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Antigen-Antibody Interactions. For the sake of time, I’m eliminating most of the math . Notes in red you can ignore, but I don’t want to take out! We’ll cover affinity, then various reactions and cool things you can do with them- this is a fairly practical chapter, after the first part.
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Antigen-Antibody Interactions • For the sake of time, I’m eliminating most of the math . • Notes in red you can ignore, but I don’t want to take out! • We’ll cover affinity, then various reactions and cool things you can do with them- this is a fairly practical chapter, after the first part.
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.
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] k1 Ag + Ab <-> Ab.Ag; k-1 Association or affinity constant “tight” binding- Ka is large, Kd is small. Seems like Kd is used more often. 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!)
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.
Old pregnancy test. It also illustrates agglutination inhibition Or conjugate of some ilicit drug
Western blot- finding 1 protein out of many in serum or cytosol
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.