CN110256554A - The preparation method of immunoglobulin subclass antibodies - Google Patents
The preparation method of immunoglobulin subclass antibodies Download PDFInfo
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- CN110256554A CN110256554A CN201910567139.7A CN201910567139A CN110256554A CN 110256554 A CN110256554 A CN 110256554A CN 201910567139 A CN201910567139 A CN 201910567139A CN 110256554 A CN110256554 A CN 110256554A
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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Abstract
The present invention provides the preparation methods of immunoglobulin subclass antibodies, comprising: IgG, six steps of inactivation of virus and preservation are concentrated in preparation IgG semifinished product, desalination of dialysing, chromatographic purifying；Wherein, repeatedly centrifugation purification is carried out to serum using neutral saturated ammonium sulfate solution and IgG semifinished product is prepared, and used DEAE cellulose, QAE cellulose, Sephadex G150 or Sephadex G200 column and carry out chromatographic purifying.The present invention combines the purity for the immunoglobulin hypotype extracted to be greatly improved using salting out method and ion exchange chromatography, and purity is improved at 5 times or more, and can remove DNA and RNA etc.；Have the characteristics that with high purity, at low cost, equipment is simple, its bioactivity can be reserved for separated object.
Description
Technical field
The present invention relates to the technical fields of immunoglobulin G, and in particular, to the preparation side of immunoglobulin subclass antibodies
Method.
Background technique
Bone-marrow-derived lymphocyte is converted into thick liquid cell under antigenic stimulus, and generation can be specifically bound anti-with corresponding antigens
Body, referred to as immunoglobulin.Immunoglobulin G (IgG) is the principal component of Immunoglobulin in Serum, accounts for about and ball is immunized in serum
The 75% of albumen total content.IgG has 4 hypotypes, i.e. IgG1, IgG2, IgG3, IgG4, these four hypotypes contain shared by the normal human
Measuring ratio is respectively 60-70%, 14-20%, 4-8% and 2-6%.IgG is internal most important antibody, has antiviral, neutralization disease
Poison, antibacterial and immunoregulatory function, IgG1, IgG3 mainly fight proteantigen, such as common virus infection.IgG2 is
The main antibody of the capsular bacterium containing polysaccharide body is fought, most slow, most easy shortage is developed.And IgG4 is related with the allergic reaction of food.
The amount of IgG2, IgG3, IgG4 hypotype can be covered by main IgG1, no matter therefore IgG total amount be low value or normal value, cannot all arrange
Except there is the case where hypotype defect, appearance can individually occur or combine in shortage.IgG is also uniquely can be by the anti-of placenta
Body plays an important role in newborn is anti-infective.Some autoantibodies, such as the LE factor of systemic loupus erythematosus, antithyroid
Globulin antibody also belongs to IgG.The IgG content in serum is detected, most common method is simple immunodiffusion method and immune ratio
Turbid method, but the latter gradually replaces the former.
Immunoglobulin IgG is the main component of serum immune globulin, accounts for about the 75% of all immunoglobulins, therefore,
Antibody isolates and purifies primarily discrete IgG purification.Thick formulation extracts Immunoglobulin IgG ammonium sulfate salting-out process multi-purpose greatly or sulphur
Acid sodium-salt analyses method, and salting out method has at low cost, and equipment is simple, the characteristics of can be reserved for its bioactivity to separated object；But it is mentioned
The product purity taken is not high.Ion exchange chromatography, which extracts the common ion-exchanger of IgG, DEAE cellulose, hands over through ion
It is purer to change the IgG that chromatography obtains, is free of other foreign proteins.Organic solvent method has quickly, simple and easy, and antibody titer is high, special
Anisotropic good advantage, and most importantly affinity of antibody is stronger；But it is suitable only for purification IgG1 and IgG2b, to IgG3
The rate of recovery and purification effect it is poor.Polyethylene glycol method reaction condition is mild, high specificity, and yield is high, and the dosage of drug is few, it is not necessary to
Desalting processing is carried out, the gained IgG active retention time is long, is adapted to the separation of unstable large biological molecule；But this
Method removes polyethylene glycol, and complicated operation.Affinity film chromatography method is a kind of more effective bioactive substance purification process, egg
White to be concentrated in purification process, after being integrated to affinity ligand, property is more stable, and activity recovery improves；Purification step
Simply, suitable for the purifying of labile protein；But it can adsorb some heteroproteins, in another elution process with know from experience fall off into
Enter separation system, costly, mechanical strength is low for carrier, and aglucon preparation is difficult, and aglucon and carrier couple condition fierceness etc..
Salting out method is that Hammarsten in 1878 is used for the first time, and haemocyanin is successfully divided into clearly by he with magnesium sulfate
Albumen and globulin two parts.With the development of technology once using neutral salt such as sodium peroxydisulfate, sodium chloride, sodium phosphate and ammonium sulfate
Come protein of saltouing, wherein with it is most wide be ammonium sulfate.There are many advantages not available for other salt for ammonium sulfate, such as in water
Chemical property is stablized；Solubility is big, and 25 DEG C of whens can reach the concentration of 4.1mol/L；The temperature coefficient variation of solubility is smaller,
Changes in solubility is little within the scope of 0-30 DEG C；And ammonium sulfate is cheap and easy to get, and subsection efect is better than other salt, and property is mild, i.e.,
The bioactivity of protein will not be influenced when making dense.Now commonly referred to as salting out method actually be mostly ammonium sulfate precipitation
Method.
Summary of the invention
For the defects in the prior art, the object of the present invention is to provide a kind of preparation sides of immunoglobulin subclass antibodies
Method, the present invention combine the purity for the immunoglobulin hypotype extracted to obtain mentioning significantly using salting out method and ion exchange chromatography
Height, purity is improved at 5 times or more, and can remove DNA and RNA etc.；It is simple with high purity, at low cost, equipment, to being divided
The characteristics of can be reserved for its bioactivity from object.
According to an aspect of the present invention, a kind of preparation method of immunoglobulin subclass antibodies, including following step are provided
It is rapid:
Step 1 prepares IgG semifinished product: taking serum to mix with the physiological saline of equivalent, it is molten that neutral saturated ammonium sulfate is added dropwise
Liquid, it is stirring while adding, make its saturation degree 45-50%, after being sufficiently mixed, after being placed at room temperature for 30-60min, 3000r/min centrifugation
20min, centrifuging and taking precipitating；After precipitating physiological saline solution, neutral saturated ammonium sulfate solution is added dropwise, it is stirring while adding,
Make its saturation degree 30-33%, after being sufficiently mixed, after being placed at room temperature for 30-60min, centrifuging and taking precipitating；Precipitating is water-soluble with physiology salt
Xie Hou, as IgG semifinished product；
Step 2, desalination of dialysing: by the IgG semifinished product in step 1 in the phosphate buffer of 0.01mol/L pH 6.8-7.4
Large beaker in dialyse about for 24 hours, liquid is changed 3-4 times, until sulfate radical-free ion in dialyzate；
Step 3, chromatographic purifying: the IgG product after step 2 is acted on crosses the purifying of DEAE cellulose chromatography；
Step 4 is concentrated IgG: the IgG product after chromatographic purifying is concentrated into 1% or more concentration；
Step 5, inactivation of virus are further gone by IgG product after low pH is incubated and put processing inactivation of viruses using nano-film filtration
Except virus；
Step 6 saves: being distributed into bottle freeze-drying and saves, or 0.01% gentamicin is added to protect in general refrigerator or low temperature refrigerator
It deposits.
It saltouts in resulting protein containing a large amount of ammonium sulfate, will affect ion-exchange chromatography, therefore before chromatographic purifying
Need " desalination purification ".
Preferably, the neutral saturated ammonium sulfate solution the preparation method comprises the following steps: by 800-850g ammonium sulfate and 1000ml
H2O is heated to 55-60 DEG C under constant stirring, and after keeping 6-10min, filters while hot, and filtrate sets ambient temperature overnight, then with
28%NH4OH tune pH to 7.0 forms neutral saturated ammonium sulfate solution.Generally remain sulfuric acid in commercial solid ammonium sulfate, makes
The pH of standby saturated ammonium sulfate solution often between 4.5-5.5, is acted on serum again so needing to be tuned into neutral saturated solution.
Preferably, the ammonium sulfate is being added dropwise using preceding with EDTA chelating agent or H2S removes the metal ion in it.
Some heavy metal ion are typically contained in ammonium sulfate, dosage is excessive easily to make protein denaturation, need to by the metal contained in it from
Son removes.Ammonium sulfate is preferred with quality the superior, is had an impact because containing a small amount of heavy metal in substandard products to protein sulfhydryl.If substandard products must
Heavy metal must be removed, H can be passed through in the solution2S is filtered after standing overnight, heating evaporation H2S, or made with EDTA chelating agent
To chelate the metal ion in it.
Preferably, it is operated in the step 1: after precipitating physiological saline solution, it is molten that neutral saturated ammonium sulfate is added dropwise
Liquid, it is stirring while adding, make its saturation degree 30-33%, after being sufficiently mixed, after being placed at room temperature for 30-60min, centrifuging and taking precipitating；Weight
Multiple the operation 2-3 times, to completely remove albumin.
Preferably, SephadexG25 or electrodialysis desalination can also be used in the step 2.
Preferably, desalination of dialysing in the step 2 is carried out using bag filter or glassine paper.
Preferably, 1%BaCl is used after dialysis desalination in the step 22Check the SO in dialyzate4 2-Or with nessler reagent
Check NH4, up to no SO4 2-Or NH4Until appearance.Nessler reagent checks NH4When take 3-4ml dialyzate, reagent adding 1-2 drop, out
It is now brick-red to think there is NH4In the presence of.
Preferably, QAE cellulose, Sephadex G150 or Sephadex G200 column layer can also be used in the step 3
Analysis purifying.
Sephadex G is cross-link dextran, and the glucan of different size model is indicated with English alphabet G, and " G " reflection is solidifying
The crosslinking degree of glue, degrees of expansion and branch's range；The subsequent Arabic number of G is 10 times that gel obtains water number, such as G-25 is
It absorbs water 2.5 grams when every gram of gel expansion.
Preferably, the purity of the IgG preparation uses zone electrophoresis method, agar two-phase double diffusion identification method, immunoelectrophoresis
Identification method or the identification of disc electrophoresis identification method.
Preferably, which can be used to prepare immunoglobulin hypotype IgG1, IgG2, IgG3, IgG4.
Compared with prior art, the present invention have it is following the utility model has the advantages that
(1) preparation method of immunoglobulin subclass antibodies of the present invention, using salting out method and ion exchange chromatography knot
The purity for closing the immunoglobulin hypotype extracted is greatly improved, and purity is improved at 5 times or more, and can remove DNA
With RNA etc.；
(2) preparation method of immunoglobulin subclass antibodies of the present invention, preparation process is simple, produces and stablizes safety, mentions
Pure significant effect；
(3) preparation method of immunoglobulin subclass antibodies of the present invention, inactivation of virus is incubated using low pH to be put inactivation and receives
Rice filtering removal, dual virus inactivating method improve the inactivation ratio of virus, ensure that the safety of product；
(4) preparation method of immunoglobulin subclass antibodies of the present invention, have it is at low cost, equipment is simple, it is easy to operate,
Its bioactivity can be reserved for separated object, have a wide range of application.
Specific embodiment
The present invention is described in detail combined with specific embodiments below.Following embodiment will be helpful to the technology of this field
Personnel further understand the present invention, but the invention is not limited in any way.It should be pointed out that the ordinary skill of this field
For personnel, without departing from the inventive concept of the premise, various modifications and improvements can be made.These belong to the present invention
Protection scope.
Embodiment 1
The present embodiment is related to a kind of preparation method of immunoglobulin subclass antibodies, comprising the following steps:
Step 1 prepares IgG semifinished product: taking serum to mix with the physiological saline of equivalent, it is molten that neutral saturated ammonium sulfate is added dropwise
Liquid, it is stirring while adding, make its saturation degree 50%, after being sufficiently mixed, after being placed at room temperature for 60min, 3000r/min is centrifuged 20min,
Centrifuging and taking precipitating；After precipitating physiological saline solution, neutral saturated ammonium sulfate solution is added dropwise, it is stirring while adding, keep it full
It is 33%, after being sufficiently mixed, after being placed at room temperature for 60min with degree, centrifuging and taking precipitating；After precipitating physiological saline solution, as IgG
Semifinished product；
Step 2, dialyse desalination: by the IgG semifinished product in step 1 in 0.01mol/L pH 7.4 phosphate buffer it is big
It dialyses about for 24 hours, changes liquid 4 times, until sulfate radical-free ion in dialyzate in beaker；
Step 3, chromatographic purifying: the IgG product after step 2 is acted on crosses the purifying of DEAE cellulose chromatography；
Step 4 is concentrated IgG: the IgG product after chromatographic purifying is concentrated into 1% or more concentration；
Step 5, inactivation of virus are further gone by IgG product after low pH is incubated and put processing inactivation of viruses using nano-film filtration
Except virus；
Step 6 saves: being distributed into bottle freeze-drying and saves.
Further, the neutral saturated ammonium sulfate solution the preparation method comprises the following steps: by 850g ammonium sulfate and 1000ml H2O
It is heated to 60 DEG C under constant stirring, and after keeping 10min, filters while hot, filtrate sets ambient temperature overnight, then with 28%NH4OH tune
PH to 7.0 forms neutral saturated ammonium sulfate solution.Generally remain sulfuric acid, the saturation sulphur of preparation in commercial solid ammonium sulfate
The pH of acid ammonium solution often between 4.5-5.5, is acted on serum again so needing to be tuned into neutral saturated solution.
Further, the ammonium sulfate is being added dropwise using preceding with the effect of EDTA chelating agent to chelate the metal ion in it.
Some heavy metal ion are typically contained in ammonium sulfate, dosage is excessive easily to make protein denaturation, need to by the metal contained in it from
Son removes.Ammonium sulfate is preferred with quality the superior, is had an impact because containing a small amount of heavy metal in substandard products to protein sulfhydryl.If substandard products must
Heavy metal must be removed
Further, it is operated in the step 1: after precipitating physiological saline solution, it is molten that neutral saturated ammonium sulfate is added dropwise
Liquid, it is stirring while adding, make its saturation degree 33%, after being sufficiently mixed, after being placed at room temperature for 60min, centrifuging and taking precipitating；Repeat the behaviour
Make 3 times, to completely remove albumin.
Further, SephadexG25 or electrodialysis desalination can also be used in the step 2.
Further, desalination of dialysing in the step 2 is carried out using bag filter or glassine paper.
Further, 1%BaCl is used after dialysis desalination in the step 22Check the SO in dialyzate4 2-, up to no SO4 2-
Until appearance.
Further, the purity of the IgG preparation is identified using zone electrophoresis method；Slide agar or acetic acid are used when identification
Fiber membrane electrophoresis.After being loaded electrophoresis, only there is a band at the migration position of IgG preparation.When operation, while available whole blood
Final proof product, various concentration (NH4)2SO4Sample of saltouing carries out electrophoresis, as a means of comparing.
Further, which can be used to prepare immunoglobulin hypotype IgG1, IgG2, IgG3, IgG4.
Embodiment 2
The present embodiment is related to a kind of preparation method of immunoglobulin subclass antibodies, comprising the following steps:
Step 1 prepares IgG semifinished product: taking serum to mix with the physiological saline of equivalent, it is molten that neutral saturated ammonium sulfate is added dropwise
Liquid, it is stirring while adding, make its saturation degree 45%, after being sufficiently mixed, after being placed at room temperature for 30min, 3000r/min is centrifuged 20min,
Centrifuging and taking precipitating；After precipitating physiological saline solution, neutral saturated ammonium sulfate solution is added dropwise, it is stirring while adding, keep it full
It is 30%, after being sufficiently mixed, after being placed at room temperature for 30min with degree, centrifuging and taking precipitating；After precipitating physiological saline solution, as IgG
Semifinished product；
Step 2, dialyse desalination: by the IgG semifinished product in step 1 in 0.01mol/L pH 6.8 phosphate buffer it is big
It dialyses about for 24 hours, changes liquid 3 times, until sulfate radical-free ion in dialyzate in beaker；
Step 3, chromatographic purifying: the IgG product after step 2 is acted on crosses the purifying of DEAE cellulose chromatography；
Step 4 is concentrated IgG: the IgG product after chromatographic purifying is concentrated into 1% or more concentration；
Step 5, inactivation of virus are further gone by IgG product after low pH is incubated and put processing inactivation of viruses using nano-film filtration
Except virus；
Step 6 saves: being distributed into bottle freeze-drying and saves.
It saltouts in resulting protein containing a large amount of ammonium sulfate, will affect ion-exchange chromatography, therefore before chromatographic purifying
Need " desalination purification ".
Further, the neutral saturated ammonium sulfate solution the preparation method comprises the following steps: by 800g ammonium sulfate and 1000ml H2O
It is heated to 55 DEG C under constant stirring, and after keeping 6min, filters while hot, filtrate sets ambient temperature overnight, then with 28%NH4OH tune pH
To 7.0, neutral saturated ammonium sulfate solution is formed.Generally remain sulfuric acid, the saturation sulfuric acid of preparation in commercial solid ammonium sulfate
The pH of ammonium salt solution often between 4.5-5.5, is acted on serum again so needing to be tuned into neutral saturated solution.
Further, the ammonium sulfate is passed through H before use is added dropwise2S is filtered after standing overnight, heating evaporation H2S,
Remove the metal ion in it.Some heavy metal ion are typically contained in ammonium sulfate, dosage is excessive easily to make protein denaturation, needs
The metal ion contained in it is removed.Ammonium sulfate is preferred with quality the superior, because containing a small amount of heavy metal in substandard products to protein
Sulfydryl has an impact.If substandard products must be driven off heavy metal.
Further, it is operated in the step 1: after precipitating physiological saline solution, neutral saturated ammonium sulfate is added dropwise
Solution, it is stirring while adding, make its saturation degree 30%, after being sufficiently mixed, after being placed at room temperature for 30min, centrifuging and taking precipitating；Repeating should
Operation 2 times, to completely remove albumin.
Further, SephadexG25 or electrodialysis desalination can also be used in the step 2.
Further, desalination of dialysing in the step 2 is carried out using bag filter or glassine paper.
Further, NH is checked to nessler reagent after dialysis desalination in the step 24, up to no NH4Until appearance.
Nessler reagent checks NH4When take 3-4ml dialyzate, reagent adding 1-2 drop occurs brick-red thinking there is NH4In the presence of.
Further, the purity of the IgG preparation is identified using zone electrophoresis method；Slide agar or acetic acid are used when identification
Fiber membrane electrophoresis.After being loaded electrophoresis, only there is a band at the migration position of IgG preparation.When operation, while available whole blood
Final proof product, various concentration (NH4)2SO4Sample of saltouing carries out electrophoresis, as a means of comparing.
Further, which can be used to prepare immunoglobulin hypotype IgG1, IgG2, IgG3, IgG4.
Embodiment 3
The present embodiment is related to a kind of preparation method of immunoglobulin subclass antibodies, comprising the following steps:
Step 1 prepares IgG semifinished product: taking serum to mix with the physiological saline of equivalent, it is molten that neutral saturated ammonium sulfate is added dropwise
Liquid, it is stirring while adding, make its saturation degree 46%, after being sufficiently mixed, after being placed at room temperature for 40min, 3000r/min is centrifuged 20min,
Centrifuging and taking precipitating；After precipitating physiological saline solution, neutral saturated ammonium sulfate solution is added dropwise, it is stirring while adding, keep it full
It is 31%, after being sufficiently mixed, after being placed at room temperature for 40min with degree, centrifuging and taking precipitating；After precipitating physiological saline solution, as IgG
Semifinished product；
Step 2, dialyse desalination: by the IgG semifinished product in step 1 in 0.01mol/L pH 6.9 phosphate buffer it is big
It dialyses in beaker about for 24 hours, changes liquid 3-4 times, until sulfate radical-free ion in dialyzate；
Step 3, chromatographic purifying: the IgG product after step 2 is acted on crosses the purifying of QAE cellulose chromatography；
Step 4 is concentrated IgG: the IgG product after chromatographic purifying is concentrated into 1% or more concentration；
Step 5, inactivation of virus are further gone by IgG product after low pH is incubated and put processing inactivation of viruses using nano-film filtration
Except virus；
Step 6 saves: 0.01% gentamicin being added to save in general refrigerator or low temperature refrigerator.
It saltouts in resulting protein containing a large amount of ammonium sulfate, will affect ion-exchange chromatography, therefore before chromatographic purifying
Need " desalination purification ".
Further, the neutral saturated ammonium sulfate solution the preparation method comprises the following steps: by 810g ammonium sulfate and 1000ml H2O
It is heated to 56 DEG C under constant stirring, and after keeping 7min, filters while hot, filtrate sets ambient temperature overnight, then with 28%NH4OH tune pH
To 7.0, neutral saturated ammonium sulfate solution is formed.Generally remain sulfuric acid, the saturation sulfuric acid of preparation in commercial solid ammonium sulfate
The pH of ammonium salt solution often between 4.5-5.5, is acted on serum again so needing to be tuned into neutral saturated solution.
Further, the ammonium sulfate be added dropwise using it is preceding with EDTA chelating agent act on chelate the metal in it from
Son.Some heavy metal ion, the excessive metal that easily makes protein denaturation, need to will contain in it of dosage are typically contained in ammonium sulfate
Ion removes.Ammonium sulfate is preferred with quality the superior, is had an impact because containing a small amount of heavy metal in substandard products to protein sulfhydryl.If substandard products
It must be driven off heavy metal.
Further, it is operated in the step 1: after precipitating physiological saline solution, neutral saturated ammonium sulfate is added dropwise
Solution, it is stirring while adding, make its saturation degree 31%, after being sufficiently mixed, after being placed at room temperature for 40min, centrifuging and taking precipitating；Repeating should
Operation 3 times, to completely remove albumin.
Further, SephadexG25 or electrodialysis desalination can also be used in the step 2.
Further, desalination of dialysing in the step 2 is carried out using bag filter or glassine paper.
Further, 1%BaCl is used after dialysis desalination in the step 22Check the SO in dialyzate4 2-, up to no SO4 2-
Until appearance.
Further, the purity of the IgG preparation is identified using agar two-phase double diffusion identification method；It is prepared in advance when identification
The anti-igg serum that heterogenous animal is obtained is immunized in the IgG.IgG and anti-igg serum are subjected to two-phase double diffusion, as IgG is purified
Then there is a precipitation line between two sample wells in words.
Further, which can be used to prepare immunoglobulin hypotype IgG1, IgG2, IgG3, IgG4.
Embodiment 4
The present embodiment is related to a kind of preparation method of immunoglobulin subclass antibodies, comprising the following steps:
Step 1 prepares IgG semifinished product: taking serum to mix with the physiological saline of equivalent, it is molten that neutral saturated ammonium sulfate is added dropwise
Liquid, it is stirring while adding, make its saturation degree 47%, after being sufficiently mixed, after being placed at room temperature for 50min, 3000r/min is centrifuged 20min,
Centrifuging and taking precipitating；After precipitating physiological saline solution, neutral saturated ammonium sulfate solution is added dropwise, it is stirring while adding, keep it full
It is 32%, after being sufficiently mixed, after being placed at room temperature for 50min with degree, centrifuging and taking precipitating；After precipitating physiological saline solution, as IgG
Semifinished product；
Step 2, dialyse desalination: by the IgG semifinished product in step 1 in 0.01mol/L pH7.0 phosphate buffer it is big
It dialyses in beaker about for 24 hours, changes liquid 3-4 times, until sulfate radical-free ion in dialyzate；
Step 3, chromatographic purifying: the IgG product after step 2 is acted on crosses Sephadex G150 column chromatographic purifying；
Step 4 is concentrated IgG: the IgG product after chromatographic purifying is concentrated into 1% or more concentration；
Step 5, inactivation of virus are further gone by IgG product after low pH is incubated and put processing inactivation of viruses using nano-film filtration
Except virus；
Step 6 saves: 0.01% gentamicin being added to save in general refrigerator or low temperature refrigerator.
It saltouts in resulting protein containing a large amount of ammonium sulfate, will affect ion-exchange chromatography, therefore before chromatographic purifying
Need " desalination purification ".
Further, the neutral saturated ammonium sulfate solution the preparation method comprises the following steps: by 820g ammonium sulfate and 1000ml H2O
It is heated to 57 DEG C under constant stirring, and after keeping 8min, filters while hot, filtrate sets ambient temperature overnight, then with 28%NH4OH tune pH
To 7.0, neutral saturated ammonium sulfate solution is formed.Generally remain sulfuric acid, the saturation sulfuric acid of preparation in commercial solid ammonium sulfate
The pH of ammonium salt solution often between 4.5-5.5, is acted on serum again so needing to be tuned into neutral saturated solution.
Further, the ammonium sulfate is passed through H before use is added dropwise2S is filtered after standing overnight, heating evaporation H2S,
Remove the metal ion in it.Some heavy metal ion are typically contained in ammonium sulfate, dosage is excessive easily to make protein denaturation, needs
The metal ion contained in it is removed.Ammonium sulfate is preferred with quality the superior, because containing a small amount of heavy metal in substandard products to protein
Sulfydryl has an impact.If substandard products must be driven off heavy metal.
Further, it is operated in the step 1: after precipitating physiological saline solution, neutral saturated ammonium sulfate is added dropwise
Solution, it is stirring while adding, make its saturation degree 32%, after being sufficiently mixed, after being placed at room temperature for 50min, centrifuging and taking precipitating；Repeating should
Operation 3 times, to completely remove albumin.
Further, SephadexG25 or electrodialysis desalination can also be used in the step 2.
Further, desalination of dialysing in the step 2 is carried out using bag filter or glassine paper.
Further, NH is checked to nessler reagent after dialysis desalination in the step 24, up to no NH4Until appearance；
Nessler reagent checks NH4When take 3-4ml dialyzate, reagent adding 1-2 drop occurs brick-red thinking there is NH4In the presence of.
Further, the purity of the IgG preparation is identified using immunoelectrophoresis identification method, in mirror timing hole plus to test sample
Product, after electrophoresis, in slot plus anti-igg serum, AGP test for 24 hours, observe result.If the IgG extracted is pure, only occur
The precipitation line of one arc, and precipitation line is located at IgG preparation area.This identification must carry out whole serum and anti serum simultaneously
Immunoelectrophoresis, as a means of comparing.
Further, which can be used to prepare immunoglobulin hypotype IgG1, IgG2, IgG3, IgG4.
Embodiment 5
The present embodiment is related to a kind of preparation method of immunoglobulin subclass antibodies, comprising the following steps:
Step 1 prepares IgG semifinished product: taking serum to mix with the physiological saline of equivalent, it is molten that neutral saturated ammonium sulfate is added dropwise
Liquid, it is stirring while adding, make its saturation degree 48%, after being sufficiently mixed, after being placed at room temperature for 60min, 3000r/min is centrifuged 20min,
Centrifuging and taking precipitating；After precipitating physiological saline solution, neutral saturated ammonium sulfate solution is added dropwise, it is stirring while adding, keep it full
It is 32%, after being sufficiently mixed, after being placed at room temperature for 60min with degree, centrifuging and taking precipitating；After precipitating physiological saline solution, as IgG
Semifinished product；
Step 2, dialyse desalination: by the IgG semifinished product in step 1 in 0.01mol/L pH 7.1 phosphate buffer it is big
It dialyses in beaker about for 24 hours, changes liquid 3-4 times, until sulfate radical-free ion in dialyzate；
Step 3, chromatographic purifying: the IgG product after step 2 is acted on crosses Sephadex G200 column chromatographic purifying；
Step 4 is concentrated IgG: the IgG product after chromatographic purifying is concentrated into 1% or more concentration；
Step 5, inactivation of virus are further gone by IgG product after low pH is incubated and put processing inactivation of viruses using nano-film filtration
Except virus；
Step 6 saves: being distributed into bottle freeze-drying and saves.
It saltouts in resulting protein containing a large amount of ammonium sulfate, will affect ion-exchange chromatography, therefore before chromatographic purifying
Need " desalination purification ".
Further, the neutral saturated ammonium sulfate solution the preparation method comprises the following steps: by 830g ammonium sulfate and 1000ml H2O
It is heated to 58 DEG C under constant stirring, and after keeping 9min, filters while hot, filtrate sets ambient temperature overnight, then with 28%NH4OH tune pH
To 7.0, neutral saturated ammonium sulfate solution is formed.Generally remain sulfuric acid, the saturation sulfuric acid of preparation in commercial solid ammonium sulfate
The pH of ammonium salt solution often between 4.5-5.5, is acted on serum again so needing to be tuned into neutral saturated solution.
Further, the ammonium sulfate be added dropwise using it is preceding with EDTA chelating agent act on chelate the metal in it from
Son.Some heavy metal ion, the excessive metal that easily makes protein denaturation, need to will contain in it of dosage are typically contained in ammonium sulfate
Ion removes.Ammonium sulfate is preferred with quality the superior, is had an impact because containing a small amount of heavy metal in substandard products to protein sulfhydryl.If substandard products
It must be driven off heavy metal.
Further, it is operated in the step 1: after precipitating physiological saline solution, neutral saturated ammonium sulfate is added dropwise
Solution, it is stirring while adding, make its saturation degree 32%, after being sufficiently mixed, after being placed at room temperature for 60min, centrifuging and taking precipitating；Repeating should
Operation 2 times, to completely remove albumin.
Further, SephadexG25 or electrodialysis desalination can also be used in the step 2.
Further, desalination of dialysing in the step 2 is carried out using bag filter or glassine paper.
Further, 1%BaCl is used after dialysis desalination in the step 22Check the SO in dialyzate4 2-, up to no SO4 2-
Until appearance.
Further, the purity of IgG preparation is using the identification of disc electrophoresis identification method, when identification with whole serum sample and
Purification sample carries out disc electrophoresis simultaneously.There are tens of zone on disc electrophoresis in whole serum sample, and the IgG purified is then only
There is a zone.
Further, which can be used to prepare immunoglobulin hypotype IgG1, IgG2, IgG3, IgG4.
Embodiment 6
The present embodiment is related to a kind of preparation method of immunoglobulin subclass antibodies, comprising the following steps:
Step 1 prepares IgG semifinished product: taking serum to mix with the physiological saline of equivalent, it is molten that neutral saturated ammonium sulfate is added dropwise
Liquid, it is stirring while adding, make its saturation degree 49%, after being sufficiently mixed, after being placed at room temperature for 35min, 3000r/min is centrifuged 20min,
Centrifuging and taking precipitating；After precipitating physiological saline solution, neutral saturated ammonium sulfate solution is added dropwise, it is stirring while adding, keep it full
It is 33%, after being sufficiently mixed, after being placed at room temperature for 35min with degree, centrifuging and taking precipitating；After precipitating physiological saline solution, as IgG
Semifinished product；
Step 2, dialyse desalination: by the IgG semifinished product in step 1 in 0.01mol/L pH 7.2 phosphate buffer it is big
It dialyses about for 24 hours, changes liquid 4 times, until sulfate radical-free ion in dialyzate in beaker；
Step 3, chromatographic purifying: the IgG product after step 2 is acted on crosses the purifying of QAE cellulose chromatography；
Step 4 is concentrated IgG: the IgG product after chromatographic purifying is concentrated into 1% or more concentration；
Step 5, inactivation of virus are further gone by IgG product after low pH is incubated and put processing inactivation of viruses using nano-film filtration
Except virus；
Step 6 saves: 0.01% gentamicin being added to save in general refrigerator or low temperature refrigerator.
It saltouts in resulting protein containing a large amount of ammonium sulfate, will affect ion-exchange chromatography, therefore before chromatographic purifying
Need " desalination purification ".
Further, the neutral saturated ammonium sulfate solution the preparation method comprises the following steps: by 840g ammonium sulfate and 1000ml H2O
It is heated to 59 DEG C under constant stirring, and after keeping 10min, filters while hot, filtrate sets ambient temperature overnight, then with 28%NH4OH tune
PH to 7.0 forms neutral saturated ammonium sulfate solution.Generally remain sulfuric acid, the saturation sulphur of preparation in commercial solid ammonium sulfate
The pH of acid ammonium solution often between 4.5-5.5, is acted on serum again so needing to be tuned into neutral saturated solution.
Further, the ammonium sulfate is passed through H using preceding in dropwise addition in the solution2S is filtered after standing overnight, is added
Thermal evaporation H2S removes the metal ion in it.Some heavy metal ion are typically contained in ammonium sulfate, dosage is excessive easily to make egg
White matter denaturation, need to remove the metal ion contained in it.Ammonium sulfate is preferred with quality the superior, because containing an a small amount of huge sum of money in substandard products
Category has an impact to protein sulfhydryl.If substandard products must be driven off heavy metal.
Further, it is operated in the step 1: after precipitating physiological saline solution, neutral saturated ammonium sulfate is added dropwise
Solution, it is stirring while adding, make its saturation degree 33%, after being sufficiently mixed, after being placed at room temperature for 35min, centrifuging and taking precipitating；Repeating should
Operation 3 times, to completely remove albumin.
Further, SephadexG25 or electrodialysis desalination can also be used in the step 2.
Further, desalination of dialysing in the step 2 is carried out using bag filter or glassine paper.
Further, NH is checked to nessler reagent after dialysis desalination in the step 24, up to no NH4Until appearance,
Nessler reagent checks NH4When take 3-4ml dialyzate, reagent adding 1-2 drop occurs brick-red thinking there is NH4In the presence of.
Further, the purity of the IgG preparation is identified using immunoelectrophoresis identification method, in mirror timing hole plus to test sample
Product, after electrophoresis, in slot plus anti-igg serum, AGP test for 24 hours, observe result.If the IgG extracted is pure, only occur
The precipitation line of one arc, and precipitation line is located at γ-globulin area.This identification must carry out whole serum simultaneously and antiserum is anti-
The immunoelectrophoresis of body, as a means of comparing.
Further, which can be used to prepare immunoglobulin hypotype IgG1, IgG2, IgG3, IgG4.
Embodiment 7
The present embodiment is related to a kind of preparation method of immunoglobulin subclass antibodies, comprising the following steps:
Step 1 prepares IgG semifinished product: taking serum to mix with the physiological saline of equivalent, it is molten that neutral saturated ammonium sulfate is added dropwise
Liquid, it is stirring while adding, make its saturation degree 50%, after being sufficiently mixed, after being placed at room temperature for 45min, 3000r/min is centrifuged 20min,
Centrifuging and taking precipitating；After precipitating physiological saline solution, neutral saturated ammonium sulfate solution is added dropwise, it is stirring while adding, keep it full
It is 30%, after being sufficiently mixed, after being placed at room temperature for 45min with degree, centrifuging and taking precipitating；After precipitating physiological saline solution, as IgG
Semifinished product；
Step 2, dialyse desalination: by the IgG semifinished product in step 1 in 0.01mol/L pH7.3 phosphate buffer it is big
It dialyses in beaker about for 24 hours, changes liquid 3-4 times, until sulfate radical-free ion in dialyzate；
Step 3, chromatographic purifying: the IgG product after step 2 is acted on crosses Sephadex G200 column chromatographic purifying；
Step 4 is concentrated IgG: the IgG product after chromatographic purifying is concentrated into 1% or more concentration；
Step 5, inactivation of virus are further gone by IgG product after low pH is incubated and put processing inactivation of viruses using nano-film filtration
Except virus；
Step 6 saves: being distributed into bottle freeze-drying and saves.
It saltouts in resulting protein containing a large amount of ammonium sulfate, will affect ion-exchange chromatography, therefore before chromatographic purifying
Need " desalination purification ".
Further, the neutral saturated ammonium sulfate solution the preparation method comprises the following steps: by 850g ammonium sulfate and 1000ml H2O
It is heated to 55 DEG C under constant stirring, and after keeping 6min, filters while hot, filtrate sets ambient temperature overnight, then with 28%NH4OH tune pH
To 7.0, neutral saturated ammonium sulfate solution is formed.Generally remain sulfuric acid, the saturation sulfuric acid of preparation in commercial solid ammonium sulfate
The pH of ammonium salt solution often between 4.5-5.5, is acted on serum again so needing to be tuned into neutral saturated solution.
Further, the ammonium sulfate be added dropwise using it is preceding with EDTA chelating agent act on chelate the metal in it from
Son.Some heavy metal ion, the excessive metal that easily makes protein denaturation, need to will contain in it of dosage are typically contained in ammonium sulfate
Ion removes.Ammonium sulfate is preferred with quality the superior, is had an impact because containing a small amount of heavy metal in substandard products to protein sulfhydryl.If substandard products
It must be driven off heavy metal.
Further, it is operated in the step 1: after precipitating physiological saline solution, neutral saturated ammonium sulfate is added dropwise
Solution, it is stirring while adding, make its saturation degree 30%, after being sufficiently mixed, after being placed at room temperature for 45min, centrifuging and taking precipitating；Repeating should
Operation 2 times, to completely remove albumin.
Further, SephadexG25 or electrodialysis desalination can also be used in the step 2.
Further, desalination of dialysing in the step 2 is carried out using bag filter or glassine paper.
Further, 1%BaCl is used after dialysis desalination in the step 22Check the SO in dialyzate4 2-, up to no SO4 2-
Until appearance.
Further, the purity of IgG preparation is using the identification of agar two-phase double diffusion identification method, and when identification is prepared in advance
The anti-igg serum that heterogenous animal is obtained is immunized in the IgG.IgG and anti-igg serum are subjected to two-phase double diffusion, as IgG is purified
Then there is a precipitation line between two sample wells in words.
Further, which can be used to prepare immunoglobulin hypotype IgG1, IgG2, IgG3, IgG4.
Embodiment 8
The present embodiment is related to a kind of preparation method of immunoglobulin subclass antibodies, comprising the following steps:
Step 1 prepares IgG semifinished product: taking serum to mix with the physiological saline of equivalent, it is molten that neutral saturated ammonium sulfate is added dropwise
Liquid, it is stirring while adding, make its saturation degree 45%, after being sufficiently mixed, after being placed at room temperature for 55min, 3000r/min is centrifuged 20min,
Centrifuging and taking precipitating；After precipitating physiological saline solution, neutral saturated ammonium sulfate solution is added dropwise, it is stirring while adding, keep it full
It is 33%, after being sufficiently mixed, after being placed at room temperature for 55min with degree, centrifuging and taking precipitating；After precipitating physiological saline solution, as IgG
Semifinished product；
Step 2, dialyse desalination: by the IgG semifinished product in step 1 in 0.01mol/L pH 7.4 phosphate buffer it is big
It dialyses in beaker about for 24 hours, changes liquid 3-4 times, until sulfate radical-free ion in dialyzate；
Step 3, chromatographic purifying: the IgG product after step 2 is acted on crosses the purifying of DEAE cellulose chromatography；
Step 4 is concentrated IgG: the IgG product after chromatographic purifying is concentrated into 1% or more concentration；
Step 5, inactivation of virus are further gone by IgG product after low pH is incubated and put processing inactivation of viruses using nano-film filtration
Except virus；
Step 6 saves: being distributed into bottle freeze-drying and saves.
It saltouts in resulting protein containing a large amount of ammonium sulfate, will affect ion-exchange chromatography, therefore before chromatographic purifying
Need " desalination purification ".
Further, the neutral saturated ammonium sulfate solution the preparation method comprises the following steps: by 800g ammonium sulfate and 1000ml H2O
It is heated to 60 DEG C under constant stirring, and after keeping 10min, filters while hot, filtrate sets ambient temperature overnight, then with 28%NH4OH tune
PH to 7.0 forms neutral saturated ammonium sulfate solution.Generally remain sulfuric acid, the saturation sulphur of preparation in commercial solid ammonium sulfate
The pH of acid ammonium solution often between 4.5-5.5, is acted on serum again so needing to be tuned into neutral saturated solution.
Further, the ammonium sulfate be added dropwise using it is preceding with EDTA chelating agent act on chelate the metal in it from
Son.Some heavy metal ion, the excessive metal that easily makes protein denaturation, need to will contain in it of dosage are typically contained in ammonium sulfate
Ion removes.Ammonium sulfate is preferred with quality the superior, is had an impact because containing a small amount of heavy metal in substandard products to protein sulfhydryl.If substandard products
It must be driven off heavy metal.
Further, it is operated in the step 1: after precipitating physiological saline solution, neutral saturated ammonium sulfate is added dropwise
Solution, it is stirring while adding, make its saturation degree 33%, after being sufficiently mixed, after being placed at room temperature for 55min, centrifuging and taking precipitating；Repeating should
Operation 2 times, to completely remove albumin.
Further, SephadexG25 or electrodialysis desalination can also be used in the step 2.
Further, desalination of dialysing in the step 2 is carried out using bag filter or glassine paper.
Further, 1%BaCl is used after dialysis desalination in the step 22Check the SO in dialyzate4 2-, up to no SO4 2-
Until appearance.
Further, the purity of IgG preparation is using the identification of disc electrophoresis identification method, when identification with whole serum sample and
Purification sample carries out disc electrophoresis simultaneously.There are tens of zone on disc electrophoresis in whole serum sample, and the IgG purified is then only
There is a zone.
Further, which can be used to prepare immunoglobulin hypotype IgG1, IgG2, IgG3, IgG4.
Embodiment 9
The present embodiment is related to a kind of preparation method of immunoglobulin subclass antibodies, comprising the following steps:
Step 1 prepares IgG semifinished product: taking serum to mix with the physiological saline of equivalent, it is molten that neutral saturated ammonium sulfate is added dropwise
Liquid, it is stirring while adding, make its saturation degree 50%, after being sufficiently mixed, after being placed at room temperature for 60min, 3000r/min is centrifuged 20min,
Centrifuging and taking precipitating；After precipitating physiological saline solution, neutral saturated ammonium sulfate solution is added dropwise, it is stirring while adding, keep it full
It is 33%, after being sufficiently mixed, after being placed at room temperature for 60min with degree, centrifuging and taking precipitating；After precipitating physiological saline solution, as IgG
Semifinished product；
Step 2, dialyse desalination: by the IgG semifinished product in step 1 in 0.01mol/L pH 6.8 phosphate buffer it is big
It dialyses about for 24 hours, changes liquid 4 times, until sulfate radical-free ion in dialyzate in beaker；
Step 3, chromatographic purifying: the IgG product after step 2 is acted on crosses the purifying of DEAE cellulose chromatography；
Step 4 is concentrated IgG: the IgG product after chromatographic purifying is concentrated into 1% or more concentration；
Step 5, inactivation of virus are further gone by IgG product after low pH is incubated and put processing inactivation of viruses using nano-film filtration
Except virus；
Step 6 saves: being distributed into bottle freeze-drying and saves.
Further, the neutral saturated ammonium sulfate solution the preparation method comprises the following steps: by 850g ammonium sulfate and 1000ml H2O
It is heated to 60 DEG C under constant stirring, and after keeping 10min, filters while hot, filtrate sets ambient temperature overnight, then with 28%NH4OH tune
PH to 7.0 forms neutral saturated ammonium sulfate solution.Generally remain sulfuric acid, the saturation sulphur of preparation in commercial solid ammonium sulfate
The pH of acid ammonium solution often between 4.5-5.5, is acted on serum again so needing to be tuned into neutral saturated solution.
Further, the ammonium sulfate be added dropwise using it is preceding with EDTA chelating agent act on chelate the metal in it from
Son.Some heavy metal ion, the excessive metal that easily makes protein denaturation, need to will contain in it of dosage are typically contained in ammonium sulfate
Ion removes.Ammonium sulfate is preferred with quality the superior, is had an impact because containing a small amount of heavy metal in substandard products to protein sulfhydryl.If substandard products
It must be driven off heavy metal
Further, it is operated in the step 1: after precipitating physiological saline solution, it is molten that neutral saturated ammonium sulfate is added dropwise
Liquid, it is stirring while adding, make its saturation degree 33%, after being sufficiently mixed, after being placed at room temperature for 60min, centrifuging and taking precipitating；Repeat the behaviour
Make 3 times, to completely remove albumin.
Further, SephadexG25 or electrodialysis desalination can also be used in the step 2.
Further, desalination of dialysing in the step 2 is carried out using bag filter or glassine paper.
Further, 1%BaCl is used after dialysis desalination in the step 22Check the SO in dialyzate4 2-, up to no SO4 2-
Until appearance.
Further, the purity of IgG preparation is using the identification of disc electrophoresis identification method, when identification with whole serum sample and
Purification sample carries out disc electrophoresis simultaneously.There are tens of zone on disc electrophoresis in whole serum sample, and the IgG purified is then only
There is a zone.
Further, which can be used to prepare immunoglobulin hypotype IgG1, IgG2, IgG3, IgG4.
Embodiment 10
The present embodiment is related to a kind of preparation method of immunoglobulin subclass antibodies, comprising the following steps:
Step 1 prepares IgG semifinished product: taking serum to mix with the physiological saline of equivalent, it is molten that neutral saturated ammonium sulfate is added dropwise
Liquid, it is stirring while adding, make its saturation degree 50%, after being sufficiently mixed, after being placed at room temperature for 60min, 3000r/min is centrifuged 20min,
Centrifuging and taking precipitating；After precipitating physiological saline solution, neutral saturated ammonium sulfate solution is added dropwise, it is stirring while adding, keep it full
It is 33%, after being sufficiently mixed, after being placed at room temperature for 60min with degree, centrifuging and taking precipitating；After precipitating physiological saline solution, as IgG
Semifinished product；
Step 2, dialyse desalination: by the IgG semifinished product in step 1 in 0.01mol/L pH 7.0 phosphate buffer it is big
It dialyses about for 24 hours, changes liquid 3 times, until sulfate radical-free ion in dialyzate in beaker；
Step 3, chromatographic purifying: the IgG product after step 2 is acted on crosses the purifying of DEAE cellulose chromatography；
Step 4 is concentrated IgG: the IgG product after chromatographic purifying is concentrated into 1% or more concentration；
Step 5, inactivation of virus are further gone by IgG product after low pH is incubated and put processing inactivation of viruses using nano-film filtration
Except virus；
Step 6 saves: being distributed into bottle freeze-drying and saves.
Further, the neutral saturated ammonium sulfate solution the preparation method comprises the following steps: by 850g ammonium sulfate and 1000ml H2O
It is heated to 60 DEG C under constant stirring, and after keeping 10min, filters while hot, filtrate sets ambient temperature overnight, then with 28%NH4OH tune
PH to 7.0 forms neutral saturated ammonium sulfate solution.Generally remain sulfuric acid, the saturation sulphur of preparation in commercial solid ammonium sulfate
The pH of acid ammonium solution often between 4.5-5.5, is acted on serum again so needing to be tuned into neutral saturated solution.
Further, the ammonium sulfate be added dropwise using it is preceding with EDTA chelating agent act on chelate the metal in it from
Son.Some heavy metal ion, the excessive metal that easily makes protein denaturation, need to will contain in it of dosage are typically contained in ammonium sulfate
Ion removes.Ammonium sulfate is preferred with quality the superior, is had an impact because containing a small amount of heavy metal in substandard products to protein sulfhydryl.If substandard products
It must be driven off heavy metal
Further, it is operated in the step 1: after precipitating physiological saline solution, it is molten that neutral saturated ammonium sulfate is added dropwise
Liquid, it is stirring while adding, make its saturation degree 33%, after being sufficiently mixed, after being placed at room temperature for 60min, centrifuging and taking precipitating；Repeat the behaviour
Make 3 times, to completely remove albumin.
Further, SephadexG25 or electrodialysis desalination can also be used in the step 2.
Further, desalination of dialysing in the step 2 is carried out using bag filter or glassine paper.
Further, 1%BaCl is used after dialysis desalination in the step 22Check the SO in dialyzate4 2-, up to no SO4 2-
Until appearance.
Further, the purity of IgG preparation is using the identification of disc electrophoresis identification method, when identification with whole serum sample and
Purification sample carries out disc electrophoresis simultaneously.There are tens of zone on disc electrophoresis in whole serum sample, and the IgG purified is then only
There is a zone.
Embodiment 11
The present embodiment is related to a kind of preparation method of immunoglobulin subclass antibodies, comprising the following steps:
Step 1 prepares IgG semifinished product: taking serum to mix with the physiological saline of equivalent, it is molten that neutral saturated ammonium sulfate is added dropwise
Liquid, it is stirring while adding, make its saturation degree 45%, after being sufficiently mixed, after being placed at room temperature for 30min, 3000r/min is centrifuged 20min,
Centrifuging and taking precipitating；After precipitating physiological saline solution, neutral saturated ammonium sulfate solution is added dropwise, it is stirring while adding, keep it full
It is 32%, after being sufficiently mixed, after being placed at room temperature for 30min with degree, centrifuging and taking precipitating；After precipitating physiological saline solution, as IgG
Semifinished product；
Step 2, dialyse desalination: by the IgG semifinished product in step 1 in 0.01mol/L pH 7.4 phosphate buffer it is big
It dialyses about for 24 hours, changes liquid 3 times, until sulfate radical-free ion in dialyzate in beaker；
Step 3, chromatographic purifying: the IgG product after step 2 is acted on crosses the purifying of DEAE cellulose chromatography；
Step 4 is concentrated IgG: the IgG product after chromatographic purifying is concentrated into 1% or more concentration；
Step 5, inactivation of virus are further gone by IgG product after low pH is incubated and put processing inactivation of viruses using nano-film filtration
Except virus；
Step 6 saves: being distributed into bottle freeze-drying and saves.
It saltouts in resulting protein containing a large amount of ammonium sulfate, will affect ion-exchange chromatography, therefore before chromatographic purifying
Need " desalination purification ".
Further, the neutral saturated ammonium sulfate solution the preparation method comprises the following steps: by 800g ammonium sulfate and 1000ml H2O
It is heated to 55 DEG C under constant stirring, and after keeping 6min, filters while hot, filtrate sets ambient temperature overnight, then with 28%NH4OH tune pH
To 7.0, neutral saturated ammonium sulfate solution is formed.Generally remain sulfuric acid, the saturation sulfuric acid of preparation in commercial solid ammonium sulfate
The pH of ammonium salt solution often between 4.5-5.5, is acted on serum again so needing to be tuned into neutral saturated solution.
Further, the ammonium sulfate is passed through H before use is added dropwise2S is filtered after standing overnight, heating evaporation H2S,
Remove the metal ion in it.Some heavy metal ion are typically contained in ammonium sulfate, dosage is excessive easily to make protein denaturation, needs
The metal ion contained in it is removed.Ammonium sulfate is preferred with quality the superior, because containing a small amount of heavy metal in substandard products to protein
Sulfydryl has an impact.If substandard products must be driven off heavy metal.
Further, it is operated in the step 1: after precipitating physiological saline solution, neutral saturated ammonium sulfate is added dropwise
Solution, it is stirring while adding, make its saturation degree 32%, after being sufficiently mixed, after being placed at room temperature for 30min, centrifuging and taking precipitating；Repeating should
Operation 3 times, to completely remove albumin.
Further, SephadexG25 or electrodialysis desalination can also be used in the step 2.
Further, desalination of dialysing in the step 2 is carried out using bag filter or glassine paper.
Further, NH is checked to nessler reagent after dialysis desalination in the step 24, up to no NH4Until appearance.
Nessler reagent checks NH4When take 3-4ml dialyzate, reagent adding 1-2 drop occurs brick-red thinking there is NH4In the presence of.
Further, the purity of the IgG preparation is identified using zone electrophoresis method；Slide agar or acetic acid are used when identification
Fiber membrane electrophoresis.After being loaded electrophoresis, only there is a band at the migration position of IgG preparation.When operation, while available whole blood
Final proof product, various concentration (NH4)2SO4Sample of saltouing carries out electrophoresis, as a means of comparing.
Further, which can be used to prepare immunoglobulin hypotype IgG1, IgG2, IgG3, IgG4.
For above-mentioned comparative example: being had the advantage that in method of the invention
(1) purity for the immunoglobulin hypotype extracted is combined to obtain mentioning significantly using salting out method and ion exchange chromatography
Height, purity is improved at 5 times or more, and can remove DNA and RNA etc.；
(2) preparation process is simple, produces and stablizes safety, and refining effect is significant；
(3) inactivation of virus is incubated using low pH puts inactivation and nanofiltration removal, and dual virus inactivating method improves going out for virus
Motility rate ensure that the safety of product；
(4) have it is at low cost, equipment is simple, it is easy to operate, its bioactivity can be reserved for separated object, have a wide range of application.
Specific embodiments of the present invention are described above.It is to be appreciated that the invention is not limited to above-mentioned
Particular implementation, those skilled in the art can make various deformations or amendments within the scope of the claims, this not shadow
Ring substantive content of the invention.
Claims (10)
1. the preparation method of immunoglobulin subclass antibodies, which comprises the following steps:
Step 1 prepares IgG semifinished product: taking serum to mix with the physiological saline of equivalent, it is molten that neutral saturated ammonium sulfate is added dropwise
Liquid, it is stirring while adding, make its saturation degree 45-50%, after being sufficiently mixed, after being placed at room temperature for 30-60min, 3000r/min centrifugation
20min, centrifuging and taking precipitating；After precipitating physiological saline solution, neutral saturated ammonium sulfate solution is added dropwise, it is stirring while adding,
Make its saturation degree 30-33%, after being sufficiently mixed, after being placed at room temperature for 30-60min, centrifuging and taking precipitating；Precipitating is water-soluble with physiology salt
Xie Hou, as IgG semifinished product；
Step 2, desalination of dialysing: by the IgG semifinished product in step 1 in the phosphate buffer of 0.01mol/L pH 6.8-7.4
Large beaker in dialyse about for 24 hours, liquid is changed 3-4 times, until sulfate radical-free ion in dialyzate；
Step 3, chromatographic purifying: the IgG product after step 2 is acted on crosses the purifying of DEAE cellulose chromatography；
Step 4 is concentrated IgG: the IgG product after chromatographic purifying is concentrated into 1% or more concentration；
Step 5, inactivation of virus are further gone by IgG product after low pH is incubated and put processing inactivation of viruses using nano-film filtration
Except virus；
Step 6 saves: being distributed into bottle freeze-drying and saves, or 0.01% gentamicin is added to protect in general refrigerator or low temperature refrigerator
It deposits.
2. the preparation method of immunoglobulin subclass antibodies according to claim 1, which is characterized in that the neutral saturation
Ammonium sulfate the preparation method comprises the following steps: by 800-850g ammonium sulfate and 1000ml H2O is heated to 55-60 under constant stirring
DEG C, and after keeping 6-10min, it filters while hot, filtrate sets ambient temperature overnight, then with 28%NH4OH tune pH to 7.0 is formed neutral full
And ammonium sulfate.
3. the preparation method of immunoglobulin subclass antibodies according to claim 1, which is characterized in that the ammonium sulfate is molten
Liquid is being added dropwise using preceding with EDTA chelating agent or H2S removes the metal ion in it.
4. the preparation method of immunoglobulin subclass antibodies according to claim 1, which is characterized in that in the step 1
Operation: after precipitating physiological saline solution, being added dropwise neutral saturated ammonium sulfate solution, stirring while adding, makes its saturation degree
30-33%, after being sufficiently mixed, after being placed at room temperature for 30-60min, centrifuging and taking precipitating；It repeats the operation 2-3 times, it is white to completely remove
Albumen.
5. the preparation method of immunoglobulin subclass antibodies according to claim 1, which is characterized in that in the step 2
SephadexG25 or electrodialysis desalination can also be used.
6. the preparation method of immunoglobulin subclass antibodies according to claim 1, which is characterized in that in the step 2
Desalination of dialysing is carried out using bag filter or glassine paper.
7. the preparation method of immunoglobulin subclass antibodies according to claim 1, which is characterized in that in the step 2
1%BaCl is used after dialysis desalination2Check the SO in dialyzate4 2-Or NH is checked with nessler reagent4, up to no SO4 2-Or NH4Occur
Until.
8. the preparation method of immunoglobulin subclass antibodies according to claim 1, which is characterized in that in the step 3
QAE cellulose, Sephadex G150 or Sephadex G200 column chromatographic purifying can also be used.
9. the preparation method of immunoglobulin subclass antibodies according to claim 1, which is characterized in that the IgG preparation
Purity using zone electrophoresis method, agar two-phase double diffusion identification method, immunoelectrophoresis identification method or disc electrophoresis identification method identification.
10. the preparation method of immunoglobulin subclass antibodies according to claim 1, which is characterized in that the preparation method
It can be used to prepare immunoglobulin hypotype IgG1, IgG2, IgG3, IgG4.
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| CN101633920A (en) * | 2009-08-24 | 2010-01-27 | 昆明理工大学 | Preparation method of monoclonal antibody of resisting treeshrew IgG |
| CN109633164A (en) * | 2018-12-14 | 2019-04-16 | 迪瑞医疗科技股份有限公司 | A kind of thyroglobulin antibody detection kit calibration object and its preparation process |
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2019
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101633920A (en) * | 2009-08-24 | 2010-01-27 | 昆明理工大学 | Preparation method of monoclonal antibody of resisting treeshrew IgG |
| CN109633164A (en) * | 2018-12-14 | 2019-04-16 | 迪瑞医疗科技股份有限公司 | A kind of thyroglobulin antibody detection kit calibration object and its preparation process |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114276437A (en) * | 2021-12-04 | 2022-04-05 | 南京岚煜生物科技有限公司 | Preparation method of compound high-value reference substance for five immune terms |
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