This document summarizes various antigen-antibody interaction techniques including: precipitation reactions, agglutination reactions, radioimmunoassay, enzyme-linked immunosorbent assay, Western blotting, immunoprecipitation, immunofluorescence, flow cytometry, and alternatives like protein A/G and biotin-avidin interactions. It describes how each technique detects and analyzes the binding between antigens and antibodies through measurements, visualizations, or separations based on properties like molecular weight, charge, or fluorescent labeling.

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]