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Neutralization and Complement
FixationMa. Jennifer R. Tiburcio, MSMT
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Neutralization
Antigenic activity is stopped (Neutralized) by
its specific antibody
Antibody renders the antigenic
microorganisms non-infective
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Types of Neutralization
Toxin Neutralization
Animal Protection Test
Antistreptolysin O titer
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Neutralization
Virus Neutralization
In vivo test
In ovo test
Pock reduction test
In tissue culture
Plaque reduction test
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Toxin Neutralization
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Streptolysin O reagent
patients serum (suspected of being infected with Beta streptococci)
Anti-Streptolysin O Antibodies
antigen will be neutralized
no hemolysis (ASO titer)
indicator cells
with without
antigen will not be neutralized
hemolysis
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1 2 3 4 5 6 7 8 9 10 11 12
1:10 1:1001:500
0.2 1 0.8 0.6 0.4 0.3 1 0.8 0.6 0.4 - -
0.8 0 0.2 0.4 0.6 0.7 0 0.2 0.4 0.6 1.5 1
0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 - 0.5
0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
50 100 125 166 250 333 500 625 833 1250 Control
Incubate for 15 minutes at 37oC
Incubate for 45 minutes at 37oC (shake the suspension after 20 minutes)
Serum
dilution
buffer
Streptolysin O
Erythrocyte
Antistreptolysin O
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Virus Neutralization
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In vivo test
Ag is of viral origin
Uses live animals and very similar to animal
protection test
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In ovo test
Same as in vivo test
Differ in the host used
Embryonated eggs are employed for theinoculation of virus and Ab mixture
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Pock Reduction Test
Pock reducing virus - titrated to produce a
determinable number of pocks during a
specific length of time in embryonated eggs
and test serum prior to innoculation
reduction in the number of pocks produced
Indicator of the quantity of Ab present
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In Tissue Culture
Most viruses maybe cultured in one or more
cell lines grown in monolayers on the surface
of a test tube walls
Cytopathogenic effect (CPE)
Ab titer recorded as reciprocal of the serum
dilution that will completely prevent the virus
from altering the cell culture
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Plaque Reduction Test
Same as pock reduction testexcept done in
tissue culture
Reduction in plaque is proportional to the
number of Ab present
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Complement Fixation
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Complement Fixation
Detects the presence of complement fixing Ab
(IgM and IgG) against soluble Ag
Bacteriolytic System
Hemolytic or Indicator System
2 systems involved
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Bacteriolytic System
Ag and Ab (one of which is known) plus a
pretitrated complement, incubated at 37
degrees centigrade
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Hemolytic or Indicator System
Sensitized sheep RBC (coated with
complement fixing anti-sheep RBC)
Detects the presence of free or unattached
complement which will lyse the sheep RBC
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Positive complement fixation
Principle:
Ag and Ab (one of which is known) plus a
fixed amount of pretitrated complement. If
the Ag and Ab are specific for each other
they will combine, the combination will take
up (fix) the added complement
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Positive Complement Fixation
Soluble Ag
Ab in patientsserum
complement
AgAbC
complex
Sensitized Redcells
No lysis
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Negative Complement fixation
Principle:
Addition of red cells sensitized with specific
hemolysin. If complement has been fixed by the
AgAb complex, none will be available for lysis ofthe sensitized red cells. If Ag and Ab are nor
specific, one of them is lacking, complement
remains free to attached to the sensitized red cell
and lyse them
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Negative Complement Fixation
Soluble Ag
(no Ab)
Patients SerumComplement Free C
Sensitized Red cells
(hemolysin binds &
activates C)
lysis
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Complement Fixation Testing
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