6. 1. Strength of Ag-Ab Interactions FIGURE 6-2 (a) Determination of Ab affinity ( Ka ) by equilibrium dialysis. Semipermeable membrane A radioactively labeled ligand that is small to pass through ( haptens, oligopeptides )

7. 1. Strength of Ag-Ab Interactions Plot of concentration of ligand in each compartment with time The difference in the two compartments:

8. 1. Strength of Ag-Ab Interactions FIGURE 6-3 (a) Scatchard plots are based on repeated equilibrium dialyses with a constant concentration of Ab and varying concentration of ligand. ( # 1 Ab has a higher affinity than Ab # 2 ) ( all Ab have the same affinity ) (equals moles of bound ligand / mole Ab ) (The binding sites per Ab ) ( c is the conc. Of free ligand )

9. 1. Strength of Ag-Ab Interactions FIGURE 6-3 (b) Scatchard plots yield a curved line whose slope is constantly changing, if Ab preparation is polyclonal. >>> antiserum # 3 has a higher affinity than #4

11. 2. Cross-Reactivity ABO blood types - The antibodies are induced by exposure to cross-reacting microbial antigens present on common intestine bacteria. - ABO blood-group antigens have subtle differences in the terminal residues of the sugars on glyco-proteins in RBC. - Providing the basis for blood typing test in blood transfusion

12. 3. Precipitation Reactions Precipitation reactions in fluids yield a precipitin curve. FIGURE 6-4 ( Lattices or large aggregates ) ( no precipitate is formed if an Ag contains only a single copy of each epitope )

13. 3. Precipitation Reactions FIGURE 6-5 Diagrammatic representation of radial & double immunodiffusion. : precipitation reactions in gels yield visible precipitin lines; no visible precipitate forms in regions of Ab or Ag excess. in the Ab-containing semisolid medium The region of equivalence -> The area is proportional to the conc. of Ag.

16. FIGURE 6-8 -The original home pregnancy test kit employed hapten inhibition ( agglutination inhibition ) to determine the presence or absence of h uman c horionic g onadotropin (HCG) >>> The kits currently on the market use ELISA-based assays . -Also used to determine the use of illegal drugs, & immunity (Ab) to virus (rubella). 4. Agglutination Reactions

17. Sensitivity of various immunoassays

19. 6. ELISA FIGURE 6-10 Variations in the enzyme-linked immunosorbent assay (ELISA) technique, similar to RIA except using an Enzyme (alkaline ⓟ, horseradish peroxidase, & β-galactosidase) : safer & less costly. to detect Ab (HIV, HCV) to detect Ag to detect Ag

20. FIGURE 6-11 The ELISPOT assay, a modification of the ELISA assay to determine qucontitatively the # of cells in a population that are producing specific Ab or cytokine. 6. ELISA -> precipitates & forms a spot only on the areas of the well where cytokine-secreting cells had been deposited.

21. FIGURE 6-12 7. Western blotting : separates the components according to their molecular weight. : the proteins in the gel are transferred to the sheet of nitrocellulose or nylon by the passage of an electric current. : probed with Ab & then radiolabeled or enzyme-linked 2 nd Ab. : a position is visualized by means of an ELISA reaction.

24. FIGURE 6-15 Separation of fluorochrome-labeled cells with the flow cytometer which uses a laser beam & light detector. : different Ag in different cells / different levels of Ag in the same type of cell -> fluorescence intensity / the size of cells. 10. Flow cytometry & Fluorescence labeled with fluorescein (green) labeled with rhodamine (red) each dot represents a cell (small electrical change) (exciting the fluorochrome) ↓ each droplet (cell) emits the fluorescence

25. 11. Alternalives to Ag-Ab Reactions Ag-Ab-Ab* ->Ag-IgG-A/G* or Ag-Ab-biotin-(a)vidin* ① Protein A (from staphylococcus) & protein G (from streptococcus) - bind to rhe Fc region of lgG molecules (k a ～ 10 8 ) - used to detect lgG molecules in the Ag-Ab complexes - used to isolate lgG molecules in the affinity columns ② Avidin (from egg whites) & streptavidin (from streptomyces avidinii) conjugated with an enzyme, fluorechrome, radioactive label) - bind to biotin (a vitamin) with higher affinity (k a ～ 10 15 ) - Ab can be labeled with (k a ～ 10 18 )

27. CLINICAL FOCUS Distribution of selected markers on some leukemic cell types (leukemia can wise at any maturational stage of any one of the hematopoietic lineages) -> Immuno phenotyping: the determination of the profile of selected cell- surface markers displayed by the leukemic cell, using “flow cytometry & mAb” [: a cute l ymphocytic l eukemia] [: c hronic l ymphocytic l eukemia]