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k 1 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]; k 1 /k -1 = [Ab.Ag]= Ka = [Ag][Ab] When [Ab.Ag]= [Ab] (i.e., ½ of the Ab is bound), then Ka= 1/[Ag] 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!) “tight” binding- Ka is large, Kd is small. Seems like Kd is used more often. Association or affinity constant
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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 plot-
slope, 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 =
[Ag][Ab]
When [Ab.Ag]= [Ab] (i.e., ½ of the Ab is bound) , then Ka= 1/[Ag]
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!)
“tight” binding- Ka is large, Kd is small. Seems like Kd is used more often.
Association or affinity constant
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
Polyclonals often ppt when monoclonals won’t
Precipitation- turning a soluble antigen into an insoluble Ab-Ag complex
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 A
Type AB Type O
Old pregnancy test. It also illustrates agglutination inhibition
Or conjugate of some ilicit drug
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 blot- finding 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 other- deflect left. No dye- no 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.
