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FIXATION TEST
(CFT)
Microbiology Pharmacy
Dhean Vetra
260110110151
Nafilah Najla
260110110152
260110110154
Alhamzah Rahmat
260110110155
Faisal F Fasa
260110110156
Fauzan Fikri
260110110157
260110110159
Ariqa Dhianur S
Nusaibah Al Hima
Aisya Amalia M
260110110160
260110110161
260110110162
260110110164
Melanie Wibowo
260110110165
Introductio
n
Result and
Interpretati
on
Principles
CF
T
Negative
Test
Component
s
Positive
Test
1. INTRODUCTION
The complement fixation test (CFT) was extensively used in syphilis
serology after being introduced by Wasserman in 1909.
1. Complement is a
protein (globulin)
present in normal
serum.
2. Whole
complement system
is made up of nine
components: C1 to
C9
4. Complement
binds to Ag-Ab
complex
3. Complement
proteins are heat
labile and are
destroyed by
heating at 56C for
20 30 minutes.
5. When the Ag is
an RBC it causes
lysis of RBCs.
2. PRINCIPLE
Complement takes part in many of the immunological reactions. It
gets absorbed during the combination of antigens and antibody.
This property of antigenantibody complex to fix the complement
is used in complement fixation test for the identification of
specific antibodies.
The haemolytic system containing sheep erythrocytes (RBC) and
its corresponding antibody (amboceptor) is used as an indicator
which shows the utilization or availability of the complement.
If the complement is fixed then there will be no lysis of sheep
erythrocytes, thus denoting a positive test.
If the complement is available then there will be haemolysis which
is a property of complement, denoting a negative test.
The test
requires five
reagents
and is
carried out
in two steps.
3. COMPONENTS OF CFT
1. Test
System
Antigen
: It may be soluble
or
particulate.
Antibody
: Human serum
(May or may not contain
Antibody towards specific
Antigen)
Complement
: It is pooled
serum obtained from 4 to 5
guinea pigs. It should be fresh
or specially preserved as the
complement activity is heat
labile (stored at -30 C in small
fractions). The complement
activity should be initially
standardized before using in the
test.
2.
Indicator
System
(Haemol
ytic
system)
Erythrocytes
: Sheep
RBC
Amboceptor(
Hemolysin)
:Rabbit
antibody to
sheep red cells
prepared by
inoculating
sheep
erythrocytes
into rabbit
under standard
immunization
protocol.
4. POSITIVE TEST
Step 1:
At 37C
Antigen + Antibody + Complement
gets fixed
(from serum)
Complement
1 Hour
Step 2:
At 37C
Fixed Complement complex + Haemolytic system
No Haemolysis
1 Hour
(Test Positive)
5. NEGATIVE TEST
Step 1:
At 37C
Step 2:
At 37C
Free Complement + Haemolytic system
Haemolysis
1 Hour (Test Negative)
6. RESULTS AND
INTERPRETATIONS:
No haemolysis is considered as a positive test.
haemolysis of erythrocytes indicative of a
negative test.
1
2
3
4
A