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

©1999 Timothy G. Standish

OuchterlonyOuchterlony

Timothy G. Standish, Ph. D.


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


SSSS

Light Chain

Light ChainSS

SS

Antibody StructureAntibody Structure

Constant Constant

Constant Constant

VV

V

V

Antigen binding site

Antigen binding

site

Heavy Chains


Antigen BindingAntigen Binding

Variable

Light

Variable

Heavy

Antigen 1Antigen 3

Antigen 2


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.


OuchterlonyOuchterlony

2

Ab

1


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


Non-identity:Non-identity:What You Really SeeWhat You Really See


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


Non-identity:Non-identity:What You Really SeeWhat You Really See


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


Identity:Identity:What You Really SeeWhat You Really See


Documents

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