The Ag-Ab
interaction is
due to lots of
non-covalent
interactions- lock
and key!
Affinity-Where we’re going
• Bottom line- be able to interpret a Ka or Kd
as tight or loose• No Scatchard this time
• Be able to interpret a Scatchard plotslope, shape, # of binding sites, etc.
k1
Ag + Ab <-> Ab.Ag;
k-1
At equilibrium the rate of formation = the rate of
dissociation, and so k1[Ag][Ab]= k-1[Ab.Ag];
k1/k-1= [Ab.Ag]= Ka = Association or affinity
constant
[Ag][Ab]
When [Ab.Ag]= [Ab] (i.e., ½ of the Ab is
bound) , then Ka= 1/[Ag]
“tight” binding- Ka is
large, Kd is small.
Seems like Kd is used
more often.
Ka units are L/mol- 10^6-10^8
Kd is dissociation constant, 1/Ka,
units mol/L, 10^-6-10^-8
Let’s look at what this means if you
have a Ka of 10^6, and [Ab] = 10^-4
M, [Ag] 10^-6M
We interrupt this
PowerPoint
presentation for a
chalk talk! (not this
time!)
Bottom line, again:
• Bottom line- be able to interpret a Ka or Kd
as tight or loose-
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 > avidity
than IgG with > affinity, b/c of pentameric
binding.
New Topic- Cross-reactivity
• Some Ab’s 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.
Practical Ag-Ab reactions
•
•
•
•
Precipitation- various types
Agglutination- various types
RIA’s
ELISA’s
Precipitation- turning a soluble
antigen into an insoluble Ab-Ag
complex
Polyclonals often ppt when
monoclonals won’t
Immunoelectrophoresis
The antigens are
electrophoresed in agarose,
then the antibody applied.
Agglutination- clumping of
RBC’s, or other particles
Blood Grouping
What blood type is it?
Type B
Type AB
Type A
Type O
Or conjugate
of some ilicit
drug
Old pregnancy
test. It also
illustrates
agglutination
inhibition
Radioimmunoassay- detecting
Hepatitis B surface Ag
VERY sensitive!
Detecting Ab’s against HIV- HIV
coat protein is the Ag
Elispot- how many
cells are making a
particular
cytokine??
Western blotfinding 1
protein out of
many in
serum or
cytosol
Indirect immunofluorescence
Detects cell
component as
cytoplasmic, rather
than nuclear
FACS machine
Fluorescence-activated
cell sorter. Julie (former
student who interns at
Stanford) says people
used bad words about this
machine at Stanford.
Rapid communication
between computer and
deflection plates. If
both dyes- deflect
right; one or the otherdeflect left. No dyeno deflection. Cells are
individually counted.
Using flow cytometery to
diagnose acute lymphocytic
leukemia
Key points
• Affinity, avidity, Ka, Kd, interpretation of
Skatchard plot.
• Types of reactions- precipitation,
agglutination, RIA, ELISA, fluorescence,
FACS, western blots.