1 / 17

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.

verna
Télécharger la présentation

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

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

More Related