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.

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

[Ag] [Ab.Ag] + [Ag]

So, after all that math Bottom line: if your Ab is in excess, almost all your antigen will be bound, while very little of your antibody is bound! 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)

Bound ag/total Ab/free ag Bound ag/total Ab r/c= K a n-K a r r/c= 0, K a r= K a n, # 1’s affinity is > #2

It seems to be ½ N = -Ko

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.

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 BType 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.