Exercise.Ⅲ Complement Fixation Test
Principle
Complement（C）has no specificity and could be activated by any antigen-antibody complex.But complement could not be activated by dissociative antigen or antibody.
Complement fixation test（CF）is a kind of reaction system that complement participated in.In CF，sheep red blood cell（SRBC）and hemolysin（anti-SRBC antibody）act as indicating system.The combination of SRBCand hemolysin could activate complement and lead to RBC destruction，hemolysis occurs.
So the five components in CF test could be divided into 2 system.1）test system：known antigen（or antibody）and unknown antibody（or antigen）.2）indicating system：SRBC and hemolysin.Test system reacts with complement at first.Then indicating system is added.No hemolysis indicates that antigen is corresponding to antibody and the immune complex of the testing system activated and used up complement.So no complement combine with indicating system and no hemolysis occurs.This is defined as CF test positive result.On the contrary，if hemolysis occurs，the CF test has negative result.
Preparation test
1.Titering of Hemolysin Unit
Materials
（1）2% SRBC：take fresh sheep blood get rid of fiber，Wash with normal saline for 2 times.Prepare to 2%concentration for usage.
（2）Hemolysin：serum of animal immunized by SRBC.
（3）Complement：take fresh guinea pig serum and dilute to 1∶30.Preserve in refrigerator for usage.
（4）Others：Normal Saline，test tube，pipette，37℃water bathe box.
Methods
（1）Add hemolysin of different dilution 0.2 ml to tubes as shown in form below，respectively.Add other reagent respectively.
（2）Mix completely and put in 37℃water bathe for 30 min.Then observe the result.
（3）Determine the highest dilution hemolysin that has complete hemolysis as 1 unit.
Experiment in the table below shows that the No.11 tube（that is，1∶9600 dilution）0.2 ml hemolysin is 1 unit.The dilution fluid contains 2 units hemolysin is often used in experiment.We could prepare the hemolysin by taking 1∶100 hemolysin 1 ml and normal saline 47 ml.
2.Titering of Complement
Materials
（1）Complement：Dilute to 1∶30（Same as the titering of hemolysin）.（
2）2 unit hemolysin.
（3）2% SRBC（Same as the titering of hemolysin）.
（4）Other reagents（Same as the titering of hemolysin）.
Methods
（1）Add 1∶30 diluted complement into tubes as form 2.
（2）Add other reagents in turn to each tube.Mix and put in 37℃water bathe for 15 to 30 min.Observe the result and decide the complement unit（Table 1）.
Table 1 Titering of Hemolysin（unit：ml）
3.Complement unit
The minimum quantity that could dissolve certain quantity RBC is determined as 1 confirmed unit.For instance，if complete hemolysis phenomena occurred since No.3 tube in form 2，we would determine that complement quantity in tube 3（0.1ml）as 1 confirmed unit.
Usually the next higher tube complement quantity rather than the confirmed unit is referred to as 1 practical unit.Take an example，if in form 2，the tube 4（0.12ml）is 1 practical unit.Then it could be expressed as below，(https://www.daowen.com)
1 confirmed unit=0.1ml 1∶30 diluted complement
1 practical unit=0.12ml 1∶30 diluted complement
1 Complement dilution
To make 0.2ml complement contains 2 practical units，we could calculate as formula below，
30∶（20.12）=X∶0.2
X=25
So we could dilute the complement original fluid for 25 fold，and use 0.2ml of it（Table 2）.
Table 2 Titering of Complement（unit：ml）
Formal test
1.Materials
（1）Complement：Dilute according to the complement unit tittering result.
（2）Antigen：Boiled typhoid bacteria fluid of 1×109/ml for 2hours.Centrifuge at 3000 rpm for 30 min.Extract supernatant as antigen.Titer antigen before experiment and use 4 unit antigen in experiment.
（3）Unknown serum：Inactivate at 56℃for 30min.Dilute to 1∶5 after inactivation.
（4）Hemolysin 2 unit.
（5）2% SRBC
2.Procedures and results
（1）Take 5 test tubes.Mark them and put into rack in turn.
（2）Add reagent as the table below（Table 3）.
Table 3 Complement Fixation Test（unit：ml）
3.Results
Tube 2、3、4、5 are control tubes，which should appear hemolysis、hemolysis、hemolysis and no hemolysis，respectively.These control results indicating that control tubes are normal.Tube 1 is experiment tube.No hemolysis indicates Positive result of complement fixation test（+）.Hemolysis indicates negative result（-）.
4.Attentions
（1）Shake gently before sheep blood usage.Avoid severe shake in case hemolysis.
（2）Do not mix up pipette of different reagents.
（3）Complement is not stable and should be kept at low temperature.Take it out from refrigerator just before adding sample.
（4）Avoid water dropping into tube during water bathe.
（5）Reaction situation of control tubes is the reference for judging the experiment credibility，as a series of factors may affect the experiment.