Complement fixation test (CFT)

Complement fixation test (CFT)

1. Dr. J. P. Saranraj
COMPLEMENT FIXATION
TEST

2. COMPLEMENT FIXATION TEST
TheComplementsystemisapartoftheimmune
systemthatenhances(complements)the
abilityofantibodiesandphagocyticcellsto
clearmicrobesanddamagedcellsfroman
organism,promotesinflammation,andattacks
thepathogen'scellmembrane.
ItispartoftheInnateimmunesystem.
TheComplementfixationtest(CFT)isoneofthe
majortraditionaltestsforthedemonstrationof
presenceofspecificantigensorantibodies.
CFTwasextensivelyusedinSyphilisserology
afterbeingintroducedbyWassermanin1909.
CFTconsistof2steps.

3. First step –Complement fixation stage
Aknownantigenandinactivatedpatient’s
serumareincubatedwithastandardized,
limitedamountofComplement (Patient’s
serumisheatedat56°Cfor30minutesto
inactivateendogenouscomplementwhichmay
disturbthetestcalibration).
Iftheserumcontainsspecific,complement
activatingantibodythecomplementwillbe
activatedorfixedbytheAntigen-antibody
complex.
Ifthereisnoantibodyinthepatient’sserum,
therewillbenoformationofantigen-antibody
complex,andthereforecomplementwillnotbe
fixed.

4. Second step –Indicator Stage
Thesecondstepdetectswhethercomplement
hasbeenutilizedinthefirststepornot.Thisis
donebyaddingtheIndicatorsystem.
IftheComplementisfixedinthefirststepdueto
thepresenceofantibodyinthepatient’sserum
therewillbenocomplementlefttofixtothe
indicatorsystem.
Ifthereisnospecificantibodyinthepatient’s
serum,therewillbenoantigen-antibody
complex,andtherefore,complementwillbe
presentfreeorunfixedinthemixture.This
unfixedcomplementwillnowreactwiththe
Antibody-coatedsheepRBCstobringabout
theirlysis.

6. Results
Positivetest:Theavailablecomplementisfixed
byAg-AbcomplexandNohemolysisofsheep
RBCsoccurs.Sothetestispositiveforpresence
ofantibodies.
Negativetest:NoAg-Abreactionoccursandthe
complementisfree.Thisfreecomplementbinds
tothecomplexofsheepRBCandit’santibodyto
causeHemolysis,causingthedevelopmentof
pinkcolor.

7. Advantages
Abilitytoscreenagainstalargenumberofviral
andbacterialinfectionsatthesametime.
Economical.
Disadvantages
Notsensitive-cannotbeusedforimmunity
screening.
Timeconsumingandlaborintensive.
Oftennon-specifice.g.Cross-reactivity
betweenHerpesSimplexVirusandVaricella
ZosterVirus.