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
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
Ab excess
Ag excess
Ag:Ab ratio
Physical form of Ag
Agglutination Tests
Lattice Formation
Agglutination/Hemagglutination
Definition - tests that have as their endpoint
the agglutination of a particulate antigen
Agglutinin/hemagglutinin
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
+
Patients RBCs
Coombs Reagent
(Antiglobulin)
Coombs (Antiglobulin)Tests
Indirect Coombs Test
Detects anti-erythrocyte antibodies in serum
Step 1
+
Patients
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
Patients sample
Agglutination/Hemagglutination Inhibition
Definition
Applications
Measurement of soluble Ag
Practical considerations
Same as agglutination test
Precipitation Tests
Lattice Formation
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
Prior to Test
Labeled
Ag
Test
+
Labeled
Ag
Patients
sample
Test
+
Solid Labeled
Ag
Phase
Patients
sample
Solid
Phase
Quantitative
Most sensitive test
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
Patients
sample
Immobilized
Ag
Solid
Phase
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
Patients
sample
Ag
Immobilized
Solid
Phase
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
Complement Fixation
Methodology
No Ag
Ag
Ag
Ag
Patients
serum