1. Principles of Immunology Antigen-Antibody Interactions 4/25/06

2. Word/Terms List Agglutinin EIA Equivalence zone FIA Immunodiffusion
Immunoelectrophoresis
RIA
Titer

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. 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.

5. 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

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

7. 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

8. Agglutination Tests
Lattice Formation

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

10. 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

11. 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

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

13. 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

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

15. Precipitation Tests
Lattice Formation

16. Radial Immunodiffusion
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

17. Immunoelectrophoresis
Method
Ags are separated by electrophoresis Ag - + Ag Ab Ag Ab
Interpretation
Precipitin arc represent individual antigens

18. Immunoelectrophoresis
Method
Interpretation
Qualitative
Relative concentration

19. Radioimmuoassays (RIA) Enzyme-Linked Immunosorbent Assays (EIA)
Lattice formation not required

20. Competitive RIA/ELISA for AgY + 
Prior to Test
Labeled 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 Y + 
Test
Patient’s
sample
Labeled Ag

21. Solid Phase Non-Competitive RIA/ELISAY Ag
Immobilized
Ab in
Patient’s
sample
Labeled
Anti-Ig
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

22. Solid Phase Non-Competitive RIA/ELISAY Ag
Immobilized
Ag in
Patient’s
sample
Labeled Ab
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

23. Tests for Cell Associated Antigens
Lattice formation not required

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

25. Immunofluorescence Y Qualitative to Semi-Quantitative Indirect
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

26. Assays Based on Complement
Lattice formation not required

27. Y Complement Fixation 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