity
10
10

Avidity
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
Coombs (Antiglobulin)Tests
Incomplete Ab
Direct Coombs Test
Detects antibodies on erythrocytes
+


Patients RBCs Coombs Reagent
(Antiglobulin)
Coombs (Antiglobulin)Tests
Indirect Coombs Test
Detects anti-erythrocyte antibodies in serum
Patients
Serum
Target
RBCs
+


Step 1
+

Coombs Reagent
(Antiglobulin)
Step 2
Coombs (Antiglobulin)Tests
Applications
Detection of anti-Rh Ab
Autoimmune hemolytic anemia
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 (Mancini)
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
Ab is placed in trough cut in the agar
Immunoelectrophoresis
Method
Interpretation
Qualitative
Relative concentration
Countercurrent electrophoresis
Method
Ag and Ab migrate toward each other by
electrophoresis
Used only when Ag and Ab have opposite charges
Qualitative
Rapid
Ag
Ab
-
+
Radioimmuoassays (RIA)
Enzyme-Linked Immunosorbent
Assays (ELISA)
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
Competitive RIA/ELISA for Ag
Method cont.
Determine amount
of labeled Ag bound
to Ab
NH
4
SO
4

anti-Ig
Immobilize the Ab
Quantitative
Most sensitive test
+

Test
+
Patients
sample
Labeled
Ag
+

Concentration determined from a standard curve
using known amounts of unlabeled Ag
Solid
Phase
Solid
Phase
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
Quantitative
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
Quantitative
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

Immunofluorescence
Flow Cytometry
Cells in suspension are labeld with fluorescent tag
Direct or Indirect Fluorescence
Cells analyzed on a flow cytometer
Flow
Tip
Laser
FL
Detector
Light
Scatter
Detector
Immunofluorescence
Flow Cytometry cont.
Data displayed
Green Fluorescence Intensity
N
u
m
b
e
r

o
f

C
e
l
l
s

Unstained cells
FITC-labeled cells
One Parameter Histogram
Red Fluorescence Intensity
G
r
e
e
n

F
l
u
o
r
e
s
c
e
n
c
e

I
n
t
e
n
s
i
t
y

Two Parameter Histogram
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
Methodology