Antigen-AntibodyAntigen-AntibodyInteractions:Interactions:Principles & ApplicationsPrinciples & Applications

- a bimolecular association - a bimolecular association

involving various noncovalent interactionsinvolving various noncovalent interactions- Is similar to an enzyme-substrate interactions, Is similar to an enzyme-substrate interactions,

but not lead to an irreversible chemical alterationbut not lead to an irreversible chemical alteration

Chapter 6Chapter 6

FIGURE 6-1- Four types of non-covalent forces operates over a very short distance ( generally 1 angstrom )- The interaction depends on a very close fit between the Ab & Ag.

1.1. Strength of Antigen-Antibody InteractionsStrength of Antigen-Antibody Interactions

2.2. Cross-ReactivityCross-Reactivity

3.3. Precipitation ReactionsPrecipitation Reactions

4.4. Agglutination ReactionsAgglutination Reactions

5.5. RadioimmunoassayRadioimmunoassay

6.6. Enzyme-Linked Immunosorbent AssayEnzyme-Linked Immunosorbent Assay

7.7. Western BlottingWestern Blotting

8.8. ImmunoprecipitationImmunoprecipitation

9.9. ImmunofluorescenceImmunofluorescence

10.10. Flow Cytometry and FluorescenceFlow Cytometry and Fluorescence

11.11. Alternatives to Antigen-Antibody ReactionsAlternatives to Antigen-Antibody Reactions

12.12. Immunoelectron MicroscopyImmunoelectron Microscopy

1. Strength of Ag-Ab Interactions1. Strength of Ag-Ab Interactions

Antibody affinity- is a quantitative measure of binding strength- combined strength of the noncovalent interactions between a binding site on an Ab & monovalent Ag

Antibody avidity- Incorporates affinity of multiple binding sites- 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 Interactions1. Strength of Ag-Ab Interactions

Forward & reverse rate constants ( k1 & k-1)Association & dissociation constants ( Ka & Kd ) for 3 ligand-Ab interaction

- High affinity complexes have high Ka values- Very stable complexes have very low values of Kd

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 Interactions1. Strength of Ag-Ab Interactions

1. Strength of Ag-Ab Interactions1. Strength of Ag-Ab Interactions

Plot of concentration of ligand in each compartment with time

The difference in the two compartments:

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 Interactions1. 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

1. Strength of Ag-Ab Interactions1. Strength of Ag-Ab Interactions

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-Reactivity2. 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

2. Cross-Reactivity2. Cross-Reactivity

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 Reactions3. Precipitation Reactions

3. Precipitation Reactions3. Precipitation Reactions

FIGURE 6-5Diagrammatic 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 Reactions3. Precipitation Reactions

FIGURE 6-6 (a)

Immunoelectrophoresis.- 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 Reactions4. Agglutination Reactions

+ + + (control)

-visible clumping by interaction between Ab & a particulate antigen suchas RBC, latex beads.-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.

FIGURE 6-8-The original home pregnancy test kit employed hapten inhibition (agglutination inhibition) to determine the presence or absence of human chorionic gonadotropin (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 Reactions4. Agglutination Reactions

Sensitivity of various immunoassays

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.

5. Radioimmuno Assay 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 6-10Variations 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

6. ELISA 6. ELISA

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 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 2nd 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 → A radiolabeled Ag-Ab complex → 0 → SDS•PAGE → autoradiography- Ag-Ab complex + 2nd 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

Fluorochromes-Fluorescein (490→517nm)-Rhodamine (515→546nm)-Phycoerythrin : absorb light of one wavelength & emit fluorescence at a longer wavelength than fluorescein.

FIGURE 6-15Separation 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.

labeled with fluorescein (green)labeled with rhodamine (red)

each dot represents a cell

(small electrical change)

(exciting the fluorochrome) ↓each droplet (cell) emitsthe fluorescence

10. Flow cytometry & 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 (ka ～ 108) - 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 (ka ～ 1015) - Ab can be labeled with (ka ～ 1018)

FIGURE 6-16

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 portion.

12. Immuno EM.

electron-denselabelsAbsorbs 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”

[:acute lymphocytic leukemia] [:chronic lymphocytic leukemia]