Immunoprecipitation
1
Definition of Immunology
 The
study of the physiological
mechanisms which enable the body to
recognize materials as foreign and to
neutralize, eliminate, or metabolize them
without injury to the host tissue.
2
Overview of Immunodiagnostic
Studies
 Antigen-antibody
•
•
•
•
reactions for diagnosis of:
infectious disease,
autoimmune disorders,
immune allergies,
and neoplastic disease.
 These
modalities also test for:
• blood groups and types,
• tissue and graft transplant matching.
3
Types of Tests
 Many
methods of varying sophistication
are used for immunodiagnostic studies
4
Types of Tests
Name of Test
Observable Reaction
Visible Change
Tests for
Clumping
Agglutination
Particulate antigen reacts with corresponding
antibody; antigen may be in form of
RBCs (hemagglutination, latex, or
charcoal coated with antigen).
Treponemal, heterophile,
and cold agglutinin
antibodies
Soluble antigen reacts with corresponding
antibody by ID or count.
Precipitates
Fungal antibodies, food
poisoning,
Quantitative
immunoglobulins IgA,
IgG
Competition between two antigen-antibody
systems (test and indicator systems)
Complement activation,
hemolysis
Viral antibodies
Fluorescent-tagged antibody reacts with
antigen-antibody complex in the
presence of ultraviolet light.
Visible microscopic
fluorescence
Antinuclear antibodies
(ANAs);
antimitochondrial
antibodies (AMAs)
Indirect EIA for quantification of an antigen
or antibody enzyme and substrate
Color change indicates
enzyme substrate
reaction.
Hepatitis and human
immunodeficiency
virus (HIV)
(screening)
Electrophoresis separation of antigen
subspecies
Detection of antibodies of
specific mobility
Confirms HIV-1
Measures either antigen or antibody in
solution through the scattering of a
light beam; antibody reagent used to
detect antigen IgA, IgG, IgM;
concurrent controls are run to establish
amount of background scatter in
reagents and test samples.
Light scatter
proportionately
increases as
numbered size of
immune complexes
increases.
Quantitative
immunoglobulins IgA,
IgM, C-reactive
protein, antistreptolysin O
recorded in mg/dL or5
IU/mL
Precipitation
Complement fixation
(CF)
Immunofluorescence
Enzyme-linked
immunosorbent
assay (ELISA)
Immunoblot (eg,
Western blot [WB])
Rate nephelometry
Antigen-antibody interaction

Antigens are substances that stimulate and
subsequently react with the products of an immune
response. They may be:






enzymes,
toxins,
microorganisms (eg, bacterial, viral, parasitic, fungal),
tumors,
or autoimmune factors.
Antibodies are proteins produced by the body's immune
system in response to an antigen or antigens. The
antigen-antibody response is the body's natural defense
against invading organisms .
6
Structure of an antibody
7
Antibody Structure
Antigen
binding
site
V
V
V
Light
Chain
Antigen
binding
site
V
SS
SS
Heavy Chains
Light
Chain
8
Classes of antibodies
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
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blood, high IgM levels indicate a current infection.
Three Distinct Phases of
Antigen/Antibody Reactions
 Primary

Phenomenon –
involves the combination of an individual binding site on an antibody
molecule with a single epitope or determinant site on an antigen
 Secondary

Phenomenon –
Lattice formation include precipitation, agglutination and complement
fixation
 Tertiary

Phenomenon –
Detected by affect on tissues or cells, Inflammation, phagocytosis,
deposition of immune complexes, immune adherence and chemotaxis
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Detection of antigen-antibody
interactions:
Primary Phenomenon e.g.
1.



Enzyme linked immuono sorben assay (ELISA)
Immuno flurescent antibody technique (IFAT)
Radio immuno assay (RIA)
Secondary Phenomenon : e.g.
2.



Agglutination tests
Complement fixation tests (CFT)
Precipitation tests
Tertiary Phenomenon : e.g.
3.

Reaction not visible, detected by affect of reaction on tissues
or cells
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Antigen Binding
Antigen 1
Antigen 3
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Ag-Ab interactions
Bonds:




Hydrogen
Ionic
Hydrophobic interactions
Van der Waals forces
Each bond is weak; many
are strong
To “hold” they must be close
and complementary
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Features of the Antigen-Antibody
Interaction
•Reversibility
Non-covalent Interactions
•Affinity
Measure of the strength of the binding
Ease of association or dissociation
•Avidity
Increase in affinity due to multivalent
binding
The summation of multiple affinities
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Binding of the epitope in
the antigen binding site
GOOD FIT
POOR FIT
antibody combining site
antigen determinant
high attraction
low repulsion
High Affinity
high repulsion
low attraction
Low Affinity
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Antibody Affinity

Affinity – the strength of the total noncovalent
interactions between a single Ag-binding site on an Ab
and a single epitope


Low affinity Abs bind Ag weakly and dissociate readily
High affinity Abs bind Ag more tightly and remain bound longer
Ag + Ab
k1
Ab-Ag
k-1
K1 = forward (association) constant
K-1 = reverse (dissociation) constant
17
Antibody Affinity
K can be determined using equilibrium dialysis
18
Antibody Avidity

Avidity – the strength of the total noncovalent
interactions between a multivalent Ab and Ag


The avidity of an antibody is a better measure of its binding
capacity within biological systems than is the affinity of its
individual binding sites
High avidity can compensate for low affinity; eg. pentameric IgM
often has lower affinity than IgG but its higher avidity (due to
presence of 10 binding sites) may enable it to bind Ag more
effectively
20
Antibody Avidity
Keq =
104
Affinity
106
Avidity
1010
Avidity
21
Effect of multivalent
interactions
antibody
IgG
IgG
IgM
effective antibody valence 1
1
2
up to 10
antigen valence
1
n
n
equilibrium constant (L/M) 104
104
107
1011
advantage of multi-valence -
-
103-fold
107-fold
affinity
avidity
avidity
definition of bindng
Fab
1
affinity
intrinsic affinity
functional affinity
22
Factors Affecting Measurement of
Ag/Ab Reactions
• Affinity
• Avidity
Ab excess
Ag excess
• Ag:Ab ratio
• Physical form of Ag
Equivalence – Lattice formation
23
Tests Based on Ag/Ab Reactions
 All
tests based on Ag/Ab reactions will
have to depend on lattice formation or they
will have to utilize ways to detect small
immune complexes
 All tests based on Ag/Ab reactions can be
used to detect either Ag or Ab
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Immunoprecipitation
 Immunoprecipitation
is the technique of
precipitating an antigen out of solution
using an antibody that specifically binds to
that particular antigen. This process can
be used to isolate and concentrate a
particular antigen from a sample
containing many thousands of different
antigens.
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Immunoprecipitation tests


The antigen and antibody are in soluble form
Combine to form a visible precipitate
 Complete precipitation occurs at certain Ab/Ag
concentrations
Antigen
Antibody
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Precipitation Curve
– antibody excess, many
antibodies coat all antigen sites- results in
false negative
 Postzone – antigen excess, antibody coats
antigen but cannot get lattice formation,
results in false negative
 Zone of Equivalence – antigen and
antibody present in optimal proportions to
bind and give visible reaction
 Prozone
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Precipitation Curve
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Precipitation Reactions
Ab + soluble Ag

Lattice
Precipitate
Precipitation techniques


Tube precipitation test
Gel diffusion
• Single radial
• Double


Immunoelectrophoresis
Countercurrent electrophoresis (CEP),
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Tube precipitation test
30
Gel diffusion

Immunodiffusion tests

Antigen and antibody
placed in separate wells
cut in gel
• Solutions diffuse and
meet between the wells

Results in line of
precipitation at zone of
optimal proportion
31
Double Diffusion
Note: A line of precipitation has formed
between the center well and wells 3 & 5.
This indicates there is antigen/antibody
specificity between the center well and
these two wells.
Usually a known antigen or known
antibody is placed in the center and test
serum is placed in the peripherial wells.
Antigens and antibodies will diffuse and
at some point optimal concentrations
will occur and if the antigen is specific
for the antibody a precipitate line will
form.
Example: Has this patient ever had rubella, or diptheria? If they have their serum
will contain antibodies against the disease.
Put patient serum in the center. Put the disease agents (antigens) in wells 1 – 5,
and allow to diffuse. A precipitation line between wells indicates that the patient
has had that disease
32
Double Diffusion
Ab and Ag diffuse towards each other
Used for determining antigenic relationship between substances
Identity
Non-identity
Partial identity
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Double Diffusion
1
2
Ab
34
Double Diffusion
35
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
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Identity:
What You Really See
37
Double Diffusion
38
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
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Non-identity:
What You Really See
40
Double Diffusion
41
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
42
Non-identity:
What You Really See
43
Radial immunodiffusion test

Radial immunodiffusion test is quantitative




Antibody is added to liquid agar that is allowed to
harden
Created a uniform antibody concentration
Antigen added to wells cut in gel
• Diffusion outward forms concentration gradient
• Ring forms at antigen-antibody precipitation
Standards can be used to construct standard curve to
establish concentration
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Single radial immunodiffusion
Ag
45
Single radial immunodiffusion
46
Single radial immunodiffusion
r
r  [Ag]
47
Single radial immunodiffusion
 Interpretation
 Quantitative

Ag
Diameter of ring
is proportional
to the
concentration
Ig levels
Ag
Ag
Ag
Diameter2

Ab in gel
Ag Concentration
48
Single radial immunodiffusion
49
Single radial
immunodiffusion
Exercise 1
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Materials
 Standard
 Single
radial immunodifusion plates
 Moisture chamber
 Micropipettes
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Specimen
 Collect
whole blood without anticoagulant
and allow to clot at room temperature.
Separate serum by centrifugation within 23 hours after collection.
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Method

Remove plates from refrigerator to room
temperature approximately 30 minutes before
filling wells. Do not open bag until ready for use.
 If excess moisture is present, remove plate from
its bag and remove cover until evaporation has
dried the surface and wells. Replace cover until
used.
 For best results, three wells should be filled with
reference sera for each plate. Location of each
should be noted.
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Preparing the standards (serial
dilution)
 Label
four microtest tubes: 1:2, 1:4, 1:8,
and 1:16.
 Using a micropipette, add 50 microliters of
Buffer to each tube.
 With a fresh pipet tip, add 50 microliters of
“Standard” to the tube labeled 1:2, Mix.
Transfer 50 microliters of the 1:2 dilution to
the tube labeled 1:4, Mix, and so on.
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Performance of Test

Deliver specimen or standard (5 µl) to well by
placing the pipette tip at the bottom of the well.
Allow the well to fill to the top of the agar
surface. Avoid bubbles to ensure proper volume
and diffusion of sample. Visualization may be
aided by placing the plate on dark background.
 Mark time of completion on plate cover and
replace cover.
 Replace plate in bag and reseal carefully.
 Incubate plates upright on a flat surface at room
temperature (20 to 24oC) over 48 hours for End
Point readings.
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Calibration Curve
 Using
the reference sera determine their
ring diameters to the nearest 0.1mm.
 Using regular graph paper plot the
concentration on the X axis and the zone
diameters squared on the Y axis for each
protein for End point readings.
 Draw a straight line of "best fit" between
the four points.
56
Results

Measure diameters of precipitin zones to within
0.1mm. Variations in incubation times of more
than 30 minutes will produce changes in
diameters, especially those at higher levels of
antigen except when plates have reached
endpoint.
 Determine the concentration of each unknown or
specimen protein from the reference curve find
the corresponding concentration.
57
Diameter2
Results
Ag Concentration
58