COMPLEMENT FIXATION
TEST
Principle
“If antigen and antibody are
specific then complement will be
fixed with this complex of antigen
Antibody and this fixation can be
checked with indictor system.
This test have two stages / system”
Class notes by Dr. Hira Hmaeed
Complement fixation stage / strong
system
Indicator stage / weak system
Complement fixation stage contain
antigen, antibodies and
complement.
Indicator stage contain sheep
R.BCS + Amboceptors. There are
certain requirements for performing
this test.
Class notes by Dr. Hira Hmaeed
Preparation of complement
“Complement is protein like
substance present in serum”. For
the complement preparation.
Take blood from guinea-pig by
cardiac puncture.
Incubate at room temperature for
some minutes
Class notes by Dr. Hira Hmaeed
Centrifuge blood at 3000 rpm for
15 min.
Cell will be in sediment and collect
supernatant.
Again centrifuge supernatant at
same speed and collect
supernatant and it will contain
complement so preserve it for test.
Class notes by Dr. Hira Hmaeed
. Sheep RBCS
Take blood of sheep from jugular
vein
Separate RBCS by centrifugation
Wash RBCS as in HA test
Stored R.BCS at 4°C to utilized in
test.
Class notes by Dr. Hira Hmaeed
Amboceptor
injecting 1-2% solution of
R.BC of sheep in ear vein
of rabbit. Antibody will be
produced by rabbit against
RBCS.
Class notes by Dr. Hira Hmaeed
METHOD OF INJECTING R.BCS
1st day 0.2ml of 2% RBC in rabbit ear
vein.
Leave 2nd day
0.4 ml 3rd day
leave 4th day
Similarly increase dose from 0.2ml upto
1ml and inject on alternate day so at
9th day we inject 1 ml.
Class notes by Dr. Hira Hmaeed
After 14th day of injection
slaughter the rabbit collect
the blood and collect serum
from that blood. As it is
active immunization so
antibodies will be formed
after 10-14 days of
exposure.
Class notes by Dr. Hira Hmaeed
But this system contain
amboceptor as well as
complement but we require only
amboceptor so destroy this
complement by heating the
serum at 56°C for 15 min. now
this serum will contain only
amboceptor so preserve it for
test. Class notes by Dr. Hira Hmaeed
STRONG SYSTEM
For this system we require
complement Ag and Ab.
Complement is already
prepared
Ag is unknown (it is collected
from infected animal tissue /
cell). Class notes by Dr. Hira Hmaeed
Ab are known to us it is taken from any
standard laboratory OR
Other method is injected vaccine of
that suspected disease in laboratory
and take Ab from laboratory animal. It
is also contained complement which is
destroy by heating.
Standardize each requirement before
performing test.
Class notes by Dr. Hira Hmaeed
TITRATION OF COMPLEMENT
Make 2 fold serial dilution of
complement
Take 50µl of NS in microtitration
plate
Add 50µl of complement in first
well and mix it then so on as in
HA test. Class notes by Dr. Hira Hmaeed
Then add 50µl of haemolytic sys
tem (Amboceptor +1% sheep
RBCS) in each well.
Incubate at 37°C for 30min, then
record results. There will be Ag-Ab
reaction and there will be
haemolysis (reddish fluid) because
of fixation of complement with Ag-
Ab complex.
Class notes by Dr. Hira Hmaeed
In some well there will be no
haemolysis e.g. 1:64 has no
haemolysis. Last well have
haemolysis and it’s reciprocal is titre
of complement, so it is 32. 32 is one
haemolytic unit but we use 8
haemolytic unit of test i.e. 32/8 = 4 so
we use 1:4 well for test.
Now take 1ml of this 1:4 and add 3ml
NS and use this solution in test.
Class notes by Dr. Hira Hmaeed
TITRATION OF AMOCEPTOR
–Prepare 2 fold serial dilution of
amboceptor
–Take serum from rabbit and form
dilution as well do earlier.
–Add 1% RBC + 50µl complement
in each well.
Class notes by Dr. Hira Hmaeed
–Incubate at 37°C for 30 min.
–There will be haemolysis
–The last highest dilution of
amoboceptor having
haemolysis is one haemolytic
unit of amboceptor. Here we
will use 4th haemolytic unit of
amboceptor.
Class notes by Dr. Hira Hmaeed
HOW WE PERFORM CF TEST
CF stage
Take microtitration plate and
add 25µl Ab + 25µl Ag the add
25µl of standard complement.
Incubate at 37°C for 30 min
Class notes by Dr. Hira Hmaeed
Reaction will take place between
Ag and Ab.
If Ag-Ab are specific then Ag-Ab
complex is form and complement is
fixed and if Ag-Ab complex or
immune complex is not form then
complement is free.
Class notes by Dr. Hira Hmaeed
Indicator stage
Indictor (Amoceptor + Sheep
RBCS) Add 25µl of
amoboceptor +25 µl of RBC.
Incubate at 37°C for 30 min.
Record results
Positive no haemolysis
Class notes by Dr. Hira Hmaeed
Negative haemolysis
In case positive test: All available
complement is fixed by the Ag-Ab
reaction no haemolysis occur so the
test is +ve. In case –ve test:
No antigen-Ab complex occur. The
complement is free and it cause RBCS
lysis in indicator stage.
Class notes by Dr. Hira Hmaeed
Class notes by Dr. Hira Hmaeed
ADVANTAGES
It is convient and rapid test
Cheap because demand on equipment
and reagent is small and large variety
of test antigen is readily available.
Ability to screen against a large no. of
viral and bacterial infection at the same
time.
Class notes by Dr. Hira Hmaeed
Class notes by Dr. Hira Hmaeed
Disadvantage
Not more sensitive
Time consuming and labour
intensive.
Class notes by Dr. Hira Hmaeed
Disease in which CFT is used as
specific test
Viral disease: e.g. Marek’s
disease, viral encephalitis in
human.
Fungal diseases. E.g. fungal
infection of histoplasma.
Class notes by Dr. Hira Hmaeed
Bacterial disease e.g.
Brucellosis, Mycoplasma
pnemoniae. Acute respiratory
disease.
Rickettsial disease and is used
to detect many chlamydial
species
Class notes by Dr. Hira Hmaeed
