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True strength of the Ab-Ag interaction within biological systems
The interaction at one site will increase the possibility
of reaction at a second site
High avidity can compensate for low affinity
( secreted pentameric IgM has a higher avidity than IgG )
1. Strength of Ag-Ab Interactions
High affinity complexes have high Ka values
Very stable complexes have very low values of Kd
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)
Antibody elicited by one Ag can cross-react with unrelated Ag.
occurs if two different Ags share identical or very similar epitope
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)
an antigen mixture is first electrophoresed to separate its components by charge
diffusion & producing lines of precipitation.
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
visible clumping by interaction between Ab & a particulate antigen suchas RBC,
-depend on the crosslinking of polyvalent antigens, similar to precipitation rxns
(lgM is a good agglutinin)
-provide a way to type bacteria with a panel of typing antisera.
-routinely performed to type RBCs for blood transfusion.
+ + + (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
Sensitivity of various immunoassays
5. Radioimmuno Assay
One of the most sensitive technique for measuring hormones, drugs, & vitamins
at conc. Of<0.001 ㎍ / ㎖ first discovered by Dr. Berson & Yalow in 1960
(1977 Novel prize to Yalow)
- The principle involves competitive binding of radiolabeled Ag and unlabeled Ag
to the limited supply of a high affinity Ab.
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
Ag-Ab attached to a synthetic bead complex >>> 0
labeling Ag with radiolabeled leucine, cysteine, or methionine
- Ag-Ab complex + 2 nd Ab attatched to a synthetic bead or magnetic beads >>> 0 or magnet
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
: absorb light of one wavelength & emit
fluorescence at a longer wavelength than
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 )
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.
(The density of class I exceeds that of class II)
Electron-dense label (ferritin or colloidal gold) is conjugated to the Fc
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]