1. Ag-Ab reactions Tests for Ag-Ab reactions

2. Nature of Ag/Ab Reactions
Source: Li, Y., Li, H., Smith-Gill, S. J., Mariuzza, R. A., Biochemistry 39, 6296, 2000
Lock and Key Concept
Non-covalent Bonds
Hydrogen bonds
Electrostatic bonds
Van der Waal forces
Hydrophobic bonds
Multiple Bonds
Reversible

3. Affinity = ∑ attractive and repulsive forces
Strength of the reaction between a single antigenic determinant and a single Ab combining site Ab Ag
High Affinity Ab Ag
Low Affinity
Affinity = ∑ attractive and repulsive forces

4. Calculation of Affinity
Ag + Ab Ag-Ab
Applying the Law of Mass Action:
Keq =
[Ag-Ab]
[Ag] x [Ab]

5. Avidity
The overall strength of binding between an Ag with many determinants and multivalent Abs Y
Keq =
104
Affinity Y
106
Avidity Y
1010
Avidity

6. 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.

7. Cross Reactivity Cross reactions
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 C
Similar epitope
Cross reactions
Anti-A Ab
Ag A
Anti-A Ab
Ag B
Shared epitope

8. Factors Affecting Measurement of Ag/Ab Reactions
Ab excess
Ag excess
Affinity
Avidity
Equivalence – Lattice formation
Ag:Ab ratio
Physical form of Ag

9. 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

10. Agglutination Tests
Lattice Formation

11. Agglutination/Hemagglutination
Definition - tests that have as their endpoint the agglutination of a particulate antigen
Agglutinin/hemagglutinin Y + ↔
Qualitative agglutination test
Ag or Ab

12. Agglutination/Hemagglutination
Quantitative agglutination test
Titer
Prozone
1/2
1/4
1/8
1/16
1/32
1/64
1/128
1/256
1/512
1/1024
Pos.
Neg.
Titer 64 8
512
<2 32
128 4
Patient 1 2 3 5 6 7

13. Agglutination/Hemagglutination
Definition
Qualitative test
Quantitative test
1/2
1/4
1/8
1/16
1/32
1/64
1/128
1/256
1/512
Applications
Blood typing
Bacterial infections
Fourfold rise in titer
Practical considerations
Easy
Semi-quantitative

14. Passive Agglutination/Hemagglutination
Definition - agglutination test done with a soluble antigen coated onto a particle Y + ↔
Applications
Measurement of antibodies to soluble antigens

15. Coombs (Antiglobulin)Tests
Incomplete Ab
Direct Coombs Test
Detects antibodies on erythrocytes + ↔ Y
Patient’s RBCs
Coombs Reagent
(Antiglobulin)

16. Coombs (Antiglobulin)Tests
Indirect Coombs Test
Detects anti-erythrocyte antibodies in serum Y
Patient’s
Serum
Target
RBCs + ↔
Step 1 + ↔ Y
Coombs Reagent
(Antiglobulin)
Step 2

17. Coombs (Antiglobulin)Tests
Applications
Detection of anti-Rh Ab
Autoimmune hemolytic anemia

18. Agglutination/Hemagglutination Inhibition
Definition - test based on the inhibition of agglutination due to competition with a soluble Ag Y + ↔
Prior to Test Y + ↔
Test
Patient’s sample

19. Agglutination/Hemagglutination Inhibition
Definition
Applications
Measurement of soluble Ag
Practical considerations
Same as agglutination test

20. Precipitation Tests
Lattice Formation

21. Radial Immunodiffusion (Mancini)
Method
Ab in gel
Ag in a well Ag
Ab in gel
Ag Concentration
Diameter2
Interpretation
Diameter of ring is proportional to the concentration
Quantitative
Ig levels

22. 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

23. Immunoelectrophoresis
Method
Interpretation
Qualitative
Relative concentration

24. 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

25. Radioimmuoassays (RIA) Enzyme-Linked Immunosorbent Assays (ELISA)
Lattice formation not required

26. Competitive RIA/ELISA for Ag
Method
Determine amount of Ab needed to bind to a known amount of labeled Ag Y + ↔
Prior to Test
Labeled Ag
Use predetermined amounts of labeled Ag and Ab and add a sample containing unlabeled Ag as a competitor Y + ↔
Test
Patient’s
sample
Labeled Ag

27. Competitive RIA/ELISA for Ag
Method cont.
Determine amount of labeled Ag bound to Ab
↓ NH4SO4
↓ anti-Ig
Immobilize the Ab Y + ↔
Test
Patient’s
sample
Labeled Ag
Solid
Phase
Concentration determined from a standard curve using known amounts of unlabeled Ag
Quantitative
Most sensitive test

28. 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 Y Ag
Immobilized
Ab in
Patient’s
sample
Labeled
Anti-Ig
Quantitative

29. 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 Y Ag
Immobilized
Ag in
Patient’s
sample
Labeled Ab
Quantitative

30. Tests for Cell Associated Antigens
Lattice formation not required

31. Immunofluorescence Direct Y
Ab to tissue Ag is labeled with fluorochrome Ag Y
Fluorochrome
Labeled Ab
Tissue Section

32. Immunofluorescence Indirect Y Qualitative to Semi-Quantitative
Ab to tissue Ag is unlabeled
Fluorochrome-labeled anti-Ig is used to detect binding of the first Ab. Ag Y
Fluorochrome
Labeled Anti-Ig
Tissue Section
Unlabeled Ab
Qualitative to Semi-Quantitative

33. 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

34. Green Fluorescence Intensity Two Parameter Histogram
Immunofluorescence
Flow Cytometry cont.
Data displayed
Red Fluorescence Intensity
Green Fluorescence Intensity
Two Parameter Histogram
One Parameter Histogram
Unstained cells
Number of Cells
FITC-labeled cells
Green Fluorescence Intensity

35. Assays Based on Complement
Lattice formation not required

36. Complement Fixation Y Methodology Ag No Ag
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
No Ag Ag Y
Patient’s
serum Ag Y