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Antigen-Antibody Interactions
For the sake of time, Im eliminating mostof the math .
Notes in red you can ignore, but I dontwant to take out!
Well cover affinity, then various reactionsand cool things you can do with them- this
is a fairly practical chapter, after the firstpart.
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The Ag-Abinteraction isdue to lots ofnon-covalentinteractions- lockand key!
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Affinity-Where were going
Bottom line- be able to interpret a Ka or Kdas tight or loose-
No Scatchard this time Be able to interpret a Scatchard plot-
slope, shape, # of binding sites, etc.
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k1
Ag + Ab Ab . Ag;
k-1
At equilibrium the rate of formation = the rate ofdissociation, and so k1[Ag][Ab]= k-1[Ab.Ag];
k1/k-1= [Ab.Ag]= Ka =[Ag][Ab]
When [Ab.Ag]= [Ab] (i.e., of the Ab isbound) , 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^-8Lets look at what this means if youhave a Ka of 10^6, and [Ab] = 10^-4M, [Ag] 10^-6M
We interrupt thisPowerPointpresentation for achalk talk! (not thistime!)
tight binding - Ka islarge, Kd is small.Seems like Kd is usedmore often.
Association or affinityconstant
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Avidity
Binding is often with multiple epitopes tomultiple antibodies- the total strength isavidity - Thus, the total binding may bestronger than the individual bindings- theremay be cooperativity, etc. IgM > aviditythan IgG with > affinity, b/c of pentameric
binding.
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New Topic- Cross-reactivity
Some Abs react to things other than the Ag that elicited them
Ex: anti-A and anti-B antibodies; Mprotein antibodies that X-react againstheart muscle.
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Practical Ag-Ab reactions
Precipitation- various types Agglutination- various types
RIAs ELISAs
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Polyclonals often ppt whenmonoclonals wont
Precipitation- turning a solubleantigen into an insoluble Ab-Agcomplex
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Immunoelectrophoresis
The antigens areelectrophoresed in agarose,then the antibody applied.
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Agglutination- clumping ofRBCs, or other particles
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Radioimmunoassay- detectingHepatitis B surface Ag
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VERY sensitive!
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Detecting Abs against HIV - HIVcoat protein is the Ag
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Western blot-finding 1
protein out ofmany in
serum orcytosol
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Indirect immunofluorescence
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Detects cellcomponent ascytoplasmic, ratherthan nuclear
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FACS machine
Fluorescence-activatedcell sorter. Julie (formerstudent who interns at
Stanford) says peopleused bad words about thismachine at Stanford.
Rapid communicationbetween computer and
deflection plates. Ifboth dyes- deflectright; one or the other-deflect left. No dye-no deflection. Cells areindividually counted.
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Using flow cytometery todiagnose acute lymphocyticleukemia
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Key points
Affinity, avidity, Ka, Kd, interpretation ofSkatchard plot.
Types of reactions- precipitation,agglutination, RIA, ELISA, fluorescence,FACS, western blots.
