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