1、Exp III Analysis of antibody-forming cells in vitroQHS Complement-binding site CH1 CH2IgM CH3，IgG CH2C1q binding siteAgAg + AbBinding to ComplementAg + Ab + CActivation of ComplementLysis of target cellsTest 1 Analysis of antibody-forming cells in vitroQHS1. Principle: AgSRBCImmunize the mice4 days

2、laterPCIgM+ SRBC Complement( QHSQuantitative Hemolytic Spectrophotometric determination )YYYYHbHbHbSpleen2. Materials: Fresh SRBC solution(1.5108/ml)SRBC-immunized miceSRBC-absorbed complementFrom Guinea pigAbsorbed by SRBC to block non-specific antibody 3. Methods: Take the spleen and wash with Han

3、ks + 5% FCSGrind the spleen on a brass net (200 mesh)Collect the cell suspension and add Hanks + 5% FCSCentrifuge at 2000rpm, 5mina. Prepare spleen cellsDiscard the supernatantAdd 5mlTris-HCl and incubate at 37oC for 5minCentrifuge at 2000rpm, 5minSacrifice the SRBC-immunized mice by cervical disloc

4、ationDiscard the supernatant and add 1.2mL Hanks + 5% FCS3. Methods: b. Ag+Ab+Complement Reaction:Spleen cellsHanks1mlAdd 1ml SRBC solutionAdd 1ml complementMixing thoroughlyIncubate at 37 for 1hCentrifuge at 3000rpm for 5minCTCompare the color change between Control and Test tube3. Methods: RBCWBCWBCWBCWBC1mm1mmCell concentration/ml = Total cell count in 4 squares /4 x 104Cell concentration/ml= Total cell count in 5 squares x