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Immunological assays
Antigen-antibody interactions
Antibody-based assays
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Affinity and specificity for antigen
Assays detect presence of antigen or antibody
diagnosis
monitoring humoral immune responsetherapeutics
analysis of interesting molecules
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Nature of antigen-antibody interaction
p. 138
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Affinity- strength of interaction between one
Ag-binding site and one epitope
Measured by various methods including
equilibrium dialysis, competition assays,
microchips (e.g., Biacor)Association constants can be
calculated
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Avidity
Multiple interactions between antibody and
antigen
IgM (with 10 antigen binding sites per molecule)
tends to have low affinity but high avidity
May be more biologically significant measure
than affinity
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p. 153
Cross-reactivity (p. 141)
May contribute to autoimmunity or be protective,
depednign on circumstances
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Precipitation- classic demonstration of
antibody-antigen interaction
Antibody and soluble antigen aggregate to form
a visible precipitate
Antibody must be bivalent (Fabs wont work)
Antigen must be multivalent
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p. 142
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Precipitation reactions in fluids (p. 142)
Precipitation reaction can be seen
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Precipitation can also be seen in gels
Antibody and antigen diffuse toward each
other and precipitate where there
is equivalence
Radial immunodiffusion
Double diffusion
Immunoelectrophoresis
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p. 143
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Agglutination- reaction between antibody and
particulate antigen
Presence of excess antibody can inhibit
agglutination (prozone effect)
Each antibody competes for epitopes; if it
binds one, it cannot link one antigen
to another
Some antibodies bind but do not agglutinate
Epitope density or availability
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Hemagglutination- red blood cells
Well #10- sheep RBC only control
1 through 9- serial twofold dilutions of anti-SRBC
p. 145
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Bacterial agglutination
If a patient has a bacterial infection, the patient
will produce specific antibodies to that
bacterium
Serum can be titered with bacterial agglutination
reactions
Dilutions of serum are tested (usually twofold)example: 1:256
dilution shows agglutination
but 1:512 does not
Titer is 256
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Titer can be monitored over time; should
decrease as patient recovers
Antisera are used to type bacteria, too
(against surface antigens, flagellar antigens, etc.)
Example: E. coli O157:H7
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Passive agglutination
Soluble antigens dont agglutinate
But if you stick them onto something else
(like a latex bead or, historically, a RBC)
you can obtain an agglutination reaction
Using beads increases sensitivity
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Agglutination inhibition
Current tests are ELISAs
p. 146
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Agglutination inhibition assays
Can test for the presence of substances in fluids(e.g., drugs in
urine)
How?
Rubella test is an agglutination inhibition assay;
rubella virus causes hemagglutination
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ELISA- enzyme-linked immunosorbent assay
Based on RIA
Nearly as sensitive
Cheaper and safer
Many detection systems have been developed
Many variations of the assay have been developed
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All ELISAs use an antibody conjugated with an
enzyme that turns a colorless substrateinto a colored
product
Direct- detects antigens using a single labeled
antibody against that antigen
Relatively few applications and permutations
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Indirect ELISA detects antibodies
(prior exposure to antigen, e.g.,HIV)
p. 149 (see also for other types of
ELISA designs)
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Chemiluminescence
ELISA technique is the same, detection system
is different
Luminol, hydrogen peroxide and horseradishperoxidase react to
emit light
Detection system is more sensitive than the
enzyme substrates
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ELISPOT- can detect secreting cells
p. 150
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The ELISA is very versatile
antigen
Antibody (mouse Fab,
Human Fc)
Complement (human)
Goat anti-human C1q
E
Enzyme-conjugatedSheep anti-goat Ab
substrate
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All IgG1 antibodies were identical except
for carbohydrate in CH2
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Western blotting
p. 151
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Immunofluorescence
Antibodies can be labeled with fluorescent dye
Can localize binding sites on cells
Dyes: Fluorescein, rhodamine, phycoerythrin
can be conjugated to Fc region of Ab
(so antigen binding is unaffected)
Absorb at one wavelength and emit at another
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Direct and indirect immunofluorescence
p. 166
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p. 166
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Versatile technique
Differentiate T cell subsets
Detecting Ag-Ab complexes
Localization of target molecules in tissue
(variation: immunohistochemical staining)
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Flow cytometry is quantitative
FACS- fluoresence-activated cell sorter
Analyze cell populations
Sort cells with different features into different
containers (e.g., T and B cells; cells that
are producing a cell-surface marker
from those that are not)
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p. 155
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Uses for flow cytometry
Percentage of a total population of cells
Measuring antigen density within a population
of cells
Multiple antibodies can be used to assess several
cell surface antigens simultaneously
Clinical analysis (tumor characterization)
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Cell culture
gene transfectionhybridoma technology
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Summary
The structure of antibodies enables them
to recognize and bind antigen and to
perform appropriate effector functions
in response
The exquisite specificity and effector activity
of antibodies makes them very usefuL
in research and diagnostics
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The organization of immunoglobulin genes
allows for the formation of over
10 billion antigen specificities
Various in vivo and invitro experimental
systems have provided significantinsights into the immune
response
and its regulation
