Ag-Ab reactions
Tests for Ag-Ab reactions
Nature of Ag/Ab Reactions
• Lock and Key Concept
http://www.med.sc.edu:85/chime2/lyso-abfr.htm
• Non-covalent Bonds
– Hydrogen bonds
– Electrostatic bonds
– Van der Waal forces
– Hydrophobic bonds
• Multiple Bonds
• Reversible
Source: Li, Y., Li, H., Smith-Gill, S. J.,
Mariuzza, R. A., Biochemistry 39, 6296, 2000
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
Calculation of Affinity
Ag + Ab
Ag-Ab
Applying the Law of Mass Action:
Keq =
[Ag-Ab]
[Ag] x [Ab]
Avidity
• The overall strength of binding between an Ag
with many determinants and multivalent Abs
Keq =
104
Affinity
106
Avidity
1010
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
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
• Quantitative agglutination test
Neg.
Pos.
1/1024
1/512
1/256
1/128
1/64
1/32
1/16
1/8
1/4
Patient
1/2
– Titer
– Prozone
Titer
1
2
3
64
8
512
4
5
<2
32
6
7
8
128
32
4
– 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
• Applications
– Blood typing
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
Coombs (Antiglobulin)Tests
• Incomplete Ab
• Direct Coombs Test
– Detects antibodies on erythrocytes
↔
+
Patient’s RBCs
Coombs Reagent
(Antiglobulin)
Coombs (Antiglobulin)Tests
• Indirect Coombs Test
– Detects anti-erythrocyte antibodies in serum
Step 1
↔
+
Patient’s
Serum
Target
RBCs
Step 2
+
↔
Coombs Reagent
(Antiglobulin)
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
+
+
↔
Patient’s sample
Agglutination/Hemagglutination Inhibition
• Definition
• Applications
– Measurement of soluble Ag
• Practical considerations
– Same as agglutination test
Precipitation Tests
Lattice Formation
Radial Immunodiffusion (Mancini)
• 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
– Ab is placed in trough cut in the agar
+
Ag
Ag
Ab
Ag
Ab
• Interpretation
– Precipitin arc represent individual antigens
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
-
+
Ag
• Qualitative
–Rapid
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
– 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
+
Competitive RIA/ELISA for Ag
• Method cont.
– Determine amount
of labeled Ag bound
to Ab
• ↓ NH4SO4
• ↓ anti-Ig
• Immobilize the Ab
Test
+
Solid Labeled
Ag
Phase
+
↔
Patient’s
sample
+
Solid
Phase
– Concentration determined from a standard curve
using known amounts of unlabeled Ag
• Quantitative
– Most sensitive test
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
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
• Quantitative
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 antiIg is used to detect binding
of the first Ab.
• Qualitative to SemiQuantitative
Unlabeled
Ab
Fluorochrome
Labeled Anti-Ig
Ag
Tissue Section
Immunofluorescence
• Flow Cytometry
– Cells in suspension are labeld with fluorescent tag
• Direct or Indirect Fluorescence
– Cells analyzed on a flow cytometer
Flow
Tip
FL
Detector
Light
Scatter
Detector
Laser
Immunofluorescence
• Flow Cytometry cont.
– Data displayed
Two Parameter Histogram
Number of Cells
Unstained cells
FITC-labeled cells
Green Fluorescence Intensity
Green Fluorescence Intensity
One Parameter Histogram
Red Fluorescence Intensity
Assays Based on Complement
Lattice formation not required
Complement Fixation
• Methodology
–
–
–
–
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