1. 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 )

2. 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. FIGURE 6-6 (a)
Immunoelectrophoresis.
- an antigen mixture is first electrophoresed to separate its components by charge
- diffusion & producing lines of precipitation.

4. 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
+ + + (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.

5. 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).
6-

6. Sensitivity of various immunoassays

7. 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.
Radioimmuno AssayRadioimmuno 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.

8. 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
ELISAELISA

9. 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.
ELISAELISA
-> precipitates & forms a spot only on the areas of the well
where cytokine-secreting cells had been deposited.

10. FIGURE 6-12
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.

11. FIGURE 6-13
Immunoprecipitates can be collected using magnetic beads
coupled to a secondary antibody.
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.

12. Immunofluorescence
mIgM-producing B cells indirectly stained with rhodamine-conjurated
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.

13. 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.
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
Flow cytometry & Fluorescence

14. 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
)

15. 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.
Immuno EM.
electron-denselabels
Absorbs electrons.

16. 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]