Antigen-Antibody Interactions

1. 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.

2. The Ag-Ab interaction is due to lots of non-covalent interactions- lock and key!

3. 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.

4. 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!)

6. Bottom line, again: • Bottom line- be able to interpret a Ka or Kd as tight or loose-

7. 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.

8. 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.

9. Practical Ag-Ab reactions • Precipitation- various types • Agglutination- various types • RIA’s • ELISA’s

10. Precipitation- turning a soluble antigen into an insoluble Ab-Ag complex Polyclonals often ppt when monoclonals won’t

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

16. Agglutination- clumping of RBC’s, or other particles

17. Old pregnancy test. It also illustrates agglutination inhibition Or conjugate of some ilicit drug

18. Radioimmunoassay- detecting Hepatitis B surface Ag

19. VERY sensitive!

20. Detecting Ab’s against HIV- HIV coat protein is the Ag

21. Elispot- how many cells are making a particular cytokine??

22. Western blot- finding 1 protein out of many in serum or cytosol

23. Indirect immunofluorescence

24. Detects cell component as cytoplasmic, rather than nuclear

25. 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.

26. Using flow cytometery to diagnose acute lymphocytic leukemia

27. Key points • Affinity, avidity, Ka, Kd, interpretation of Skatchard plot. • Types of reactions- precipitation, agglutination, RIA, ELISA, fluorescence, FACS, western blots.