5. n & cf (reuploaded)

8/13/2019 5. N & CF (Reuploaded)

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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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5. n & cf (reuploaded)

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