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
Fab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
Ag + AbBinding to Complement
Ag + Ab + C
Activation of Complement
Lysis of target cells
Test 1 Analysis of antibody-forming cells in vitro——QHS
1. Principle: 1. Principle:
Ag
SRBC Immunize the mice4 days later4 days later
PCPC
IgM
+ SRBC Com+ SRBC Complementplement
Quantitation by spectrophotometry
( QHS——Quantitative Hemolytic Spectrophotometric determination )
Hb
Hb
Hb
Spleen
Test 1 Analysis of antibody-forming cells in vitro——QHS
2. Materials: 2. Materials:
Fresh SRBC solution(1.5×108/ml)
SRBC-immunized mice
SRBC-absorbed complement From Guigea pig
Absorbed by SRBC to block non-specific antibody
3. Methods: 3. Methods: Take the spleen and wash with H
ank’s + 5% FCS
Grind the spleen on a brass net (200 mesh)
Collect the cell suspension and add Hank’s + 5% FCS
Centrifuge at 2000rpm, 5min
a. Prepare spleen cells
Discard the supernatant
Add 4.5ml DDW and mix hardly for 45sAdd 4.5ml DDW and mix hardly for 45s
Add 0.5ml 9% NaCl quicklyquickly
Centrifuge at 2000rpm, 5min
Discard the supernatant and add 5ml Hank’s + 5% FCS
Count the cells
Adjust the concentration to 2×107/ml
Sacrifice the SRBC-immunized mice by cervical dislocation
Centrifuge at 2000rpm, 5minadd 1mL Hank’s + 5% FCS
3. Methods: 3. Methods:
b. Ag+Ab+Complement Reaction:
Spleen cellsHank’s
1ml
Add 1ml SRBC solution
Add 1ml complement
Mixing thoroughly
Incubate at 37℃ for 1h
Centrifuge at 3000rpm for 5min
Analyze the supernatant by spectrophotometry at 413nm
C T
3. Methods: 3. Methods:
RBC
WBC WBC
WBCWBC
1mm
1mm
Cell concentration/ml = Total cell count in 4 squares /4 x 104
Cell concentration/ml= Total cell count in 5 squares x 5 x 104
Test 1 Analysis of antibody-forming cells in vitro——QHS
4. Attentions: 4. Attentions:
Fresh SRBC solution
Keep the spleen cells alive
Test 2 Third immunization
Test 2 Third immunization
Methods:Uncoagulated SRBCUncoagulated SRBC
Add 3ml N.S and mixAdd 3ml N.S and mix
2000rpm, 5min2000rpm, 5min
2000rpm, 5min2000rpm, 5min
Discard the supernantDiscard the supernant
Discard the supernantDiscard the supernant
Make 20%Make 20% 、、 25%25% 、、 30%30% 、、40% SRBC solution40% SRBC solution
Take 0.1ml to immunTake 0.1ml to immunize the mice subcutaize the mice subcutaneouslyneously
Add 3ml N.S and mixAdd 3ml N.S and mix