Antigen-Antibody Interactions

1
Antigen-Antibody Interactions

• Another chapter where well cover some things in
  more detail than well test on them- youll want
  to pay attention to the bottom line, particularly
  with the math that we go over.
• Well cover affinity, then various reactions and
  cool things you can do with them- this is a
  fairly practical chapter, after the first part.

2
The Ag-Ab interaction is due to lots of
non-covalent interactions- lock and key!
3
Affinity-Where were going

• Bottom line- be able to interpret a Ka or Kd as
  tight or loose-
• Be able to interpret a Skatchard plot- slope,
  shape, of binding sites, etc.

4
At equilibrium the rate of formation the rate
of dissociation, and so k1AgAb
k-1Ab.Ag K1/k-1 Ab.Ag Ka association
constant AgAb
k1 Ag Ab lt-gt Ab.Ag
k-1
When Ab.Ag Ab, then Ka 1/Ag
Ka units are L/mol- 106-108 Kd is dissociation
constant, 1/Ka, units mol/L, 10-6-10-8 Lets
look at what this means if you have a Ka of 106,
and Ab 10-4 M, Ag 10-6M
We interrupt this PowerPoint presentation for a
chalk talk!
5
Ag
Ab.Ag Ag
6
So, after all that math
Skatchard plot A way to plot the relationship
between bound and free antigen, as Ag levels are
increase, that yields Ka and the number of
binding sites of the antibody and the makeup of
the antibody (monoclonal or polyclonal)
Bottom line if your Ab is in excess, almost all
your antigen will be bound, while very little of
your antibody is bound!
7
r/c Kan-Ka r r/c 0, Ka r Kan,
1s affinity is gt 2
Bound ag/total Ab/free ag
Bound ag/total Ab
8
It seems to be r/c _at_ ½ N -Ko
9
Bottom line, again

• Bottom line- be able to interpret a Ka or Kd as
  tight or loose-
• Be able to interpret a Skatchard plot- slope,
  shape, of binding sites, etc.

10
Avidity

• Binding is often with multiple epitopes to
  multiple antibodies- the total strength is
  avidity- Thus, the total binding may be stronger
  than the individual bindings- there may be
  cooperativity, etc. IgM gt avidity than IgG with
  gt affinity, b/c of pentameric binding.

11
New Topic- Cross-reactivity

• Some Abs react to things other than the Ag that
  elicited them
• Ex anti-A and anti-B antibodies M protein
  antibodies that X-react against heart muscle.

12
Practical Ag-Ab reactions

• Precipitation- various types
• Agglutination- various types
• RIAs
• ELISAs

13
Precipitation- turning a soluble antigen into an
insoluble Ab-Ag complex
Polyclonals often ppt when monoclonals wont
14
(No Transcript)
15
(No Transcript)
16
(No Transcript)
17
Immunoelectrophoresis The antigens are
electrophoresed in agarose, then the antibody
applied.
18
(No Transcript)
19
Agglutination- clumping of RBCs, or other
particles
20
Old pregnancy test
21
Radioimmunoassay- detecting Hepatitis B surface Ag
22
VERY sensitive!
23
Detecting Abs against HIV- HIV coat protein is
the Ag
24
Elispot- how many cells are making a particular
cytokine??
25
Western blot- finding 1 protein out of many in
serum or cytosol
26
Indirect immunofluorescence
27
Detects cell component as cytoplasmic, rather
than nuclear
28
FACS machine Fluorescence-activated cell sorter.
Julie says people used bad words about this
machine at Stanford.
Rapid communication between computer and
deflection plates. If both dyes- deflect rt one
or the other- deflect left. No dye- no
deflection. Cells are individually counted.
29
Using flow cytometery to diagnose acute
lymphocytic leukemia
30
Key points

• Affinity, avidity, Ka, Kd, interpretation of
  Skatchard plot.
• Types of reactions- ppt, agglutination, RIA,
  ELISA, fluorescence, FACS, western blots.