The immunoglobulin extraction technique can be applied to (1) infer the body's humoral immune function, and (2) diagnose high and low Ig caused by certain diseases.
Operation method
Immunoglobulin extraction technology
Principle
With the development and needs of immunology, the purification of immunoglobulin and its components has become essential. There are many methods of purification, there is a single method, but most of them use a combination of more than two steps, especially the purification of ammonium sulfate as the basis, and then through the chromatographic column method to improve the purity of immunoglobulin and its components is most commonly used. Ammonium sulfate solution dehydrates protein colloids and neutralizes their charge to precipitate them (called salting out). Different concentrations of ammonium sulfate salting out proteins have different compositions, and this principle is utilized to extract the desired immunoglobulin components. Salt dialysis can only be used for crude extraction, and in order to obtain purified immunoglobulin components, they must be further separated by chromatography. Immunoglobulins include IgG, IgM, IgA, IgE and IgD.
Materials and Instruments
Animal serum Colostrum Bile Move 1. Take 20 ml of serum, add 20 ml of saline, then add drop by drop (NH4) 2SO4 saturated solution 10ml, make into 20% (NH4) 2SO4 solution, stirring while adding, mix thoroughly, and then leave for 30min. 2. Centrifuge at 3000r/min for 20min, discard the precipitate to remove fibrin. 3. In the supernatant add (NH4) 2SO4 saturated solution 30ml, make into 50% (NH4) 2SO4 solution, mix thoroughly and leave for 30min. 4. Centrifuge at 3000r/min for 20min, discard the supernatant. 5. Add 20 ml of saline to the precipitate to dissolve it, then add (NH4) 2SO4 saturated solution 10ml, make 33% (NH4) 2SO4 solution, mix thoroughly and leave for 30min. 6. Centrifuge at 3000r/min for 20min and discard the supernatant to remove albumin. Repeat step 5 for 2--3 times. 7. 10ml of saline dissolve the precipitate and put it into dialysis bag. 8. Remove salt, dialyze in normal water overnight, then dialyze in saline at 4°C for 24h, with several fluid changes in between. Check SO42- in dialysate with 1% BaCl2 or check NH4 with Nasher's reagent (take 3--4ml dialysate, add 1--2 drops of reagent, brick red color is considered to be the presence of NH4), until no SO42- or NH4 appears. Until there is no SO42- or NH4 present. SephadexG25 or electrodialysis can also be used to remove salt. 9. Centrifuge to remove precipitation (to remove impurity protein), the supernatant is crude IgG (i.e., γ-globulin, such as 36% saturated ammonium sulfate precipitation of serum products that is euglobulin, Euglobin, containing γ-globulin). 10. Through the DEAE - cellulose chromatography column. (See Chromatography Techniques for column loading procedure). Elute with 0.01 Mol/L pH 7.4 PBS (0.03 Mol/L NaCl) and collect the eluate. Sephadex G150 or G200 columns can also be used. 11. Protein and its quantitative identification. 12. The purity of IgG can be identified by one of the following methods. ① Slide agar or acetate membrane electrophoresis can be used. After adding the sample electrophoresis, only in the migration site of γ-globulin appears a band. Operation, at the same time, can be used to whole serum samples, different concentrations of (NH4) 2SO4 saline samples for electrophoresis, in order to compare. ②Agar duplex double diffusion identification Prepare anti-IgG serum obtained by immunizing heterozygous animals with this IgG. The IgG and anti-IgG serum are subjected to biphasic double diffusion, and a precipitation line appears between the two sample wells if the IgG is purified. ③Immunoelectrophoresis identification Add the sample to be tested in the wells, and after electrophoresis, add anti-IgG serum in the tank, agar diffusion for 24h, and observe the results. If the extracted IgG is pure, only one curved precipitation line appears, and the precipitation line is located in the γ-globulin region. Immunoelectrophoresis of whole serum and antiserum antibodies must be performed simultaneously for this identification for comparison. ④Disk electrophoresis identification Disc electrophoresis is performed on both whole serum and purified samples. Whole serum samples show tens of bands on disc electrophoresis, while purified IgG shows only one band. 13. Concentration and preservation of IgG Concentration of IgG Preservation of IgG Generally concentrated to a concentration of 1% or more, and then divided into vials lyophilized and preserved, or add 0.01% thimerosal in the ordinary refrigerator or low-temperature refrigerator preservation, pay attention to prevent repeated freezing and thawing. Caveat Material preparation and reagent preparation1. Animal serum2.Ammonium sulfate saturated solutionAmmonium sulfate 800g~850gH2O1000mlHeat until most of the solute is dissolved, filter while hot, leave at room temperature overnight, then add 28% of the solute to the mixture.NH4OH to adjust pH to 7.0 (no pH adjustment is also possible).Note: Ammonium sulfate is preferable to the best quality, because the inferior product contains a small amount of heavy metals that have an effect on protein sulfhydryl groups. If the heavy metals must be removed from the inferior product, the solution can be passed through theH2S, let it stand overnight and then filter it, and evaporate the H2S by heating.3.0.01Mol/L pH7.4PBS solutionLiquid A: 0.10Mol/LNaH2PO4Liquid A: 0.10Mol/L NaH 2 PO 4NaH2PO4-; 2H2O15.60gAdd H2O to 1000.mlLiquid B: 0.10Mol/LNa2HPO4LiquidNa2HPO4-12H2O35.80gAdd H2O to 1,000mlJust take 19ml of liquid A and 81ml of liquid B and add water to 1000ml.4.1%BaCl2solution5.Nano solutionHgI 115.00gKI 80.00gAddH2Oto 500.00mlDissolve and filter, then add 20% NaOH 500.00ml and mix.6. 0.50 Mol/L of HCl solution and 0.50 Mol/L of NaOH solution.7. Elution solution: 0.03 Mol/L NaCl solution.8. Dialysis bag (or cellophane). Common Problems The application of ammonium sulfate chromatography and DEAE-cellulose chromatography to purify IgG is not only complicated and time-consuming, the volume of the sample is greatly increased in the process, but also the denaturation is serious when dialysis is performed, which is easy to cause a decrease in the potency of the antibody, and so on. In order to overcome this deficiency, especially when a large amount of IgG is extracted, the following simple methods are often adopted. 1. DEAE-cellulose direct extraction method The sample is taken directly through the DEAE-cellulose column. The following is the simplest beaker method. ① to a certain amount of DEAE52 into a beaker, add 0.01Mol / L pH8.0 PBS buffer, let stand for 30min, go to the supernatant fine particles, and then repeat. (ii) Filtered by using a Büchner funnel (with two layers of filter paper inside). ③ Add the components in the ratio of 5g wet weight of DEAE-cellulose plus 1ml serum and 3ml distilled water mixture and mix thoroughly. ④ Place in 4°C for 1h, stirring several times in between. ⑤ Filtering was performed with a Brinell funnel, and then the cellulose was rinsed with 0.01 Mol/L pH8.0 PB buffer and filtered. The filtrate is the extracted IgG solution. 2.DEAE--SephadexA--50 is a weakly alkaline anion exchanger, which can adsorb acidic proteins after changing Cl-type to OH-type by NaOH, except for γ--G, which is a neutral protein. --G is a neutral protein, the rest are acidic proteins. When the pH of the solution is 6.5, acidic proteins are adsorbed by DEAE--SephadexA--50, and only γ--G remains in the solution. Therefore, this principle can be used to extract γ--G. The process of this extraction method is greatly shortened, the volume of the sample before and after purification does not change much, and the resulting γ--G has no denaturation phenomenon. After four treatments, its purity is also better. The disadvantage is that the collection volume is small and the loss is large. ① Treatment of DEAE-SephadexA-50. Take several grams of DEAE--SephadexA--50, suspend it in distilled water, pour off the upper layer of small particles after 1h, then treat it with 0.50Mol/L NaOH for 1h, wash it with distilled water to neutral, then treat it with 0.5 Mol/L HCl for 0.5h, washed to neutral, and finally equilibrated and drained with 0.01Mol/L pH6.5 PB solution. ② Take a certain amount of serum, add an equal amount of 0.01 Mol/L pH6.5 PB liquid, and then add 1/4 of the liquid volume of filter-dried DEAE--SephadexA--50, mix, leave at 4°C for 1h, stirring from time to time, and filtration, Collect the filtrate. (iii) Then treat the filtrate with the same weight of DEAE--SephadexA--50. Repeat the process three times. (Total of four times throughout the process). The filtrate was obtained as γ--G products. ④ Recovery of DEAE--SephadexA--50. Elute with 0.5Mol/L Na2HPO4, filter until the filtrate is free of protein (OD280﹤0.04), and then wash with distilled water until neutral to be recycled. For more product details, please visit Aladdin Scientific website.
Ammonium sulfate NH4OH PBS NaH2PO4 NaH2PO4 2H2O BaCl2 HgI KI HCl NaOH PB DEAE52 Dextran gel G200 Sodium sulfate anhydrous Borate buffer
Centrifuge Dialysis bag Cryogenic refrigerator Electrophoresis tank Electrophoresis apparatus