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Ag-Ab Reactions Tests For Ag-Ab Reactions
Ag-Ab Reactions Tests For Ag-Ab Reactions
Ag-Ab Reactions Tests For Ag-Ab Reactions
• 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
Ab Ab
Ag Ag
Ag + Ab Ag-Ab
[Ag-Ab]
Keq =
[Ag] x [Ab]
Avidity
• The overall strength of binding between an Ag
with many determinants and multivalent Abs
Cross reactions
Ag A Ag B Ag C
• Affinity
• Avidity Ab excess Ag excess
• Ag:Ab ratio
• Physical form of Ag
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
1/1024
1/256
1/512
1/128
1/16
1/64
1/32
Neg.
Pos.
1/8
1/4
1/2
Patient Titer
1 64
2 8
3 512
4 <2
5 32
6 128
7 32
8 4
Agglutination/Hemagglutination
• Definition
• Qualitative test
1/256
1/128
1/512
1/16
1/64
1/32
1/8
• Quantitative test
1/4
1/2
• Applications
– Blood typing
– Bacterial infections
–Fourfold rise in titer
• Practical considerations
– Easy
– Semi-quantitative
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
+ ↔
Step 1
+ ↔
Patient’s Target
Serum 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 Ag Ag Ag
– Ag in a well
• Interpretation
– Diameter of ring is
proportional to the
concentration Diameter2
• Quantitative
– Ig levels
Ag Concentration
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 Ab
• Qualitative
–Rapid
Radioimmuoassays (RIA)
Enzyme-Linked Immunosorbent
Assays (ELISA)
Fluorochrome
Labeled Ab
Ag
Tissue Section
Immunofluorescence
• Indirect
– Ab to tissue Ag is
unlabeled Fluorochrome
Labeled Anti-Ig
– Fluorochrome-labeled anti- Unlabeled
Ab
Ig is used to detect binding
of the first Ab.
Ag
• Qualitative to Semi- Tissue Section
Quantitative
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
FITC-labeled cells
Ag No Ag
Ag
Patient’s
serum
Ag