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©1999 Timothy G. Standish
OuchterlonyOuchterlony
Timothy G. Standish, Ph. D.
©1999 Timothy G. Standish
Characterization of ProteinsCharacterization 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
©1999 Timothy G. Standish
SSSS
Light Chain
Light ChainSS
SS
Antibody StructureAntibody Structure
Constant Constant
Constant Constant
VV
V
V
Antigen binding site
Antigen binding
site
Heavy Chains
©1999 Timothy G. Standish
Antigen BindingAntigen Binding
Variable
Light
Variable
Heavy
Antigen 1Antigen 3
Antigen 2
©1999 Timothy G. Standish
Classes of ImmunoglogulinsClasses of ImmunoglogulinsIgG - 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.
©1999 Timothy G. Standish
OuchterlonyOuchterlony
2
Ab
1
©1999 Timothy G. Standish
Ouchterlony:Ouchterlony:Non-identityNon-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
X Y
Anti XAnti Y
©1999 Timothy G. Standish
Non-identity:Non-identity:What You Really SeeWhat You Really See
©1999 Timothy G. Standish
Ouchterlony:Ouchterlony:Partial IdentityPartial 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
X Y
Anti XAnti Y
©1999 Timothy G. Standish
Non-identity:Non-identity:What You Really SeeWhat You Really See
©1999 Timothy G. Standish
Ouchterlony:Ouchterlony:IdentityIdentity
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
X X
Anti X
©1999 Timothy G. Standish
Identity:Identity:What You Really SeeWhat You Really See
©1999 Timothy G. Standish