Antigen-Antibody Interactions: Principles & Applications <ul><li>- a bimolecular association </li></ul><ul><li>involving various noncovalent interactions </li></ul><ul><li>Is similar to an enzyme-substrate interactions, </li></ul><ul><li>but not lead to an irreversible chemical alteration </li></ul>Chapter 6
FIGURE 6-1 <ul><li>Four types of non-covalent forces operates over a very short distance </li></ul><ul><li>( generally 1 angstrom ) </li></ul><ul><li>- The interaction depends on a very close fit between the Ab & Ag. </li></ul>
<ul><li>Strength of Antigen-Antibody Interactions </li></ul><ul><li>Cross-Reactivity </li></ul><ul><li>Precipitation Reactions </li></ul><ul><li>Agglutination Reactions </li></ul><ul><li>Radioimmunoassay </li></ul><ul><li>Enzyme-Linked Immunosorbent Assay </li></ul><ul><li>Western Blotting </li></ul><ul><li>Immunoprecipitation </li></ul><ul><li>Immunofluorescence </li></ul><ul><li>Flow Cytometry and Fluorescence </li></ul><ul><li>Alternatives to Antigen-Antibody Reactions </li></ul><ul><li>Immunoelectron Microscopy </li></ul>
1. Strength of Ag-Ab Interactions <ul><li>Antibody affinity </li></ul><ul><li>is a quantitative measure of binding strength </li></ul><ul><li>combined strength of the noncovalent interactions </li></ul><ul><li>between a binding site on an Ab & monovalent Ag </li></ul><ul><li>Antibody avidity </li></ul><ul><li>Incorporates affinity of multiple binding sites </li></ul><ul><li>True strength of the Ab-Ag interaction within biological systems </li></ul><ul><li>The interaction at one site will increase the possibility </li></ul><ul><li>of reaction at a second site </li></ul><ul><li>High avidity can compensate for low affinity </li></ul><ul><li>( secreted pentameric IgM has a higher avidity than IgG ) </li></ul>
1. Strength of Ag-Ab Interactions <ul><li>High affinity complexes have high Ka values </li></ul><ul><li>Very stable complexes have very low values of Kd </li></ul>Forward & reverse rate constants ( k1 & k-1) Association & dissociation constants ( Ka & Kd ) for 3 ligand-Ab interaction
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 )
1. Strength of Ag-Ab Interactions Plot of concentration of ligand in each compartment with time The difference in the two compartments:
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 )
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
2. Cross-Reactivity CROSSREACTIVITY (i) Cowpox antigens in vaccinia virus are cross-reactive to smallpox antigens in variola virus (∵ share similar or identical epitope ) (ii) Rabies & JE vaccine >>> encephalitis ( 뇌염 ) (: rabbit brain antigen contaminated vs human brain Ag ) (iii) Streptococcus pyogenes infection >>> heart & Kidney damage following infection (: cell wall proteins called M antigens vs Myocardial & skeletal muscle proteins ). (iv) Original antigenic sin. - The existence of long-lived lymphocytes & crossreactivity - Vaccination with one strain of flu elicited Ab responses to another flu strain. (v) cross-reacting bacterial Ag vs glycoproteins on RBC) <ul><li>Antibody elicited by one Ag can cross-react with unrelated Ag. </li></ul><ul><li>occurs if two different Ags share identical or very similar epitope </li></ul>
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
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 )
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.
3. Precipitation Reactions FIGURE 6-6 (a) <ul><li>Immunoelectrophoresis. </li></ul><ul><li>an antigen mixture is first electrophoresed to separate its components by charge </li></ul><ul><li>diffusion & producing lines of precipitation. </li></ul>
FIGURE 6-7 Demonstration of humaglutination using Ab against sheep red blood cells (SRBCs): a constant # of SRBCs plus serial two-fold dilutions of anti-SRBC serum 4. Agglutination Reactions <ul><li>visible clumping by interaction between Ab & a particulate antigen suchas RBC, </li></ul><ul><li>latex beads. </li></ul><ul><li>-depend on the crosslinking of polyvalent antigens, similar to precipitation rxns </li></ul><ul><li>(lgM is a good agglutinin) </li></ul><ul><li>-provide a way to type bacteria with a panel of typing antisera. </li></ul><ul><li>-routinely performed to type RBCs for blood transfusion. </li></ul>+ + + (control)
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
5. Radioimmuno Assay <ul><li>One of the most sensitive technique for measuring hormones, drugs, & vitamins </li></ul><ul><li>at conc. Of<0.001 ㎍ / ㎖ first discovered by Dr. Berson & Yalow in 1960 </li></ul><ul><li>(1977 Novel prize to Yalow) </li></ul><ul><li>- The principle involves competitive binding of radiolabeled Ag and unlabeled Ag </li></ul><ul><li>to the limited supply of a high affinity Ab. </li></ul>FIGURE 6-9 A solid-phase radioimmunoassay (RIA) to detect hepatitis B virus in blood samples & A standard curve to determine the conc. of HBsAg in unknown serum.
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
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.
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.
FIGURE 6-13 Immunoprecipitates can be collected using magnetic beads coupled to a secondary antibody. 8. Immunoprecipitation <ul><li>Ag-Ab attached to a synthetic bead complex >>> 0 </li></ul><ul><li>labeling Ag with radiolabeled leucine, cysteine, or methionine </li></ul><ul><li>-> A radiolabeled Ag-Ab complex -> 0 -> SDS•PAGE -> autoradiography </li></ul><ul><li>- Ag-Ab complex + 2 nd Ab attatched to a synthetic bead or magnetic beads >>> 0 or magnet </li></ul>EM showing a cell with magnetic beads attached to its surface via antibodies.
9. Immunofluorescence mIgM-producing B cells indirectly stained with rhodamine-conjugated secondary Ab under a fluorescence microscope . FIGURE 6-14 <ul><li>Fluorochromes </li></ul><ul><li>Fluorescein (490->517nm) </li></ul><ul><li>Rhodamine (515->546nm) </li></ul><ul><li>Phycoerythrin </li></ul><ul><li>: absorb light of one wavelength & emit </li></ul><ul><li>fluorescence at a longer wavelength than </li></ul><ul><li>fluorescein. </li></ul>
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
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 )
FIGURE 6-16 <ul><li>An immunoelectronmicrograph of the surface of a B-cell lymphoma was stained with two antibodies (Ab against class II MHC labeled sith 30nm gold particles, & another Ab against class I MHC w/ 15nm gold particles. </li></ul><ul><li>(The density of class I exceeds that of class II) </li></ul><ul><li>Electron-dense label (ferritin or colloidal gold) is conjugated to the Fc </li></ul><ul><li>portion. </li></ul>12. Immuno EM. electron-denselabels Absorbs electrons.
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]