1. Lecture 14 Antibody-Antigen Reactions

2. Binding of the epitope in the antigen binding site
POOR FIT
GOOD FIT
antibody combining site
Study Guide
What constitutes a good fit in an antigen binding site?
How does antibody affinity relate to fit?
What are the forces that are attractive and repulsive?
What is actually "fitting" into the combining site?
antigen determinant
high attraction
low repulsion
high repulsion
low attraction

3. Antibody Avidity
Multiple interactions between antigen binding sites and epitopes
Greater than additive
Similar to Velcro™
More relevant to biologic systems than affinity
Study Guide
How does avidity differ from affinity?
Do you like rabbits?

4. Effect of multivalent interactions
antibody
Fab
IgG
IgG
IgM
effective antibody valence 1 1 2
up to 10
antigen valence 1 1 n n
equilibrium constant (L/M) 10 4 10 4 10 7 10 11
Study Guide
What is the advantage of making IgM antibody?
advantage of multi-valence - - 10 3
-fold 10 7
-fold
definition of bindng
affinity
affinity
avidity
avidity
intrinsic affinity
functional affinity

5. Biological Consequences of Antibody Affinity/Avidity
Neutralization of toxins
Complement activation
Immune elimination of antigen
Virus neutralization
More intense immune complex disease in animals
higher levels of circulating antigen-antibody complexes
more intense localization of immune complexes on basement membranes.
more severe impairment of organ function
Study Guide
What are the biological consequences of producing low avidity antibody?

6. Cross-Reaction Immunogen Reactivity with rabbit antiserum A B C D
Immunize
Immunogen
anti-A
anti-B
anti-C
anti-D
Study Guide
How can the immune response to one microorganism react with a microorganism of a different genus and species?
What is an epitope?
Cross-reaction is due to the sharing of epitopes between two different antigens. Antibodies made against shared epitopes will react with both antigens.
This is often the cause of “false positives” in immunological tests.
Reactivity with
rabbit
antiserum A B C D A E F G
Cross-reactive antigen

7. Primary Interactions Between Antigen and Antibody
Determined by the interaction between epitopes and antigen-binding sites
Independent of biochemical and biologic phenomena that may follow
Depend only on the quantity and quality (affinity/avidity) of antibodies
The basis for ELISA, RIA, IFA assays

8. Enzyme (ELISA) Immunoassay
Enzyme conjugated to anti-Ig antibody (“second antibody”)
Add
substrate
for enzyme
anti-antigen
antibody
Study Guide
Radioimmunoassays (RIA) and Immunfluorescence Assays (IFA) depend on the detection of a radionuclide or a fluorescent probe attached to the second antibody, instead of the enzyme. In those cases, no substrate is needed.
How would you use this assay to measure the concentration or detect the presence of an antigen?
Antigen

9. Immune Precipitation Antigen Antibody Study Guide
Why do soluble antigens and antibodies precipitate?
Antigen
Antibody

10. Radial Immunodiffusion
Ab-containing gel
Precipitin
ring
Ag/8
Ag/4
Ag/2
Ag/1
D2 Ring
Antigen Concentration

11. Agglutination RBC RBC RBC IgM Antibody RBC RBC RBC RBC RBC
Study Guide
What is the difference between precipitation and agglutination?
IgG Antibody
RBC
RBC
RBC

12. Complement activation
A system of plasma proteins that interact with
Antigen/antibody complexes
Pathogen surface motifs (alternative and lectin pathways
Activation of complement results in
Chemo-attraction of inflammatory cells
Peptide mediators of inflammation (anaphylatoxins)
Increased blood vessel permeability
Smooth muscle contraction
Mast cell degranulation
Opsonization of pathogens (enhances phagocytosis)
Killing of pathogens (membrane attack complex)
Can you identify examples of receptor-ligand interactions that occur during complement activation?

13. Overview of the Complement Cascade

14. Effector Actions of Complement

15. Terms to remember Affinity Agglutination Avidity Chemotaxis Complement
Classical pathway
Alternative (properdin) pathway
Cross-reaction
Epitope/Antigenic Determinant
Opsonization
Precipitation
Second antibody Specificity
Valence