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Ouchterlony. Timothy G. Standish, Ph. D. Characterization of Proteins. Some proteins can be identified by their intrinsic properties - like enzyme activity - others are more difficult to characterize.

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ouchterlony

Ouchterlony

Timothy G. Standish, Ph. D.

characterization of proteins
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
antibody structure

Antigen binding site

Antigen binding site

V

V

V

V

Constant

Constant

Constant

Constant

SS

SS

Light

Chain

Light Chain

SS

SS

Antibody Structure

Heavy Chains

antigen binding

Antigen 3

Variable

Heavy

Variable

Light

Antigen Binding

Antigen 1

Antigen 2

classes of immunoglogulins

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.

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.

Classes of Immunoglogulins
ouchterlony non identity

Anti Y

Anti X

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.

X

Y

Anti X Y

ouchterlony partial identity

Anti Y

Anti X

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.

X

Y

Anti X Y

ouchterlony identity

Anti X

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.

X

X

Anti X Y

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The

End