Principles and Applications of Chemiluminescence

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					Ch. 6.    Antigen-antibody interactions:

     Principles and applications

     Strength of Ag-Ab interactions

     - Affinity is a quantitative measure of binding
           strength

     - Avidity incorporates affinity of multiple
           binding sites


                       Ch. 6
Assays detect presence of antigen or antibody

     diagnosis
     monitoring humoral immune response
     therapeutics
     analysis of interesting molecules

If looking for Ag, use known Ab

If looking for Ab, use known Ag


                      Ch. 6
        p. 146




Ch. 6
Affinity- strength of interaction between one
      Ag-binding site and one epitope

Measured by various methods including
    equilibrium dialysis
    competition assays
    microchips

Association constants can be calculated




                      Ch. 6
Ab avidity

Involves multiple interactions between
antibody and antigen

Both Ab and Ag must be bivalent or
multivalent

IgM (with 10 antigen-binding sites per
molecule): low affinity but high avidity

Avidity may be a more biologically
significant measure than affinity
                      Ch. 6
Cross-reactivity




  p. 149

                   Ch. 6
Precipitation reactions - classic demonstration of
     antibody-antigen interaction

Antibody and soluble antigen form a lattice that
     eventually develops into a visible
     precipitate

Antibody must be bivalent
     (monovalent Fabs won’t work)

Antigen must be bivalent or multivalent



                       Ch. 6
p. 142,
5th ed.




          Ch. 6
Precipitation reactions in fluids (p. 142, 5th ed.)




                                              p. 150 for
                                              SPR app.




Precipitation reaction can be seen
                        Ch. 6
Precipitation can also be seen in gels

Antibody and antigen diffuse toward each
    other and precipitate where there
    is equivalence

Radial immunodiffusion – Ab in gel
Double diffusion – Ab and Ag diffuse
Immunoelectrophoresis
    - Electrophoresis first
    - Then precipitation in gel
                  Ch. 6
Ch. 6   p. 152
Ch. 6
        p. 153




Ch. 6
        p. 152



Ch. 6
Agglutination- interaction between antibody and
     a particulate antigen resulting in visible
     clumping

Presence of excess antibody can inhibit
     agglutination (prozone effect)

- Each antibody “competes” for epitopes
- Some antibodies bind but do not agglutinate
- Epitope density or availability



                      Ch. 6
p. 153




         Ch. 6
Bacterial agglutination to diagnose infection

If a patient has a bacterial infection, the patient
      will produce specific antibodies to that
      bacterium

Serum can be titered with bacterial agglutination
    reactions

Dilutions of serum are tested (usually twofold)
      example: 1:256 dilution shows agglutination
      but 1:512 does not

Titer is 256
                        Ch. 6
Titer can be monitored over time;

Convalescent serum will have higher titer than
    acute one (“Paired serum samples”)

Antisera are used to type bacteria, too

(against surface antigens, flagellar antigens, etc.)

Example: E. coli O157:H7




                       Ch. 6
Passive agglutination

Useful with soluble Ag’s that don’t agglutinate

But if you stick them onto something else
      (like a latex bead or, historically, a RBC)
      you can obtain an agglutination reaction

Using synthetic beads increases sensitivity




                        Ch. 6
Agglutination inhibition assays

Can test for the presence of substances in fluids
     (e.g., HCG or drugs in urine)


Rubella test is an agglutination inhibition assay;
    rubella virus causes hemagglutination

Ab in person’s serum inhibits hemagglutination


                       Ch. 6
 Agglutination inhibition (or, Hapten inhibition)




p. 146,
5th ed.
          Current tests are ELISAs
                         Ch. 6
Principle of radioimmunoassay (RIA):

Competitive binding of radiolabelled Ag and
    unlabelled Ag to a high-affinity Ab

Highly sensitive

Quantitative

Radioactive iodine or tritium used

Many variations of the assay have been developed


                        Ch. 6
p. 156


         Ch. 6
Ch. 6
ELISA- enzyme-linked immunosorbent assay

Similar in principle to RIA

Nearly as sensitive
Cheaper and safer

Many detection systems have been developed

Many variations of the assay have been developed




                         Ch. 6
All ELISAs use an antibody conjugated
with an enzyme that turns a colorless
substrate into a colored product

Many variations:
    Direct
    Indirect
    Sandwich
    Competitive

Qualitative and quantitative to detect
presence
     of Ag or Ab
                     Ch. 6
p. 157


         Ch. 6
Chemiluminescence

Most sensitive assay available!

Luminol, hydrogen peroxide and horseradish
    peroxidase react to emit light

Ab.HRP + Ag  Ab.HRP.Ag (luminol, H2O2)
     light

Expose photographic film or luminometer


                     Ch. 6
        p. 158




Ch. 6
Ch. 6
ELISPOT




      Ch. 6
        Western
        blotting




        p. 159




Ch. 6
        p. 160




Ch. 6
Ch. 6
Immunofluorescence: to visualize Ag on
         or within cells

Antibodies can be labeled with fluorescent dye

Can localize binding sites on cells

Dyes: Fluorescein, rhodamine, phycoerythrin
     can be conjugated to Fc region of Ab
     (so antigen binding is unaffected)

Absorb light at one wavelength (UV) and emit light
    at another wavelength (e.g., red or green)

                        Ch. 6
    Immunofluorescence




p. 161

                 Ch. 6
p. 166
         Ch. 6
Versatile technique:

- Differentiate T cell subsets

- Detect Ag-Ab complexes

- Localize of target molecules in tissue

(variation: immunohistochemical staining)




                       Ch. 6
Flow cytometry is quantitative

FACS- fluoresence-activated cell sorter

Analyze cell populations

Sort cells with different features into different
     containers (e.g., T and B cells; cells that
     are producing a cell-surface marker
     vs. those that are not)



                        Ch. 6
        p. 162




Ch. 6
p. 163

         Ch. 6
Uses for flow cytometry:

Percentage of a total population of cells

Measuring antigen density within a population
    of cells

Multiple antibodies can be used to assess several
     cell surface antigens simultaneously

Clinical analysis (leukemia typing)



                       Ch. 6
Immunoelectron microscopy




    p. 164
                  Ch. 6
Summary

The structure of antibodies enables them
    to recognize and bind antigen and to
    perform appropriate effector functions

The exquisite specificity and effector activity
    of antibodies makes them very useful
    in research and diagnostics



                      Ch. 6
The organization of immunoglobulin genes
    allows for the formation of over
    10 billion antigen specificities

Various in vivo and in vitro experimental
     systems have provided significant
     insights into the immune response
     and its regulation




                    Ch. 6

				
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