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Exp Ⅲ 1. Analysis of antibody-forming cells in vitro——QHS 2. Antibody preparation(3)

Exp Ⅲ 1. Analysis of antibody-forming cells in vitro——QHS 2. Antibody preparation(3). Test 1 Analysis of antibody-forming cells in vitro——QHS. Complement-binding site. 结合抗原之后. 结合抗原之前. Fab 段. C H1. 暴露的 C1q 结合位点. C1q 结合位点被屏障. C H2. Fc 段. IgM CH3 区， IgG CH2 区. Ag + Ab.

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Exp Ⅲ 1. Analysis of antibody-forming cells in vitro——QHS 2. Antibody preparation(3)

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  1. Exp Ⅲ 1. Analysis of antibody-forming cells in vitro——QHS 2. Antibody preparation(3)

  2. Test 1 Analysis of antibody-forming cells in vitro——QHS

  3. Complement-binding site

  4. 结合抗原之后 结合抗原之前 Fab段 CH1 暴露的C1q结合位点 C1q 结合位点被屏障 CH2 Fc段 IgM CH3区，IgG CH2区

  5. Ag + Ab Binding to Complement Ag + Ab + C Activation of Complement Lysis of target cells

  6. Y Hb Y Y Hb Y Hb ( QHS——Quantitative Hemolytic Spectrophotometric determination ) 1. Principle: Test 1 Analysis of antibody-forming cells in vitro——QHS Ag Spleen IgM 4 days later PC Immunize the mice SRBC + SRBC Complement Quantitation by spectrophotometry

  7. 2. Materials: Test 1 Analysis of antibody-forming cells in vitro——QHS • Fresh SRBC solution(1.5×108/ml) • SRBC-immunized mice • SRBC-absorbed complement • From Guigea pig • Absorbed by SRBC to block non-specific antibody

  8. Sacrifice the SRBC-immunized mice by cervical dislocation Take the spleen and wash with Hank’s + 5% FCS 3. Methods: Grind the spleen on a brass net (200 mesh) Collect the cell suspension and add Hank’s + 5% FCS a. Prepare spleen cells Centrifuge at 2000rpm, 5min Discard the supernatant Add 4.5ml DDW and mix hardly for 45s Adjust the concentration to 2×107/ml Add 0.5ml 9% NaCl quickly Centrifuge at 2000rpm, 5min Count the cells Discard the supernatant and add 5mlHank’s + 5% FCS add 1mL Hank’s + 5% FCS Centrifuge at 2000rpm, 5min

  9. C T 1ml 3. Methods: Hank’s Spleen cells Add 1ml SRBC solution b. Ag+Ab+Complement Reaction: Add 1ml complement Mixing thoroughly Incubate at 37℃ for 1h Centrifuge at 3000rpm for 5min Analyze the supernatant by spectrophotometry at 413nm

  10. 3. Methods: 1mm WBC WBC 1mm RBC WBC WBC Cell concentration/ml = Total cell count in 5 squares x 5 x 104 Cell concentration/ml = Total cell count in 4 squares /4 x 104

  11. 4. Attentions: Test 1 Analysis of antibody-forming cells in vitro——QHS • Fresh SRBC solution • Keep the spleen cells alive

  12. Test 2 Third immunization

  13. Test 2 Third immunization Uncoagulated SRBC Methods: Add 3ml N.S and mix 2000rpm, 5min Discard the supernant Add 3ml N.S and mix 2000rpm, 5min Discard the supernant Take 0.1ml to immunize the mice subcutaneously Make 20%、25%、30%、40% SRBC solution

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