Lecture 14 Antibody-Antigen Reactions

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

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?

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

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?

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

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

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

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

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

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

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?

Overview of the Complement Cascade

Effector Actions of Complement

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