Antigen-Antibody Interactions

For the sake of time, Im eliminating most of the math . Notes in red you can ignore, but I dont want to take out! Well 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 were 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] tight binding- Ka is large, Kd is small. Seems like Kd is used more often. We interrupt this PowerPoint presentation for a chalk talk! (not this time!)

When [Ab.Ag]= [Ab] (i.e., 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

Lets look at what this means if you have a Ka of 10^6, and [Ab] = 10^-4 M, [Ag] 10^-6M

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 Abs 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 RIAs ELISAs

Precipitation- turning a soluble antigen into an insoluble Ab-Ag complex

Polyclonals often ppt when monoclonals wont

Immunoelectrophoresis
The antigens are electrophoresed in agarose, then the antibody applied.

Agglutination- clumping of RBCs, or other particles

Or conjugate of some ilicit drug

Old pregnancy test. It also illustrates agglutination inhibition

Radioimmunoassay- detecting Hepatitis B surface Ag

VERY sensitive!

Detecting Abs 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.