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					 Immunology 2008
     Lecture 5
Antibody Structure II
     9 October
most (not all) Ig,
 especially IgG
             Definitions


Gamma-globulin – electrophoretic mobility

Immunoglobulin – chain & domain structure

Antibody – binds Ag
            Electrophoresis of human serum.




Immunoelectrophoresis (IEP) of human serum
  Measuring Antibody/Antigen Reactions
Binding is invisible: how to detect & quantitate?

1) Precipitation: Ouchterlony

2) Solid-state binding, labelled antibody: RIA/ELISA

3) Liquid-phase binding: Equilibrium Dialysis

4) Binding of complement: Complement Fixation
                            (hemolysis)
             “inhibitor”


See Handout #4
                  50% end-point ~1:40
                    (“titer” ~1:40)




Plot of RIA/ELISA Results
                 binding: aX & aY




See Handout #2
See Handout #2
answer next week…
  Measuring Antibody/Antigen Reactions
Binding is invisible: how to detect & quantitate?


1) Precipitation: Ouchterlony
2) Binding of labelled antigen: RIA/ELISA

3) Binding of labelled antigen: Equilibrium Dialysis

4) Binding of complement: Complement Fixation
                Equilibrium Dialysis

           ● Analyze the binding of Ab to Ag

              ● Determine valency (> 2??)
                and strength of binding…




See Appendix 5, p.168
Requirements for Equilibrium Dialysis:
 1) Purified Ab (known molar conc.)

 2) Monovalent, dialyzable (& detectable) hapten
                                        measure
                                      free hapten




                          More sites or stronger binding
                           pull free hapten to the left.
Ab   + H            AbH     K1
AbH + H             AbH2    K2
AbH2 + H            AbH3    K3
   etc...
 Assumption: Binding sites are
  equivalent and independent

S + H            SH     Keq
                         [SH]
By definition:   Keq = --------
                        [S][H]
                  r
Substitute &     --- = Kn       -    Kr
  re-write:
                  c

       This is a straight line of the form:
                   Y = b - mX
●Curvature: Heterogeneity (measured as “a”)
●X-intercept: n=2
●For homogeneous IgG Ab, will get a straight line, n=2, slope = -Keq
●For IgM & some IgA, n > 2
AFFINITY: Strength of binding of a single antibody
 combining site with a single epitope: Keq


AVIDITY: Strength of binding of a multivalent antibody
 to a multivalent antigen; a measure of the ability of
 antibodies to form stable complexes with their
 antigens
   Multivalent binding:
Low affinity with multivalent binding
     may yield   high avidity
Avidity varies with affinity, geometry of
     antigen, flexibility of Ab, etc.
Handout #3
 Heterogeneity of Abs
 1) Specificity (absorption
   & affinity purification)
 *2) Affinity/avidity




 Problem: What would these
  two fractions look like if
     analyzed by RIA?


Handout #3
 Know assays & examples from notes,
     handouts & problems sets


Review Ouchterlony assay & simulator
(used for Ig Genetics next week)


          Q&A/Reviews
          …on request
            MONDAY
 Antibody Structure III, Chapter 4

          TUESDAY
Complement, Chapter 5, Appendix 6
Immunoglobulin Genetics, Chapter 6
Fin

				
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