(1) Preparation of materials and reagents
1. Animal Serum
2. Ammonium sulfate saturated solution
Ammonium sulfate 800g ~ 850g
H2O 1 000ml
Heat until most of the solutes are dissolved, filter while hot, and leave to room temperature overnight, and then adjust the pH to 7.0 with 28% NH4OH (it can be adjusted without adjusting the pH). Note: The quality of ammonium sulfate is better, because the defective products contain a small amount of heavy metals on the protein sulfhydryl group. If heavy metals must be removed from the defective product, H2S can be passed through the solution, filtered after standing overnight, and H2S can be evaporated by heating.
3.0.01Mol / L pH7.4PB solution
Liquid A: 0.10Mol / L NaH2PO4 liquid
NaH2PO4 • 2H2O 15.60g plus H2O to 1 000.ml
Liquid B: 0.10Mol / L Na2HPO4 liquid
Na2HPO4 • 12H2O 35.80g plus H2O to 1 000ml
Take 19ml of liquid A and 81ml of liquid B and add water to 1000ml.
4.1% BaCl2 solution
5. Nessler's solution
HgI 115.00g
KI 80.00g plus H2O to 500.00ml
After melting, filter, and then add 20% NaOH 500.00ml and mix.
6.0.50 Mol / L HCl solution and 0.50 Mol / L NaOH solution.
7. Eluent: 0.03Mol / L NaCl solution.
8. Dialysis bag (or cellophane).
(2) Operation method
1. Take 20ml of serum, add 20ml of normal saline, then add 10ml of (NH4) 2SO4 saturated solution drop by drop to make 20% (NH4) 2SO4 solution, stir while adding, mix thoroughly, and let stand for 30min.
2. Centrifuge at 3000r / min for 20min, discard the precipitate to remove fibrin.
3. Add 30ml of (NH4) 2SO4 saturated solution to the supernatant to make 50% (NH4) 2SO4 solution, mix thoroughly and let stand for 30min.
4. Centrifuge at 3000r / min for 20min and discard the supernatant.
5. Add 20ml of physiological saline to the precipitate to dissolve it, and then add 10ml of (NH4) 2SO4 saturated solution to make 33% (NH4) 2SO4 solution. After fully mixed, let stand for 30min.
6. Centrifuge at 3000r / min for 20min and discard the supernatant to remove albumin. Repeat step 5, 2 ~ 3 times.
7. Dissolve the precipitate with 10ml of physiological saline and put it in a dialysis bag.
8. Dialysis and desalination, dialysis in normal water overnight, and then dialyzed in normal saline at 4 ℃ for 24h, changing fluid several times in the middle.
Check SO42- in dialysate with 1% BaCl2 or NH4 + with Nessler's reagent (take 3 ~ 4ml dialysate, add 1 ~ 2 drops of reagent, brick red means NH4 + is present) until no SO42- or NH4 + appears until. Sephadex G25 or electrodialysis can also be used for desalting.
9. Centrifugation to precipitate (removal of miscellaneous proteins), the supernatant is the crude extracted IgG (ie, gamma globulin, such as the serum product precipitated with 36% saturated ammonium sulfate, which is Euglobin, Euglobin, containing gamma globulin) .
10. Pass the DEAE-cellulose chromatography column. (See chromatography technique for packing process). It was eluted with 0.01 Mol / L pH 7.4 PBS (0.03 Mol / L NaCl), and the eluate was collected.
Sephadex G150 or G200 columns can also be used.
11. Protein and its quantitative identification (see Section 8 of this chapter).
12. The purity of IgG can be identified by one of the following methods.
⑴ Zone electrophoresis: slide agar or cellulose acetate membrane electrophoresis can be used. After sample loading electrophoresis, only one band appeared at the migration site of γ-globulin. During operation, electrophoresis can be performed on whole serum samples and salted-out samples of different concentrations (NH4) 2SO4 for comparison.
⑵ Biphasic double-diffusion identification of agar: prepare anti-IgG serum obtained by immunizing the heterogeneous animal with the IgG in advance. Biphasic and double diffusion of IgG and anti-IgG serum, if IgG is purified, a precipitation line appears between the two sample wells.
(3) Identification by immunoelectrophoresis: (see Chapter 3 for immunoelectrophoresis). The sample to be tested is added to the well, after electrophoresis, anti-IgG serum is added to the well, and the agar is diffused for 24 hours, and the result is observed. If the extracted IgG is pure, only one curved precipitation line appears, and the precipitation line is located in the γ-globulin region. This identification must be performed simultaneously with immunoelectrophoresis of whole serum and antiserum antibodies for comparison.
â‘· Disc electrophoresis identification: Carry out disc electrophoresis simultaneously with whole serum samples and purified samples. There are dozens of bands on the whole serum sample on the disk electrophoresis, while the purified IgG has only one band.
13. Concentration and storage of IgG
â‘´ Concentration of IgG: please refer to Section 9 of this chapter.
⑵ Preservation of IgG: It is generally concentrated to a concentration of more than 1%, and then packed into vials for lyophilization, or added with 0.01% thimerosal in ordinary refrigerators or low-temperature refrigerators, taking care to prevent repeated freezing and thawing.
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