医学免疫学教学资料：Exp3 Complement fixation test

1、Exp III 1. Complement fixation test (CFT) 2. Preparation of Antibody (Third time immunization) 1. Complement fixation Test(CFT)Complement-binding siteFab段Fc段C1q 结合位点被屏障结合抗原之前 CH1 CH2暴露的C1q结合位点结合抗原之后IgM CH3区，IgG CH2区 PrincipleTest 1 Determination of the titer of hemolysin PrincipleTest 1 Determinatio

2、n of the titer of hemolysinAg + AbComplementAg + Ab + CActivation of Complement PrincipleTest 1 Determination of the titer of hemolysinActivation of ComplementLysisDefinition Complement fixation reaction: Ag-Ab reaction in the presence of complement and an indicator system SRBC and hemolysin (anti-S

3、RBC Ab) Two systems, five components in CFT:Test system: known Ag or Ab, unknown Ab or Ag and quantitative complement.Indicator system: SRBC and hemolysinPrincipleAg+Unknown serum1 2Complement12Step 1 Formation of Ag-Ab complexStep 2Complement fixation and exhaustionSRBC+hemolysinStep 3Lysis of SRBC

4、12AgYPatientsserum YYYYYYYYAbPositiveNo AbNegativeAgAgAgAg: Lytic products from typhiodTest serum: 56 30minComplement: from guinea pig2% SRBCHemolysinMaterialMethods12345TestSerum controlAg controlComplement controlSRBCcontrolTest serum0.2mL0.2mL0.2mL0.2mL0.2mL0.2mL0.2mL0.2mL0.2mL0.2mL0.4mL0.8mLAgCo

5、mplement N.SShake，incubate in water bath at 37 for 15minHemolysinSRBC0.2mL0.2mL0.2mL0.2mL0.2mL0.2mL0.2mL0.2mL0.2mL Shake，incubate in water bath at 37 for 15minAnticipated results1Hemolysis: clear, red No hemolysis: turbid, ruddyHemolysisNo hemolysis2. TestSerum controlAg controlComplement controlSRBC control？HemolysisHemolysisHemolysisNo hemolysisShake SRBC before usePipettes for different reagents should not be confusedAttentions2. Third immunizationUncoagulated SRBCAdd 3ml N.S and mix2000rpm, 5min2000rpm, 5minDiscard the supernatantDiscard the supernata