Download Ouchterlony Procedure

Ouchterlony
Timothy G. Standish, Ph. D.
©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
©1999 Timothy G. Standish
Antibody Structure
Antigen
binding
site
V
V
V
Light
Chain
Antigen
binding
site
V
SS
SS
Light
Chain
Heavy Chains
©1999 Timothy G. Standish
Antigen Binding
Antigen 1
Antigen 3
©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.
©1999 Timothy G. Standish
Ouchterlony
1
2
Ab
©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
X
indicating that
either X and Y
are different or
that well 3 has
a mixture of
two
antibodies
in it.
Y
Anti X Y
Anti X
Anti Y
©1999 Timothy G. Standish
Non-identity:
What You Really See
©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.
X
Y
Anti X Y
Anti X
Anti Y
©1999 Timothy G. Standish
Non-identity:
What You Really See
©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.
X
X
Anti X Y
Anti X
©1999 Timothy G. Standish
Identity:
What You Really See
©1999 Timothy G. Standish
©1999 Timothy G. Standish