Complement fixation test

Principle

• Detects antibody (or antigen) by its ability to fix (consume) complement when an antigen–antibody complex forms.
• Test system: patient serum (heat-inactivated at 56 °C for 30 minutes to destroy its own complement) + specific antigen + measured amount of guinea pig complement.
• Indicator system: sheep red cells sensitised with anti-sheep red cell antibody (haemolysin, amboceptor).
• If antibody is present, complement is used up and the indicator cells are not lysed: no haemolysis = positive.
• If antibody is absent, free complement lyses the indicator cells: haemolysis = negative.

Specimen

• Serum, preferably paired acute and convalescent samples 10–14 days apart; also CSF for some tests.

Procedure

• Titrate haemolysin and complement beforehand to use a standard dose (commonly two exact units of complement).
• Incubate serial serum dilutions with antigen and complement (e.g. overnight at 4 °C or 1 hour at 37 °C, per protocol).
• Add sensitised sheep cells, incubate at 37 °C, then read the degree of haemolysis.

Results

• Titre: highest serum dilution showing no or minimal haemolysis, according to the laboratory's end-point criterion.
• Fourfold rise between paired sera indicates recent infection.

Quality control and pitfalls

• Anticomplementary control (serum without antigen) detects sera that fix complement on their own, e.g. from immune complexes, bacterial contamination, heparin or haemolysis; such results are invalid.
• Antigen control, cell control, complement and haemolysin controls are essential.
• Incomplete heat inactivation or incorrect complement dose gives false results.
• Labour-intensive and needs many reagents; largely replaced by ELISA.

Clinical use

• Historically the Wassermann test for syphilis; still used in some reference labs for fungal serology (Coccidioides, Histoplasma), Q fever, and some viral and Mycoplasma infections.