1. 2. Basic Immunologic Procedures
Terry Kotrla, MS, MT(ASCP)BB

2. Introduction Detection of antigen/antibody reactions difficult
Can measure EITHER antigen or antibody.
Sensitization is the binding of a specific antibody to its’ specific antigen
Sensitization cannot be visualized
Multitude of laboratory methods have been developed to make this reaction visible

3. Factors Which Affect Reactions
Concentrations of reactants
Temperature
Length of incubation
pH of test system

4. Three Distinct Phases of Antigen/Antibody Reactions
Primary Phenomenon – Sensitization
Secondary Phenomenon – Lattice formation
Tertiary Phenomenon – Detected by affect on tissues or cells.

5. Primary phenomenon
Sensitization – binding of antibody to antigen – not visible

6. Primary phenomenon
Sensitization – binding of single antibody to single antigen site
These tests are
Difficult
Complex
Expensive
Require special equipment
Time consuming

7. Primary Phenomenon Tests
Techniques include:
Immunofluorescence
Radioimmunoassay
Enzyme immunoassay

8. Secondary Phenomenon
Sensitization taken a step further to lattice formation
Fab of Antibody molecule binds to two separate antigens on adjacent antigens
If antigen on large structures such as RBCs causes agglutination.
If both antibody and antigen are soluble results in precipitation

9. Secondary Phenomenon Reactions

10. Secondary Phenomenon These tests are: Downside is Easy to perform
Less expensive
Less time consuming
Do not require special equipment
Downside is
Less sensitive
Less specific
More interference

11. Secondary Phenomenon Examples of tests: Precipitation Agglutination
Complement Fixation

12. Tertiary Phenomenon
Reaction not visible, detected by affect of reaction on tissues or cells.
Tests include:
Inflammation
Phagocytosis
Deposition of immune complexes
Immune adherence
Chemotaxis

13. Phagocytosis

14. Secondary Phenomena Most Frequently Utilized
Precipitation – soluble antibody reacts with soluble antigen
Agglutination – particulate antigens bound together by antibody to form visible complex
Complement Fixation – antibody binding to antigen triggers activation of complement, results in cell lysis

15. Antigen-Antibody Binding
Union of antigen and antibody requires
Affinity
Avidity
Affinity and avidity determined by Law of Mass Action

16. Antibody Affinity Describes the strength of a single Ag-Ab bond.
As Ag and Ab come close together a chemical bond forms which is weak and can dissociate.
How well the Ab fits the Ag will determine stability of bond, “lock and key” fit has strongest affinity.
Ab may react with structurally similar Ags, results in cross reactivity.
Most antibodies have a high affinity for their antigens.

17. Affinity

18. Avidity Describes the combined strength of multiple Ag-Ab bonds.
Initially bond is easily broken, but multiple bindings at the same time the dissociation is overcome by the sheer number of bonds remaining.
Avidity is influenced by both the valence of the antibody and the valence of the antigen.

19. Avidity

20. Affinity versus Affinity
Affinity refers to the strength of binding between a single antigenic determinant and an individual antibody combining site whereas avidity refers to the overall strength of binding between multivalent antigens and antibodies.

21. IgM and IgG Most frequently detected immunoglobulins.
IgM has low affinity but high avidity due to 10 binding sites.
IgG has 2 strong binding sites, high affinity and avidity.

22. Law of Mass Action
Governs the reversibility of the antigen-antibody reaction.
Reversible reaction, visible reaction occurs when the rate of binding exceeds the rate of dissociation.
As affinity and avidity increases, strengthens reaction.

23. Precipitation Curve Depends on concentration of Ag and Ab.
Prozone – antibody excess, many antibodies coat all antigen sites- results in false negative
Postzone – antigen excess, antibody coats antigen but cannot get lattice formation, results in false negative
Zone of Equivalence – antigen and antibody present in optimal proportions to bind and give visible reaction

25. Precipitation Curve

26. Precipitation Curve