1. Immunology ANTIBODIES we have ~10 12 antibodies made against foreign viruses, bacteria, parasites (vaccines) antibodies combine with foreign antigens to inactivate foreign species antigenic determinants (epitopes) recognized by antibody (unique shape, 5-10 AA sequence) antibody containing serum called ANTISERUM Antibody (IgG) - antigen (small peptide from HIV)

2. ANTIBODIES structure “Y shape” two identical light chains, two identical heavy chains N-termini of light and heavy come together to form antigen binding site (unique AA here!) light heavy Hypervariable region antigen binds here Immunology

3. Western Blot IMMUNE SYSTEM antibodies found in the gamma globulin fraction of serum called immunoglobulins (Ig) - have IgG, IgA, IgD, IgE, IgM major class of Ig is IgG purified foreign proteins can act as antigens and after injection into a differing species they can stimulate antibody formation EX: inject rabbit IgG into goat to cause goat to make anti-rabbit IgG antibodies EX: vaccines - inject infant with attenuated, non-infective polio virus to cause baby to make antibodies to the antigen and acquire immunity to POLIO

4. Immunoassays - double diffusion specific Ag-Ab complexes undergo precipitation rxns that are visible in agar gel cut small wells in agar and place small amounts of Ag and Ab in wells Ag and Ab diffuse outward toward each other at rates related to [], size and shape milky white line of precipitation is seen where Ag contacts Ab

5. Immunoassay - Western Blot Abs used in Western blots to identify specific protein use of protein gels to identify purification steps purified foreign proteins can serve as antigens because they will stimulate Ab formation when injected into animals

6. Western Blot

7. Immunoassay - ELISA (enzyme linked immunosorbent assay)

8. ELISA (enzyme linked immunosorbent assay)

9. ELISA Prep of microtiter plate Adsorb Ag to plate make serial dilutions block addition sites with TBS-gelatin (blocking agent) remove liquid in specific order!!! Pay attention to the details!! reapply TBS-gelatin - specific order!!! Ab reaction add Ab to each well blocking step remove liquid in specific order!! addition of solutions with subsequent removal of liquid in specific order!! Color development add color development to each well observe blue color change pH of solution in well by adding HCl - observe yellow color

10. ELISA TMB Soluble Colorless substrate TMB Insoluble Blue Product TMB Insoluble Yellow Product HCl