# Complement Fixation and Neutralisation Tests > Complement fixation and neutralisation tests for NEET-PG Microbiology: the haemolytic indicator, reading a positive CFT, paired sera and plaque reduction tests. - Canonical URL: https://prepelephant.com/topics/neet-pg/microbiology/complement-fixation-and-neutralisation-tests - Exam / course: NEET-PG · Subject: Microbiology - Publisher: PrepElephant (https://prepelephant.com) — Prepared and reviewed by the PrepElephant Academic Review Team - First published: 2026-10-14 - Last updated: 2026-10-14 - Reference basis: Ananthanarayan and Paniker's Textbook of Microbiology; Murray's Medical Microbiology; Topley and Wilson's Microbiology and Microbial Infections - How to cite: "Complement Fixation and Neutralisation Tests", PrepElephant, https://prepelephant.com/topics/neet-pg/microbiology/complement-fixation-and-neutralisation-tests ## Direct answer The complement fixation test is a serological assay in which patient antibody and its specific antigen together consume added complement, so no free complement remains to lyse indicator sheep erythrocytes. Absence of haemolysis therefore means a positive result. Neutralisation tests use a different principle, measuring antibody that blocks a virus or toxin from infecting cells, mice or embryonated eggs. ## What you must remember - **Principle:** antigen-antibody complexes fix complement, so free complement is left only when no specific antibody is present to consume it. - **Haemolytic system:** sheep erythrocytes sensitised with rabbit anti-sheep antibody, called haemolysin, form the indicator, which lyses only when free complement is present. - **Positive CFT:** no haemolysis, so the red cells settle as a button at the bottom of the tube. - **Negative CFT:** complete haemolysis, showing that complement was not fixed and the serum lacked the specific antibody. - **Paired sera:** a fourfold or greater rise in titre between acute and convalescent samples, taken two to four weeks apart, indicates recent infection. - **Uses:** historically influenza, Mycoplasma pneumoniae, Q fever, Coccidioides and other fungal and viral infections, although ELISA has largely replaced it. - **Neutralisation:** patient serum mixed with virus prevents infection, and the plaque reduction neutralisation test is the reference method for dengue and flavivirus serology. ## A stepwise approach to the complement fixation test The test is a sequence of incubations, and the logic of each step follows from the reversed reading at the end. The steps below describe the usual laboratory order. 1. **Inactivate the serum:** patient serum is heated at 56 degrees Celsius for 30 minutes so that its own complement is destroyed before testing. 2. **Fix the complement:** patient serum, antigen and a standard amount of complement are mixed and incubated, so complement is consumed if specific antibody is present. 3. **Add the indicator:** sheep erythrocytes coated with haemolysin are added to the mixture and incubated again. 4. **Read the haemolysis:** a clear tube with settled red cells is positive, while a red, haemolysed tube is negative. 5. **Titrate:** serial dilutions are tested, and the highest dilution that still fixes complement is recorded as the titre. The indicator reacts only when complement is free, so the reading is the reverse of what most students expect. A clear tube means fixation has occurred, and that is the positive result. ## Where the exam tries to catch you Complement fixation questions are often built on the reversed reading, so the logic of the indicator is the point to check. - **Trap:** reading haemolysis as a positive complement fixation result — **Correct:** haemolysis means complement was free and the result is negative, while settled red cells in a clear tube mean the test is positive. - **Trap:** using one acute serum sample to prove recent infection — **Correct:** a fourfold rise between acute and convalescent sera is needed, since a single titre shows only exposure at some point. - **Trap:** thinking neutralisation tests detect antigen — **Correct:** they measure functional antibody that blocks infectivity, which is why the plaque reduction test is the reference method for dengue. ## Frequently asked questions ### Why is patient serum heated before a complement fixation test? Patient serum is heated at 56 degrees Celsius for 30 minutes to inactivate its own complement. Endogenous complement would otherwise fix to the antigen-antibody complex and distort the reading, so only the added standard complement is consumed. ### What does a fourfold rise in complement fixation titre indicate? A fourfold or greater rise in complement fixation titre between paired acute and convalescent sera indicates recent infection with the organism. A single titre shows only past or current exposure, so paired samples are needed for a diagnosis. ### What is the haemolytic system in complement fixation testing? The haemolytic system is the indicator made of sheep erythrocytes sensitised with rabbit anti-sheep antibody, called haemolysin. When free complement is present, it lyses these red cells, which tells the reader that no fixation has occurred in the test tube. ### Why is the plaque reduction neutralisation test used in dengue serology? The plaque reduction neutralisation test measures how much patient serum reduces plaque formation by dengue virus in cell culture. It is regarded as the reference method for flavivirus antibody because it detects functional neutralising antibody and helps distinguish cross-reactive flaviviruses.