Antigens and antibodies interact…
• They associate:
• They dissociate:
[Ag] + [Ab] 6 [AgCAb]
[AgCAb] 6 [Ag] + [Ab]
• The velocity of the association:
v1 %[Ag] [Ab]
• The velocity of the disssociation:
v2 % [AgCAb]
• Proportionality can be converted to
equality (by introducing a velocity constant)
Antigens and antibodies interact…
Thus, v1 = k1 [Ag] [Ab]
At equilibrium, v1 = v2;
Rearranging,
and
thus,
v2 = k2 [AgCAb]
k1 [Ag] [Ab] = k2 [AgCAb]
k1 / k2 = [AgCAb] / [Ag] [Ab]
k1 / k2 = (necessarily a constant) is set equal to Ka, the
association constant between antigen and antibody.
Ka measures the affinity of a specific antibody for a specific
antigen.
(Kd, the dissociation constant is the inverse of Ka. Kd were the values seen
with affinity maturation due to somatic hypermutation.)
Antigen precipitin complex…
a visible, three-dimensional network or lattice that will eventually
settle from solution as an “antigen-precipitin” complex.
• The antibody
must be
bivalent; a
precipitate will
not form with
monovalent
Fab fragments.
• The antigen
must be either
bivalent or
polyvalent; that
is, it must have
at least two
copies of the
same epitope
Ag-Ab precipitins….
• Seeing a
complex
requires
that the
Ag-Ab
complex
exist at
milligrams
/ milliliter
Numerous techniques make Ag’s visible
• See more techniques:
http://www.alzforum.org/res/com/ant/glossary.asp
Affinity versus avidity… (from the “Glossary” in your text...)
“Affinity: The strength with which a ligand
interacts with a binding site. It is represented
quantitatively by the affinity constant Ka.”
(For, “ligand” read epitope; for “binding site” read paratope.)
“Avidity: The strength of antigen-antibody binding
when multiple epitopes on an antigen interact
with multiple binding sites of an antibody.”
“Bonus effect” and those poor monoclonal Ab’s…
Agglutination:
(once again from the “Glossary”...)
• “Agglutination[:] The aggregation or clumping
of particles (e. g., latex beads) or cells (e. g.,
red blood cells).”
•
Hmmm, that’s a bit spare.
• What happens here is that a bead or cell gets
coated with an antigen of interest. Then, the
cell(bead)-Ag is reacted with Ab. If the Ab is
specific for the Ag, a mesh will form. This
complex is more readily observed then are
precipitates.
Two examples of agglutination…
• ABO blood groups…
• The ABO blood-group antigens are glycoproteins
expressed on red blood cells...
• A type O individual ... has anti-A and anti-B antibodies...
• but has no antigen on the blood cell to elicit
these antibodies!!!
• Cross reactivity (CRM) is the basis for the presence of
these blood-group antibodies, which are induced in an
individual not by exposure to red blood cell antigens but by
exposure to cross-reacting microbial antigens present on
common intestinal bacteria.
!!!!
ABO blood groups continued…
• “Antibodies to the A, B, and O antigens, called
isohemagglutinins, are usually of the IgM class. An
individual with blood type A, for example, recognizes Blike epitopes on intestinal microorganisms and produces
isohemagglutinins to the B-like epitopes. This same
individual does not respond to A-like epitopes on the
same intestinal microorganisms because these A-like
epitopes are too similar to self and a state of selftolerance to these epitopes should exist.”
• sooooo, [conversely] your ability to resist some
microorganisms is affected by lacking antibodies to
microbial antigens that would react with your own blood
cells;
– Such is the case if you are type A, or type B, or type AB.
– The missing antibodies are lost by clonal deletion because the
antigens look like “self.”
The second example…
• Imagine that one suspects that the Ag exists in
some other solution (say, the urine of an individual.)
• Then, if the Ab is reacted with the urine and there
is Ag in the urine, then the Ab receptors become
saturated with Ag.
• When the Ab is next reacted with cell(bead)
complex which is coated with the same Ag, there
will be no agglutination (thus called agglutination
inhibition) because the Ab will already have reacted
with the Ag (in the urine.)
• This protocol is used in drug testing and has been
used in some pregnancy tests.
Overview:
• There are
techniques for
suspended Ag’s
that produce
agglutinins
• There are
techniques for
soluble Ag’s that
produce
precipitins
• Modern
techniques
increase
sensitivity:
An important refinement…. ELISA
• The fundamental feature of the Enzyme Linked ImmunoSorbent Assay
is to have an enzyme linked to an antibody and to have that enzyme be
able to generate some visible product.
• Note that because AbE has a constant region as the epitope to which it
reacts, AbE can be used to detect any antibody of a particular isotype.
That is to say, AbE once prepared can be used in multiple ELISA
assays.
• Antibodies at concentrations of 0.1 to 10 ng·ml-1 can be detected.
And another refinement: FACS