CN104193825B - Novel IgM-IgG polymer and polymerization method thereof - Google Patents
Novel IgM-IgG polymer and polymerization method thereof Download PDFInfo
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Abstract
The invention discloses a novel IgM-IgG polymer and a polymerization method thereof. The novel IgM-IgG polymer is characterized by being as shown in the structural formula (1) in the specification, or in the structural formula (2) in the specification, wherein in the formulae, n ranges from 1 to 5. The polymerization method of the novel IgM-IgG polymer comprises the following steps: vulcanizing SATA/hydroxylamine, activating amino by using Sulfo-SMCC, and finally connecting IgM immune globulin with IgG immune globulin. The relative molecular weight of the polymer is about 110-170kd, and as the polymer is water-soluble protein, the polymer can be preserved for a long time at low temperature under the protection of glycerinum. By adopting the polymer, non-specific reaction can be effectively inhibited, so that the anti-interference capability of an in-vitro immunoreaction system is improved, and the normal operation of the in-vitro immunoreaction is ensured.
Description
Technical field
The present invention relates to another kind of IgM-IgG polymer and its polymerization, anti-particularly to a kind of enhancing ion vitro immunization
Answer IgM-IgG polymer and its polymerization of the capacity of resisting disturbance of system, belong to molecular chemistry synthesis field.
Background technology
Antigen（antigen）Refer to produce the material of specific immune response in body, after it enters body, can pierce
Sharp body produces antibody（antibody）, activated cell immunity.The molecular weight of antigen is generally higher than 5000, and the half of small molecule is anti-
Former（hapten）Must be combined with macro-molecular protein rear just can cause body produce specific antibody.The reactivity of antigen depends on
In antigenic determinant（antigenic determinant）, or epi-position（epitope）.According to antigen molecular size and its egg
White matter structure, an antigen molecule can be with different determinants.
Antibody refers to the immunoglobulin being combined with antigen-specific（Immunoglobulin, Ig）, it is divided into five classes, that is,
IgG, IgM, IgA, IgD and IgE, that relevant with immunoassay is IgG and IgM.The IgG monomer of each Y type shape has two antigens
Binding site or valency, molecular weight is 150000.And IgM is by the pentamer of five monomer compositions, there are 10 antigenic valences,
But the impact due to locus, only shows as five antigenic valences, the molecular weight of IgM is 950000.Ig molecule is by 4
Peptide chain forms：Article two, light chain, relative molecular weight 24000～27000；Article two, heavy chain, relative molecular weight 55000～70000.Light chain
Pass through 4 pairs of disulfide bond and heavy chain between（-S-S-）It is connected, form complete immunoglobulin molecules.
The series antibody producing for the same body of same antigen difference determinant is referred to as polyclonal antibody.And pin
The antibody that the same body of same antigen, same determinant is produced is referred to as monoclonal antibody.Antibody animal derived
Refer to the animal species that antibody produces.
Using antibody antigen association reaction principle thus detecting that the content of determinand has been widely used in in-vitro diagnosis
Technology many decades.And color-developing compounds add the Enzyme-multiplied immune technique producing so that sensitivity has obtained large increase.Isotope
The addition of tracer and the radioimmunology that produces, make sensitivity more step a stage, but consequent Spent Radioactive
The possible injury that the process of thing and lonizing radiation cause to human body, hampers the development further of this technology.Eighties of last century nine
The Chemiluminescence immunoassay that the ten's grew up, the chain being caused in antibody antigen association reaction using enzyme or other labels
Formula chemical reaction, measures the photon of release in chemical energy transformation process, thus obtaining the content of determinand.Due to this chain type
Learn reaction and can amplify photon signal so that measurement sensitivity obtains is greatly improved in geometry order of magnitude ground, expand line simultaneously
Thus improving the accuracy of high low side, this has established theoretical basiss in immune detection for the chemiluminescence to property scope.But, by
In the multiformity of each detection sample, its inclusions is each different, and many small molecules or homologue can disturb normal antibody
Antigen-specific reacts, thus causing the non-specific binding of antibody to react.Due to chemiluminescent hypersensitivity, if can not be effective
This non-specific responding of suppression, then inevitably produce false positive, false constipation of YIN type fruit, particularly in the false sun of linear low side
Inaccuracy.
Content of the invention
The present invention a kind of IgM-IgG polymer and its polymerization are provided it is therefore an objective to when reducing immunoassay small molecule or
The interference of homologue.
For reaching above-mentioned purpose, the first technical scheme that the present invention adopts is：A kind of new IgM-IgG polymer,
Meet structural formula（1）It is shown,
（1）；
Or structural formula（2）It is shown,
（2）；
In formula, n=1 ~ 5.
The present invention adopt second technical scheme be：A kind of polymerization of new IgM-IgG polymer, successively
It is made up of the following step：
The crosslinking of Part I, IgM and S- acetylthio-acetate succinimide ester
The first step, IgM immunoglobulin is dissolved in the IgM immunity ball obtaining in PBS cocktail buffer that concentration is 1mg/ml
Albumen buffer, is then charged in bag filter, under the conditions of 2 ~ 8 DEG C, dialyses 8 ~ 15 hours in described PBS cocktail buffer；
Wherein, described PBS cocktail buffer is 7.4 for 10mmol/L and pH phosphate buffer and 0.15mol/L sodium chloride
Mixed solution；
Second step, the IgM immunoglobulin buffer after dialysing in the removal first step, then to the IgM immunity after dialysis
Being slowly added into freshly prepared concentration in globulin buffer is 10 ~ 50mmol/L S- acetylthio-acetate succinimide ester
Dimethyl sulphoxide solution, concussion mix, at 15 ~ 35 DEG C reaction 25 ~ 35 minutes reactant liquor；After reaction terminates, by reactant liquor
Load in bag filter, under the conditions of 2 ~ 8 DEG C, dialyse 8 ~ 15 hours in described PBS cocktail buffer；Wherein, after described dialysis
IgM immunoglobulin buffer in IgM immunoglobulin and described S- acetylthio-acetate succinimide ester mole
Than for 1:10~1:30；IgM immunoglobulin and described concentration in IgM immunoglobulin buffer after described dialysis is 10 ~
S- acetylthio-acetate succinyl in the dimethyl sulphoxide solution of 50mmol/L S- acetylthio-acetate succinimide ester is sub-
The product that the reaction of amine ester generates is IgM-SATA, its structure such as formula（3）Shown, obtain IgM immunoglobulin just poly- liquid,
（3）；
3rd step, is slowly added into freshly prepared hydroxylamine solution in the first poly- liquid of IgM immunoglobulin obtaining to second step,
After mixing, react 1.5 ~ 2.5 hours at 15 ~ 35 DEG C；After reaction terminates, reactant liquor is loaded in bag filter, in 2 ~ 8 DEG C of conditions
Under, dialyse 8 ~ 15 hours in described PBS cocktail buffer, obtain poly- liquid in IgM immunoglobulin；Wherein, described IgM immunity
The reaction volume ratio of globulin just poly- liquid and described hydroxylamine solution is for 10:1；Described IgM immunoglobulin just poly- liquid and described hydroxyl
There is the reaction of deacylation base in the azanol in amine aqueous solution, the product of generation is IgM-SH, its structure such as formula（4）It is shown,
（4）；
Part II, IgG and 4- (N- maleimidomehyl) hexamethylene -1- carboxylic acid sulfonic group succinimide ester
Crosslinked
The first step, IgG immunoglobulin is dissolved in obtain in described PBS cocktail buffer concentration be 1mg/ml IgG exempt from
Epidemic disease globulin buffer, is then charged in bag filter, and under the conditions of 2 ~ 8 DEG C, in described PBS cocktail buffer, dialysis 8 ~ 15 is little
When；
Second step, the IgG immunoglobulin buffer after dialysing in the removal first step, the then IgG immunity after dialysis
Be slowly added in globulin buffer freshly prepared concentration be 1.5mg/ml 4- (N- maleimidomehyl) hexamethylene -1-
The PBS solution of carboxylic acid sulfonic group succinimide ester, concussion mixes, and reaction at 15 ~ 35 DEG C obtains reactant liquor in 25 ~ 35 minutes；Reaction
After end, reactant liquor is loaded in bag filter, under the conditions of 2 ~ 8 DEG C, dialyses 8 ~ 15 hours in described PBS cocktail buffer,
Obtain IgG immunoglobulin just poly- liquid；Wherein, the IgG immunoglobulin in the IgG immunoglobulin buffer after described dialysis
With described 4- (N- maleimidomehyl) hexamethylene -1- carboxylic acid sulfonic group succinimide ester PBS solution in 4- (N-
Maleimidomehyl) mol ratio between hexamethylene -1- carboxylic acid sulfonic group succinimide ester is 1:20；
The IgG immunoglobulin in IgG immunoglobulin buffer after described dialysis and described 4- (N- maleimide
Amine methyl) hexamethylene -1- carboxylic acid sulfonic group succinimide ester PBS solution in 4- (N- maleimidomehyl) hexamethylene
The IgG that the product that the reaction of alkane -1- carboxylic acid sulfonic group succinimide ester generates activates for maleimide, its structure such as formula（5）
It is shown,
（5）；
Part III, the crosslinking of poly- liquid and the first poly- liquid of IgG immunoglobulin in IgM immunoglobulin
The first step, by poly- liquid in described IgM immunoglobulin, just poly- liquid loads in bag filter with described IgG immunoglobulin
And mix, under the conditions of 2 ~ 8 DEG C, react 1.5 ~ 2.5 hours；Then dialyse 8～15 hours in described PBS cocktail buffer,
Obtain IgM-IgG immunoglobulin polymer dope；Wherein, the IgM immune globulin in poly- liquid in described IgM immunoglobulin
The mol ratio of the IgG immunoglobulin in the first poly- liquid of white and described IgG immunoglobulin is 1:1 ~ 5, described IgM immunoglobulin
In IgM immunoglobulin in poly- liquid react with the IgG immunoglobulin in described IgG immunoglobulin just poly- liquid, generation
Product is IgM- maleimide-IgG, its structure such as formula（6）It is shown,
（6）；
Second step, the described IgM-IgG immunoglobulin polymer dope obtaining is crossed sephadex G 200 post, then
With described PBS cocktail buffer eluting, flow velocity is 0.25ml/ minute, is in charge of the solution after collecting eluting, and surveys OD280nm suction
Light value, by each pipe solution mixing of the first peak recording, more mixed solution is concentrated into about 2mg/ml（Fixed with OD280
Value）, this is the IgM-IgG final polymer product of purification.
The present invention adopt the third technical scheme be：A kind of polymerization of new IgM-IgG polymer, successively
It is made up of the following step：
The crosslinking of Part I, IgG and S- acetylthio-acetate succinimide ester
The first step, IgG immunoglobulin is dissolved in the IgG immunity ball obtaining in PBS cocktail buffer that concentration is 1mg/ml
Albumen buffer, is then charged in bag filter, under the conditions of 2 ~ 8 DEG C, dialyses 8 ~ 15 hours in described PBS cocktail buffer；
Wherein, described PBS cocktail buffer is 7.4 for 10mmol/L and pH phosphate buffer and 0.15mol/L sodium chloride
Mixed solution；
Second step, the IgG immunoglobulin buffer after dialysing in the removal first step, then to the IgG immunity after dialysis
Being slowly added into freshly prepared concentration in globulin buffer is 10 ~ 50mmol/L S- acetylthio-acetate succinimide ester
Dimethyl sulphoxide solution, concussion mix, at 15 ~ 35 DEG C reaction 25 ~ 35 minutes reactant liquor；After reaction terminates, by reactant liquor
Load in bag filter, under the conditions of 2 ~ 8 DEG C, dialyse 8 ~ 15 hours in described PBS cocktail buffer；Wherein, after described dialysis
IgG immunoglobulin buffer in IgG immunoglobulin and described S- acetylthio-acetate succinimide ester mole
Than for 1:10~1:30；IgG immunoglobulin and described concentration in IgG immunoglobulin buffer after described dialysis is 10 ~
S- acetylthio-acetate succinyl in the dimethyl sulphoxide solution of 50mmol/L S- acetylthio-acetate succinimide ester is sub-
The product that the reaction of amine ester generates is IgG-SATA, its structure such as formula（7）Shown, obtain IgG immunoglobulin just poly- liquid,
（7）；
3rd step, is slowly added into freshly prepared hydroxylamine solution in the first poly- liquid of IgG immunoglobulin obtaining to second step,
After mixing, react 1.5 ~ 2.5 hours at 15 ~ 35 DEG C；After reaction terminates, reactant liquor is loaded in bag filter, in 2 ~ 8 DEG C of conditions
Under, dialyse 8 ~ 15 hours in described PBS cocktail buffer, obtain poly- liquid in IgG immunoglobulin；Wherein, described IgG immunity
The reaction volume ratio of globulin just poly- liquid and described hydroxylamine solution is for 10:1；Described IgG immunoglobulin just poly- liquid and described hydroxyl
There is the reaction of deacylation base in the azanol in amine aqueous solution, the product of generation is IgG-SH, its structure such as formula（8）It is shown,
（8）；
Part II, IgM and 4- (N- maleimidomehyl) hexamethylene -1- carboxylic acid sulfonic group succinimide ester
Crosslinked
The first step, IgM immunoglobulin is dissolved in obtain in described PBS cocktail buffer concentration be 1mg/ml IgM exempt from
Epidemic disease globulin buffer, is then charged in bag filter, and under the conditions of 2 ~ 8 DEG C, in described PBS cocktail buffer, dialysis 8 ~ 15 is little
When；
Second step, the IgM immunoglobulin buffer after dialysing in the removal first step, the then IgM immunity after dialysis
Be slowly added in globulin buffer freshly prepared concentration be 1.5mg/ml 4- (N- maleimidomehyl) hexamethylene -1-
The PBS solution of carboxylic acid sulfonic group succinimide ester, concussion mixes, and reaction at 15 ~ 35 DEG C obtains reactant liquor in 25 ~ 35 minutes；Reaction
After end, reactant liquor is loaded in bag filter, under the conditions of 2 ~ 8 DEG C, dialyses 8 ~ 15 hours in described PBS cocktail buffer,
Obtain IgM immunoglobulin just poly- liquid；Wherein, the IgM immunoglobulin in the IgM immunoglobulin buffer after described dialysis
With described 4- (N- maleimidomehyl) hexamethylene -1- carboxylic acid sulfonic group succinimide ester PBS solution in 4- (N-
Maleimidomehyl) mol ratio between hexamethylene -1- carboxylic acid sulfonic group succinimide ester is 1:20；
The IgM immunoglobulin in IgM immunoglobulin buffer after described dialysis and described 4- (N- maleimide
Amine methyl) hexamethylene -1- carboxylic acid sulfonic group succinimide ester PBS solution in 4- (N- maleimidomehyl) hexamethylene
The IgM that the product that the reaction of alkane -1- carboxylic acid sulfonic group succinimide ester generates activates for maleimide, its structure such as formula（9）
It is shown,
（9）；
Part III, the crosslinking of poly- liquid and the first poly- liquid of IgM immunoglobulin in IgG immunoglobulin
The first step, by poly- liquid in described IgG immunoglobulin, just poly- liquid loads in bag filter with described IgM immunoglobulin
And mix, under the conditions of 2 ~ 8 DEG C, react 1.5 ~ 2.5 hours；Then dialyse 8～15 hours in described PBS cocktail buffer,
Obtain IgM-IgG immunoglobulin polymer dope；Wherein, the IgG immune globulin in poly- liquid in described IgG immunoglobulin
The mol ratio of the IgM immunoglobulin in the first poly- liquid of white and described IgM immunoglobulin is 1:1 ~ 5, described IgG immunoglobulin
In IgG immunoglobulin in poly- liquid react with the IgM immunoglobulin in described IgM immunoglobulin just poly- liquid, generation
Product is IgG- maleimide-IgM, its structure such as formula（10）It is shown,
（10）；
Second step, the described IgM-IgG immunoglobulin polymer dope obtaining is crossed sephadex G 200 post, then
With described PBS cocktail buffer eluting, flow velocity is 0.25ml/ minute, is in charge of the solution after collecting eluting, surveys OD280nm extinction
Value, by each pipe solution mixing of first peak, more mixed solution is concentrated into about 2mg/ml（With OD280 definite value）, this is
The IgM-IgG final polymer product of purification.
Relevant content in technique scheme is explained as follows：
1st, in such scheme, by glycerol mix homogeneously equal with its volume for described IgM-IgG final polymer product, so
After can preserve under the conditions of subzero 20 DEG C.
2nd, in such scheme, the English of described S- acetylthio-acetate succinimide ester is entitledN-succinimidyl
S-acetylthioacetate, abbreviation SATA, purchased from Pierce company of the U.S..
3rd, in such scheme, the English entitled Dimethyl sulfoxide, abbreviation DMSO of described dimethyl sulfoxide.
4th, in such scheme, described azanol English name is Hydroxylamine, and the English name of ethylenediaminetetraacetic acid is
Ethylene Diamine Tetraacetic Acid or EDTA.
5th, in such scheme, described 4- (N- maleimidomehyl) hexamethylene -1- carboxylic acid sulfonic group succinimide ester
English name be sulfosuccinimidyl 4- [N- maleimidomethyl] cyclohexane-1-carboxylate,
Abbreviation Sulfo SMCC, purchased from Thermo Scientific company of the U.S..
6th, in such scheme, described sephadex G 200 post, English name Sephadex G200, purchased from Pharmacia
Company.
Because technique scheme is used, the present invention compared with prior art has following advantages and effect：
What prior art was commonly used is the common immunity adding in reaction system with detection antibody same animals source property
Globulin, by the principle of the non-specific material of competitive Adsorption, at utmost suppresses the generation of non-specific responding, but due to molecule
The reason space structure, when many, its effect is unsatisfactory.The polymerization of IgG-IgM polymer of the present invention is SATA/ hydroxyl
Amine sulfuration, Sulfo SMCC activation amino, and finally realize IgM immunoglobulin and be connected with IgG immunoglobulin.Obtain
IgM-IgG polymer relative molecular weight is about 110～170kd, water soluble protein, under glycerol protection, can protect prolonged cold
Deposit, this polymer can suppress non-specific responding effectively, thus improving the capacity of resisting disturbance of response system it is ensured that ion vitro immunization is anti-
That answers is normally carried out；And, in the preparation method of IgM-IgG polymer of the present invention, the pH value of reaction system, between 7 ~ 8, enters
And the possibility of immune globulin antibody degeneration can be reduced to greatest extent, namely immune globulin antibody is kept to live to greatest extent
Property.
Brief description
Accompanying drawing 1 detects polydextran gel eluting principle condition for ultraviolet light light absorption method；
Accompanying drawing 2 is SDS-polyacrylamide amide（ SDS-PAGE）Gel electrophoresiss are to determine different proportion IgM
Purification situation with IgG cross-linking products；
Accompanying drawing 3 is the IgM-IgG polymer and IgG monomer impact schematic diagram to standard curve.
Specific embodiment
Below in conjunction with the accompanying drawings and embodiment the invention will be further described：
Embodiment one：A kind of new IgM-IgG polymer and its polymerization
It is made up of the following step successively：
Part I, IgM and S- acetylthio-acetate succinimide ester【N-succinimidyl S-
acetylthioacetate(SATA)】（Purchased from Pierce company of the U.S.）Crosslinking
The first step, IgM immunoglobulin is dissolved in the IgM immunity ball obtaining in PBS cocktail buffer that concentration is 1mg/ml
Albumen buffer, is then charged in bag filter, under the conditions of 2 ~ 8 DEG C, dialyses 8 ~ 15 hours in described PBS cocktail buffer；
Wherein, described PBS cocktail buffer is 7.4 for 10mmol/L and pH phosphate buffer and 0.15mol/L sodium chloride
Mixed solution；
Second step, the IgM immunoglobulin buffer after dialysing in the removal first step, then to the IgM immunity after dialysis
Being slowly added into freshly prepared concentration in globulin buffer is 10 ~ 50mmol/L S- acetylthio-acetate succinimide ester
Dimethyl sulphoxide solution, concussion mix, at 15 ~ 35 DEG C reaction 25 ~ 35 minutes reactant liquor；After reaction terminates, by reactant liquor
Load in bag filter, under the conditions of 2 ~ 8 DEG C, dialyse 8 ~ 15 hours in described PBS cocktail buffer；Wherein, after described dialysis
IgM immunoglobulin buffer in IgM immunoglobulin and described S- acetylthio-acetate succinimide ester mole
Than for 1:10~1:30；IgM immunoglobulin and described concentration in IgM immunoglobulin buffer after described dialysis is 10 ~
S- acetylthio-acetate succinyl in the dimethyl sulphoxide solution of 50mmol/L S- acetylthio-acetate succinimide ester is sub-
The product that the reaction of amine ester generates is IgM-SATA, its structure such as formula（3）Shown, obtain IgM immunoglobulin just poly- liquid,
（3）；
3rd step, is slowly added into freshly prepared hydroxylamine solution in the first poly- liquid of IgM immunoglobulin obtaining to second step,
After mixing, react 1.5 ~ 2.5 hours at 15 ~ 35 DEG C；After reaction terminates, reactant liquor is loaded in bag filter, in 2 ~ 8 DEG C of conditions
Under, dialyse 8 ~ 15 hours in described PBS cocktail buffer, obtain poly- liquid in IgM immunoglobulin；Wherein, described IgM immunity
The reaction volume ratio of globulin just poly- liquid and described hydroxylamine solution is for 10:1；Described IgM immunoglobulin just poly- liquid and described hydroxyl
There is the reaction of deacylation base in the azanol in amine aqueous solution, the product of generation is IgM-SH, its structure such as formula（4）It is shown,
（4）；
Part II, IgG and 4- (N- maleimidomehyl) hexamethylene -1- carboxylic acid sulfonic group succinimide ester（Purchase
From Thermo Scientific company of the U.S.）Crosslinking
The first step, IgG immunoglobulin is dissolved in obtain in described PBS cocktail buffer concentration be 1mg/ml IgG exempt from
Epidemic disease globulin buffer, is then charged in bag filter, and under the conditions of 2 ~ 8 DEG C, in described PBS cocktail buffer, dialysis 8 ~ 15 is little
When；
Second step, the IgG immunoglobulin buffer after dialysing in the removal first step, the then IgG immunity after dialysis
Be slowly added in globulin buffer freshly prepared concentration be 1.5mg/ml 4- (N- maleimidomehyl) hexamethylene -1-
The PBS solution of carboxylic acid sulfonic group succinimide ester, concussion mixes, and reaction at 15 ~ 35 DEG C obtains reactant liquor in 25 ~ 35 minutes；Reaction
After end, reactant liquor is loaded in bag filter, under the conditions of 2 ~ 8 DEG C, dialyses 8 ~ 15 hours in described PBS cocktail buffer,
Obtain IgG immunoglobulin just poly- liquid；Wherein, the IgG immunoglobulin in the IgG immunoglobulin buffer after described dialysis
With described 4- (N- maleimidomehyl) hexamethylene -1- carboxylic acid sulfonic group succinimide ester PBS solution in 4- (N-
Maleimidomehyl) mol ratio between hexamethylene -1- carboxylic acid sulfonic group succinimide ester is 1:20；
The IgG immunoglobulin in IgG immunoglobulin buffer after described dialysis and described 4- (N- maleimide
Amine methyl) hexamethylene -1- carboxylic acid sulfonic group succinimide ester PBS solution in 4- (N- maleimidomehyl) hexamethylene
The IgG that the product that the reaction of alkane -1- carboxylic acid sulfonic group succinimide ester generates activates for maleimide, its structure such as formula（5）
It is shown,
（5）；
Part III, the crosslinking of poly- liquid and the first poly- liquid of IgG immunoglobulin in IgM immunoglobulin
The first step, by poly- liquid in described IgM immunoglobulin, just poly- liquid loads in bag filter with described IgG immunoglobulin
And mix, under the conditions of 2 ~ 8 DEG C, react 1.5 ~ 2.5 hours；Then dialyse 8～15 hours in described PBS cocktail buffer,
Obtain IgM-IgG immunoglobulin polymer dope；Wherein, the IgM immune globulin in poly- liquid in described IgM immunoglobulin
The mol ratio of the IgG immunoglobulin in the first poly- liquid of white and described IgG immunoglobulin is 1:1 ~ 5, described IgM immunoglobulin
In IgM immunoglobulin in poly- liquid react with the IgG immunoglobulin in described IgG immunoglobulin just poly- liquid, generation
Product is IgM- maleimide-IgG, its structure such as formula（6）It is shown,
（6）；
Second step, the described IgM-IgG immunoglobulin polymer dope obtaining is crossed sephadex G 200 post, then
With described PBS cocktail buffer eluting, flow velocity is 0.25ml/ minute, is in charge of the solution after collecting eluting, and surveys OD280nm suction
Light value, by each pipe solution mixing of the first peak recording, more mixed solution is concentrated into about 2mg/ml（Fixed with OD280
Value）, this is the IgM-IgG final polymer product of purification.
Points for attention：
First, weigh during powdered chemical medicine it should be noted that gesture slowly closes exhaust system if necessary with true as far as possible
Protect and weigh accurately.
Whether the chemical drugss weighing needed for the 2nd, checking are all in useful life.
3rd, check whether some special chemical medicines make moist or lump.
4th, after all chemical drugss weigh and finish, arrange weighing room to keep cleaning.
5th, during the solution such as Deca SATA, azanol, Sulfo SMCC, need to vibrate uniform Deca.
6th, pre-operational check：
Whether electronic weighing scale surface pallet is clean.
Whether weighing scoop is clean.
Whether required reagent is all in useful life.
Verification method：
1st, ultraviolet light absorbance method（OD280nm measures）
Have maximum absorption band, concentration and extinction using aromatic amino acid contained by protein at ultraviolet light 280nm wavelength
Spend the principle being directly proportional, the protein concentration of each sample can be detected, also include first peak product after purification（The i.e. IgM- of purification
IgG final polymer product）Determination, referring to shown in accompanying drawing 1.
2nd, SDS-polyacrylamide amide（SDS-PAGE）Gel electrophoresiss are to determine the crosslinked, pure of polymerizate
Change situation, referring to shown in accompanying drawing 2.
Wherein, in accompanying drawing 2, the 2nd road is IgM and IgG with 1：After 5 ratio crosslinkings, product purification rear electrophoresis result；4th road is
IgM and IgG are with 1：After 2 ratio crosslinkings, product purification rear electrophoresis result.The two all can substantially see heavy chain, light chain two band, molecule
Amount about 55kd and 27kd about.Standard protein molecular weight（Unit：kd）：14、20、31、43、66、97.
3rd, functional experiment：Polymer is added antibody antigen reaction system（Chemoluminescence method）To verify it in system
Capacity of resisting disturbance.To add common homologous immunoglobulin, and it is not added with any immunoglobulin for comparison.
Result：
This reaction system adopts double antibody sandwich method as detection meanss.IgM antibody therein is big mouse, and IgG
Antibody is globefish mouse, or IgM antibody is globefish mouse, and IgG antibody is big mouse.
Double antibody sandwich method：The immobilization of antigen or antibody and the labelling of antigen or antibody.In conjunction with surface of solid phase carriers
Antigen or antibody still keep its immunologic competence, the antigen of labelling or antibody both to retain its immunologic competence, again retain labelling
The catalysis activity of thing.Reacted with the antigen of surface of solid phase carriers or antibody by inspection specimen.Make solid phase carrier with the method for washing
The antigen antibody complex of upper formation is separated with other materials in liquid.Add antigen or the antibody of labelling, by reaction
In conjunction with solid phase carrier.After adding the substrate of label, substrate is catalyzed and produces chain reaction and launch photon, and photon produces
Raw amount is directly related with the amount of tested substance in specimen, carries out qualitative or quantitative analysis according to measuring light subunit relatively.Mark
The catalytic efficiency of note thing is very high, is greatly exaggerated the result of immunoreation, makes assay method reach very high sensitivity.
Competition law：Principle is antigen in specimen and the competition of a certain amount of enzyme-labelled antigen is combined with insolubilized antibody.In specimen
Amount of antigen content is the more, few in conjunction with the enzyme-labelled antigen in solid phase, and last colour developing is also more shallow, or in chemiluminescence reaction
The photon producing is fewer.
Result is as shown in following four form and accompanying drawing 3：
Table 1：Standard control（No immunoglobulin）（RLU*）
| Standard antigen mg/ml | Averagely | % | ||||
| 0 | 32468 | 24636 | 30318 | 27639 | 28765 | 4.19% |
| 0.1 | 32568 | 38393 | 31211 | 39052 | 35381 | 5.15% |
| 1 | 92647 | 103899 | 97602 | 116498 | 102662 | 14.95% |
| 10 | 746740 | 707113 | 653492 | 639643 | 686747 | 100.00% |
Table 2：IgG monomer（0.1mg/ml）(RLU*)
| Standard antigen mg/ml | Averagely | % | ||
| 0 | 27589 | 29844 | 28717 | 5.80% |
| 0.1 | 45114 | 53578 | 49346 | 8.57% |
| 1 | 86934 | 90208 | 88571 | 15.39% |
| 10 | 596739 | 554476 | 575608 | 100.00% |
Table 3：IgM-IgG （Non- purification）（0.1mg/ml）(RLU*)
| Standard antigen mg/ml | Averagely | % | ||
| 0 | 30880 | 26943 | 28912 | 5.14% |
| 0.1 | 39147 | 41531 | 40339 | 7.17% |
| 1 | 76993 | 80222 | 78608 | 13.98% |
| 10 | 532999 | 591673 | 562336 | 100.00% |
Table 4：IgM-IgG （Purification）（0.1mg/ml）(RLU*)
Note：What RLU represented is light subunit relatively.
Can see from the result of above five forms and accompanying drawing 3：
Three oblique lines are had from top to bottom, going up one most is IgM-IgG polymer in accompanying drawing 3（Purification）, centre is standard
Comparison（No immunoglobulin）Next root is IgG monomer and IgM-IgG polymer（Purification）Both threads coincidence line.
For the standard curve being come by standard antigen：The response system adding purification IgM-IgG polymer has
The curve of big slope.The sensitiveest in low concentration region, and the range of linearity is the widest.
All difficulty samples, in the standard reaction system not having immunoglobulin to add, show false positive results.But
In all reaction systems adding different immunoglobulins, the overwhelming majority in these samples all shows negative essence.And
Here wherein adds the effect of the suppression nonspecific immunity reaction of purification IgM-IgG best.Generally, each material is anti-interference
Ability：IgM-IgG (purification)>IgM-IgG (non-purification)>IgG monomer.
Conclusion：IgG-IgM polymer can strengthen the capacity of resisting disturbance of external immunoreation system effectively.
Embodiment two：A kind of new IgM-IgG polymer and its polymerization
It is made up of the following step successively：
Part I, IgG and S- acetylthio-acetate succinimide ester（Purchased from Pierce company of the U.S.）Crosslinking
The first step, IgG immunoglobulin is dissolved in the IgG immunity ball obtaining in PBS cocktail buffer that concentration is 1mg/ml
Albumen buffer, is then charged in bag filter, under the conditions of 2 ~ 8 DEG C, dialyses 8 ~ 15 hours in described PBS cocktail buffer；
Wherein, described PBS cocktail buffer is 7.4 for 10mmol/L and pH phosphate buffer and 0.15mol/L sodium chloride
Mixed solution；
Second step, the IgG immunoglobulin buffer after dialysing in the removal first step, then to the IgG immunity after dialysis
Being slowly added into freshly prepared concentration in globulin buffer is 10 ~ 50mmol/L S- acetylthio-acetate succinimide ester
Dimethyl sulphoxide solution, concussion mix, at 15 ~ 35 DEG C reaction 25 ~ 35 minutes reactant liquor；After reaction terminates, by reactant liquor
Load in bag filter, under the conditions of 2 ~ 8 DEG C, dialyse 8 ~ 15 hours in described PBS cocktail buffer；Wherein, after described dialysis
IgG immunoglobulin buffer in IgG immunoglobulin and described S- acetylthio-acetate succinimide ester mole
Than for 1:10~1:30；IgG immunoglobulin and described concentration in IgG immunoglobulin buffer after described dialysis is 10 ~
S- acetylthio-acetate succinyl in the dimethyl sulphoxide solution of 50mmol/L S- acetylthio-acetate succinimide ester is sub-
The product that the reaction of amine ester generates is IgG-SATA, its structure such as formula（7）Shown, obtain IgG immunoglobulin just poly- liquid,
（7）；
3rd step, is slowly added into freshly prepared hydroxylamine solution in the first poly- liquid of IgG immunoglobulin obtaining to second step,
After mixing, react 1.5 ~ 2.5 hours at 15 ~ 35 DEG C；After reaction terminates, reactant liquor is loaded in bag filter, in 2 ~ 8 DEG C of conditions
Under, dialyse 8 ~ 15 hours in described PBS cocktail buffer, obtain poly- liquid in IgG immunoglobulin；Wherein, described IgG immunity
The reaction volume ratio of globulin just poly- liquid and described hydroxylamine solution is for 10:1；Described IgG immunoglobulin just poly- liquid and described hydroxyl
There is the reaction of deacylation base in the azanol in amine aqueous solution, the product of generation is IgG-SH, its structure such as formula（8）It is shown,
（8）；
Part II, IgM and 4- (N- maleimidomehyl) hexamethylene -1- carboxylic acid sulfonic group succinimide ester（Purchase
From Thermo Scientific company of the U.S.）Crosslinking
The first step, IgM immunoglobulin is dissolved in obtain in described PBS cocktail buffer concentration be 1mg/ml IgM exempt from
Epidemic disease globulin buffer, is then charged in bag filter, and under the conditions of 2 ~ 8 DEG C, in described PBS cocktail buffer, dialysis 8 ~ 15 is little
When；
Second step, the IgM immunoglobulin buffer after dialysing in the removal first step, the then IgM immunity after dialysis
Be slowly added in globulin buffer freshly prepared concentration be 1.5mg/ml 4- (N- maleimidomehyl) hexamethylene -1-
The PBS solution of carboxylic acid sulfonic group succinimide ester, concussion mixes, and reaction at 15 ~ 35 DEG C obtains reactant liquor in 25 ~ 35 minutes；Reaction
After end, reactant liquor is loaded in bag filter, under the conditions of 2 ~ 8 DEG C, dialyses 8 ~ 15 hours in described PBS cocktail buffer,
Obtain IgM immunoglobulin just poly- liquid；Wherein, the IgM immunoglobulin in the IgM immunoglobulin buffer after described dialysis
With described 4- (N- maleimidomehyl) hexamethylene -1- carboxylic acid sulfonic group succinimide ester PBS solution in 4- (N-
Maleimidomehyl) mol ratio between hexamethylene -1- carboxylic acid sulfonic group succinimide ester is 1:20；
The IgM immunoglobulin in IgM immunoglobulin buffer after described dialysis and described 4- (N- maleimide
Amine methyl) hexamethylene -1- carboxylic acid sulfonic group succinimide ester PBS solution in 4- (N- maleimidomehyl) hexamethylene
The IgM that the product that the reaction of alkane -1- carboxylic acid sulfonic group succinimide ester generates activates for maleimide, its structure such as formula（9）
It is shown,
（9）；
Part III, the crosslinking of poly- liquid and the first poly- liquid of IgM immunoglobulin in IgG immunoglobulin
The first step, by poly- liquid in described IgG immunoglobulin, just poly- liquid loads in bag filter with described IgM immunoglobulin
And mix, under the conditions of 2 ~ 8 DEG C, react 1.5 ~ 2.5 hours；Then dialyse 8～15 hours in described PBS cocktail buffer,
Obtain IgM-IgG immunoglobulin polymer dope；Wherein, the IgG immune globulin in poly- liquid in described IgG immunoglobulin
The mol ratio of the IgM immunoglobulin in the first poly- liquid of white and described IgM immunoglobulin is 1:1 ~ 5, described IgG immunoglobulin
In IgG immunoglobulin in poly- liquid react with the IgM immunoglobulin in described IgM immunoglobulin just poly- liquid, generation
Product is IgG- maleimide-IgM, its structure such as formula（10）It is shown,
（10）；
Second step, the described IgM-IgG immunoglobulin polymer dope obtaining is crossed sephadex G 200 post, then
With described PBS cocktail buffer eluting, flow velocity is 0.25ml/ minute, is in charge of the solution after collecting eluting, surveys OD280nm extinction
Value, by each pipe solution mixing of first peak, more mixed solution is concentrated into about 2mg/ml（With OD280 definite value）, this is
The IgM-IgG final polymer product of purification.
Above-described embodiment only technology design to illustrate the invention and feature, its object is to allow person skilled in the art
Scholar will appreciate that present disclosure and implements according to this, can not be limited the scope of the invention with this.All according to the present invention
Equivalence changes or modification that spirit is made, all should be included within the scope of the present invention.
Claims (7)
1. a kind of IgM-IgG polymer it is characterised in that：Described IgM-IgG polymer meets structural formula（1）It is shown,
（1）；
Or structural formula（2）It is shown,
（2）；
In formula, n=1 ~ 5.
2. a kind of polymerization of IgM-IgG polymer it is characterised in that：It is made up of the following step successively：
The crosslinking of Part I, IgM and S- acetylthio-acetate succinimide ester
The first step, IgM immunoglobulin is dissolved in the IgM immunoglobulin obtaining in PBS cocktail buffer that concentration is 1mg/ml
Buffer, is then charged in bag filter, under the conditions of 2 ~ 8 DEG C, dialyses 8 ~ 15 hours in described PBS cocktail buffer；Wherein,
Described PBS cocktail buffer is that the phosphate buffer that 10mmol/L and pH are 7.4 is molten with the mixing of 0.15mol/L sodium chloride
Liquid；
Second step, the IgM immunoglobulin buffer after dialysing in the removal first step, then to the IgM immune globulin after dialysis
It is slowly added into freshly prepared concentration for the two of 10 ~ 50mmol/L S- acetylthio-acetate succinimide ester in white buffer
Methyl sulfoxide solution, concussion mixes, and reaction at 15 ~ 35 DEG C obtains reactant liquor in 25 ~ 35 minutes；After reaction terminates, reactant liquor is loaded
In bag filter, under the conditions of 2 ~ 8 DEG C, dialyse 8 ~ 15 hours in described PBS cocktail buffer；Wherein, the IgM after described dialysis
IgM immunoglobulin in immunoglobulin buffer is 1 with the mol ratio of described S- acetylthio-acetate succinimide ester:
10~1:30；IgM immunoglobulin and described concentration in IgM immunoglobulin buffer after described dialysis is 10 ~
S- acetylthio-acetate succinyl in the dimethyl sulphoxide solution of 50mmol/L S- acetylthio-acetate succinimide ester is sub-
The product that the reaction of amine ester generates is IgM-SATA, its structure such as formula（3）Shown, obtain IgM immunoglobulin just poly- liquid,
（3）；
3rd step, is slowly added into freshly prepared hydroxylamine solution in the first poly- liquid of IgM immunoglobulin obtaining to second step, mixes
Afterwards, react 1.5 ~ 2.5 hours at 15 ~ 35 DEG C, wherein, described hydroxylamine solution refers to containing 0.5mol/L azanol, 25mmol/L second
Ethylenediamine tetraacetic acid (EDTA), 10mmol/L and pH value are 7.4 phosphate buffer and the mixed solution of 0.15mol/L sodium chloride；Instead
After should terminating, reactant liquor is loaded in bag filter, under the conditions of 2 ~ 8 DEG C, in described PBS cocktail buffer, dialysis 8 ~ 15 is little
When, obtain poly- liquid in IgM immunoglobulin；Wherein, the reactant of the first poly- liquid of described IgM immunoglobulin and described hydroxylamine solution
Long-pending ratio is 10:1；Just poly- liquid and the azanol in described hydroxylamine solution deacylation base to react to described IgM immunoglobulin occur, generation
Product is IgM-SH, its structure such as formula（4）It is shown,
（4）；
Part II, IgG and 4- (N- maleimidomehyl) hexamethylene -1- carboxylic acid sulfonic group succinimide ester crosslinking
The first step, IgG immunoglobulin is dissolved in the IgG immunity ball obtaining in described PBS cocktail buffer that concentration is 1mg/ml
Albumen buffer, is then charged in bag filter, under the conditions of 2 ~ 8 DEG C, dialyses 8 ~ 15 hours in described PBS cocktail buffer；
Second step, the IgG immunoglobulin buffer after dialysing in the removal first step, the then IgG immune globulin after dialysis
Be slowly added in white buffer freshly prepared concentration be 1.5mg/ml 4- (N- maleimidomehyl) hexamethylene -1- carboxylic acid
The PBS solution of sulfonic group succinimide ester, concussion mixes, and reaction at 15 ~ 35 DEG C obtains reactant liquor in 25 ~ 35 minutes；Reaction terminates
Afterwards, reactant liquor is loaded in bag filter, under the conditions of 2 ~ 8 DEG C, dialyse 8 ~ 15 hours in described PBS cocktail buffer, obtain
IgG immunoglobulin just poly- liquid；Wherein, the IgG immunoglobulin in the IgG immunoglobulin buffer after described dialysis and institute
State 4- (N- maleimidomehyl) hexamethylene -1- carboxylic acid sulfonic group succinimide ester PBS solution in 4- (N- Malaysia
Acid imide methyl) mol ratio between hexamethylene -1- carboxylic acid sulfonic group succinimide ester is 1:20；
The IgG immunoglobulin in IgG immunoglobulin buffer after described dialysis and described 4- (N- maleimide first
Base) hexamethylene -1- carboxylic acid sulfonic group succinimide ester PBS solution in 4- (N- maleimidomehyl) hexamethylene -1-
The IgG that the product that the reaction of carboxylic acid sulfonic group succinimide ester generates activates for maleimide, its structure such as formula（5）It is shown,
（5）；
Part III, the crosslinking of poly- liquid and the first poly- liquid of IgG immunoglobulin in IgM immunoglobulin
The first step, by poly- liquid in described IgM immunoglobulin, just poly- liquid loads in bag filter and mixes with described IgG immunoglobulin
Even, under the conditions of 2 ~ 8 DEG C, react 1.5 ~ 2.5 hours；Then dialyse 8～15 hours in described PBS cocktail buffer, obtain
IgM-IgG immunoglobulin polymer dope；Wherein, the IgM immunoglobulin in poly- liquid in described IgM immunoglobulin and
The mol ratio of the IgG immunoglobulin in the first poly- liquid of described IgG immunoglobulin is 1:1 ~ 5, poly- in described IgM immunoglobulin
IgG immunoglobulin in IgM immunoglobulin in liquid and the first poly- liquid of described IgG immunoglobulin reacts, the product of generation
For IgM- maleimide-IgG, its structure such as formula（6）It is shown,
（6）；
Second step, the described IgM-IgG immunoglobulin polymer dope obtaining is crossed sephadex G 200 post, then with institute
State PBS cocktail buffer eluting, flow velocity is 0.25ml/ minute, be in charge of the solution after collecting eluting, and survey OD280nm light absorption value,
By each pipe solution mixing of the first peak recording, more mixed solution is concentrated into 2mg/ml, this is the IgM- of purification
IgG final polymer product.
3. IgM-IgG polymer according to claim 2 polymerization it is characterised in that：Described IgM-IgG is gathered
The compound end-product glycerol mix homogeneously equal with its volume, then preserves under the conditions of subzero 20 DEG C.
4. IgM-IgG polymer according to claim 2 polymerization it is characterised in that：Described IgM immune globulin
Bai Wei great mouse, described IgG immunoglobulin is globefish mouse, or described IgM immunoglobulin is globefish mouse, described
IgG immunoglobulin is big mouse.
5. a kind of polymerization of IgM-IgG polymer it is characterised in that：It is made up of the following step successively：
The crosslinking of Part I, IgG and S- acetylthio-acetate succinimide ester
The first step, IgG immunoglobulin is dissolved in the IgG immunoglobulin obtaining in PBS cocktail buffer that concentration is 1mg/ml
Buffer, is then charged in bag filter, under the conditions of 2 ~ 8 DEG C, dialyses 8 ~ 15 hours in described PBS cocktail buffer；Wherein,
Described PBS cocktail buffer is that the phosphate buffer that 10mmol/L and pH are 7.4 is molten with the mixing of 0.15mol/L sodium chloride
Liquid；
Second step, the IgG immunoglobulin buffer after dialysing in the removal first step, then to the IgG immune globulin after dialysis
It is slowly added into freshly prepared concentration for the two of 10 ~ 50mmol/L S- acetylthio-acetate succinimide ester in white buffer
Methyl sulfoxide solution, concussion mixes, and reaction at 15 ~ 35 DEG C obtains reactant liquor in 25 ~ 35 minutes；After reaction terminates, reactant liquor is loaded
In bag filter, under the conditions of 2 ~ 8 DEG C, dialyse 8 ~ 15 hours in described PBS cocktail buffer；Wherein, the IgG after described dialysis
IgG immunoglobulin in immunoglobulin buffer is 1 with the mol ratio of described S- acetylthio-acetate succinimide ester:
10~1:30；IgG immunoglobulin and described concentration in IgG immunoglobulin buffer after described dialysis is 10 ~
S- acetylthio-acetate succinyl in the dimethyl sulphoxide solution of 50mmol/L S- acetylthio-acetate succinimide ester is sub-
The product that the reaction of amine ester generates is IgG-SATA, its structure such as formula（7）Shown, obtain IgG immunoglobulin just poly- liquid,
（7）；
3rd step, is slowly added into freshly prepared hydroxylamine solution in the first poly- liquid of IgG immunoglobulin obtaining to second step, mixes
Afterwards, react 1.5 ~ 2.5 hours at 15 ~ 35 DEG C；After reaction terminates, reactant liquor is loaded in bag filter, under the conditions of 2 ~ 8 DEG C,
Dialyse 8 ~ 15 hours in described PBS cocktail buffer, obtain poly- liquid in IgG immunoglobulin；Wherein, described IgG immune globulin
The reaction volume of Bai Chuju liquid and described hydroxylamine solution is than for 10:1；Just poly- liquid is molten with described azanol for described IgG immunoglobulin
There is the reaction of deacylation base in the azanol in liquid, the product of generation is IgG-SH, its structure such as formula（8）It is shown,
（8）；
Part II, IgM and 4- (N- maleimidomehyl) hexamethylene -1- carboxylic acid sulfonic group succinimide ester crosslinking
The first step, IgM immunoglobulin is dissolved in the IgM immunity ball obtaining in described PBS cocktail buffer that concentration is 1mg/ml
Albumen buffer, is then charged in bag filter, under the conditions of 2 ~ 8 DEG C, dialyses 8 ~ 15 hours in described PBS cocktail buffer；
Second step, the IgM immunoglobulin buffer after dialysing in the removal first step, the then IgM immune globulin after dialysis
Be slowly added in white buffer freshly prepared concentration be 1.5mg/ml 4- (N- maleimidomehyl) hexamethylene -1- carboxylic acid
The PBS solution of sulfonic group succinimide ester, concussion mixes, and reaction at 15 ~ 35 DEG C obtains reactant liquor in 25 ~ 35 minutes；Reaction terminates
Afterwards, reactant liquor is loaded in bag filter, under the conditions of 2 ~ 8 DEG C, dialyse 8 ~ 15 hours in described PBS cocktail buffer, obtain
IgM immunoglobulin just poly- liquid；Wherein, the IgM immunoglobulin in the IgM immunoglobulin buffer after described dialysis and institute
State 4- (N- maleimidomehyl) hexamethylene -1- carboxylic acid sulfonic group succinimide ester PBS solution in 4- (N- Malaysia
Acid imide methyl) mol ratio between hexamethylene -1- carboxylic acid sulfonic group succinimide ester is 1:20；
The IgM immunoglobulin in IgM immunoglobulin buffer after described dialysis and described 4- (N- maleimide first
Base) hexamethylene -1- carboxylic acid sulfonic group succinimide ester PBS solution in 4- (N- maleimidomehyl) hexamethylene -1-
The IgM that the product that the reaction of carboxylic acid sulfonic group succinimide ester generates activates for maleimide, its structure such as formula（9）It is shown,
（9）；
Part III, the crosslinking of poly- liquid and the first poly- liquid of IgM immunoglobulin in IgG immunoglobulin
The first step, by poly- liquid in described IgG immunoglobulin, just poly- liquid loads in bag filter and mixes with described IgM immunoglobulin
Even, under the conditions of 2 ~ 8 DEG C, react 1.5 ~ 2.5 hours；Then dialyse 8～15 hours in described PBS cocktail buffer, obtain
IgM-IgG immunoglobulin polymer dope；Wherein, the IgG immunoglobulin in poly- liquid in described IgG immunoglobulin and
The mol ratio of the IgM immunoglobulin in the first poly- liquid of described IgM immunoglobulin is 1:1 ~ 5, poly- in described IgG immunoglobulin
IgM immunoglobulin in IgG immunoglobulin in liquid and the first poly- liquid of described IgM immunoglobulin reacts, the product of generation
For IgG- maleimide-IgM, its structure such as formula（10）It is shown,
（10）；
Second step, the described IgM-IgG immunoglobulin polymer dope obtaining is crossed sephadex G 200 post, then with institute
State PBS cocktail buffer eluting, flow velocity is 0.25ml/ minute, be in charge of the solution after collecting eluting, survey OD280nm light absorption value, will
Each pipe solution mixing of first peak, more mixed solution is concentrated into 2mg/ml, this is the IgM-IgG polymer of purification
End-product.
6. IgM-IgG polymer according to claim 5 polymerization it is characterised in that：Described IgM-IgG is gathered
The compound end-product glycerol mix homogeneously equal with its volume, then preserves under the conditions of subzero 20 DEG C.
7. IgM-IgG polymer according to claim 5 polymerization it is characterised in that：Described IgM immune globulin
Bai Wei great mouse, described IgG immunoglobulin is globefish mouse, or described IgM immunoglobulin is globefish mouse, described
IgG immunoglobulin is big mouse.
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