1. ©1999 Timothy G. Standish Ouchterlony Timothy G. Standish, Ph. D.

2. ©1999 Timothy G. Standish Characterization of Proteins Some proteins can be identified by their intrinsic properties - like enzyme activity - others are more difficult to characterize. Because antibodies are reasonably specific about what antigen they react or bind to, they can be used to distinguish protein antigens The Ochterlony procedure is one of several ways in which antibodies are used to characterize proteins

3. ©1999 Timothy G. Standish SS Light Chain Light Chain SS Antibody Structure Constant V V V V Antigen binding site Heavy Chains

4. ©1999 Timothy G. Standish Antigen Binding Variable Light Variable Heavy Antigen 1 Antigen 3 Antigen 2

5. ©1999 Timothy G. Standish Classes of Immunoglogulins IgG - A monomer - Most abundant antibody in blood. IgG easily leaves the circulatory system to fight infection and crosses the placenta conferring passive immunity to a fetus. IgD - A monomer - Found on the surface of B cells probably allowing recognition of antigens thus triggering differentiation into plasma and memory B cells IgE - A monomer - The least common antibody. The tails attach to mast cells and basophils. When antigens bind, they signal release of histamine. IgA - A dimer - Produced by cells in the mucus membranes to prevent attachment of pathogens. IgA is also found in many body secretions including milk. IgM - A pentamer - First antibody to appear following exposure to an antigen. Because it declines rapidly in the blood, high IgM levels indicate a current infection.

6. ©1999 Timothy G. Standish Ouchterlony 2 Ab 1

7. ©1999 Timothy G. Standish Ouchterlony: Non-identity If wells 1 and 2 contain two separate antigens and well 3 has antibodies to both antigens, a reaction of non-identity results indicating that either X and Y are different or that well 3 has a mixture of two antibodies in it. Anti X Y XY Anti X Anti Y

8. ©1999 Timothy G. Standish Non-identity: What You Really See

9. ©1999 Timothy G. Standish Ouchterlony: Partial Identity If wells 1 and 2 contain two separate antigens and well 3 has antibodies to both antigens, a reaction of partial identity may result if all antibodies react with X, but only some react with Y. indicating that X and Y are related in some way. Anti X Y XY Anti X Anti Y

10. ©1999 Timothy G. Standish Non-identity: What You Really See

11. ©1999 Timothy G. Standish Ouchterlony: Identity If wells 1 and 2 contain two identical antigens and well 3 has antibodies to that antigen, a reaction of identity results as all antibodies react with what is in both antigen wells indicating that the contents of both wells are closely related. Anti X Y XX Anti X

12. ©1999 Timothy G. Standish Identity: What You Really See

13. ©1999 Timothy G. Standish