Complement Fixation Test

May 18, 2019

It is one of the immunological medical tests that can be used to detect the presence of either specific antibody or specific antigen in a patient’s serum.
This test is based on whether complement fixation occurs or not.
It is used for various purposes such as diagnosing infections caused by microbes that are not easily detected by culture methods and also in rheumatic diseases.
But it has been largely superseded by other serological tests like ELISA and DNA-based methods of pathogen detection particularly PCR.
Though this test use has been reduced, it has still value in virology in which CFT provides a retrospective diagnosis because complement fixing antibodies appears in blood late (1-2 weeks after infection).

Principle

Antigen- antibody complex fixes the complement.
The coupling of complement with Ag-Ab complex does not have any visible effect such as agglutination or precipitation.
So, it is necessary to use an indicator system consisting of sheep red cells coated with anti-sheep red cell antibody i.e. also called amboceptor.
Complement lyses antibody coated red cells.
Similar effect (lysis) occurs when these Ag- Ab and complement complexes are formed on bacteria.
The antigen must be quantitated which may be in soluble form (cardiolipin antigen in W.R.).
The test serum is inactivated by heating at 56⁰C for half an hour before the test so that the complement activity of the test serum can be destroyed.
This helps to remove anti-complementary effect of some non-specific inhibitors in the serum.

Image source: researchgate

Standardization of complement

The source of complement is freshly drawn guinea pig serum.
The guinea pig serum is titrated to find out complement activity.
One unit or minimum haemolytic dose (MHD) of complement is defined as the smallest amount (highest dilution) of guinea pig serum that will cause complete lysis of arbitrarily selected one unit volume washed sheep red cells.
It occurs in the presence of excess haemolysin (amboceptor) in a fixed time at a fixed temperature (30-60 minutes at 37⁰C).

Titration of amboceptor

The amboceptor is titrated for determination of haemolysin activity.
For titration of complement, amboceptor and for CFT, normal saline with added calcium and magnesium ions is used as diluent.

Procedure of CFT

The classical example of CFT is Wassermann reaction (WR), which was used once for diagnosis of syphilis and now obsolete.
The reaction consists of two steps:

i)Test system

Antigen (cardiolipin) and antibody (inactivated patient’s serum) are mixed and a measured amount of complement (two units, usually guinea pig) is added.
It is then incubated at 370C for one hour.
If the antigen and antibody match, the complement will be utilized (fixed).
But if they do not match, the complement will remain free.

ii)Indicator system

It consists of sensitized red blood cells (sheep erythrocytes coated with 4MHD haemolysin) which is added to the test mixture and incubated at 37⁰C for 30 minutes in water-bath.

iii)Observation

Absence of haemolysis indicates that complement was used up during the initial reaction.
Presence of haemolysis indicates persistence of complement, which is taken as negative result.

Other complement dependent serological tests