CN107118277A - A kind of monoclonal antibody - Google Patents
A kind of monoclonal antibody Download PDFInfo
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- CN107118277A CN107118277A CN201710565929.2A CN201710565929A CN107118277A CN 107118277 A CN107118277 A CN 107118277A CN 201710565929 A CN201710565929 A CN 201710565929A CN 107118277 A CN107118277 A CN 107118277A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/36—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood coagulation factors
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/745—Blood coagulation or fibrinolysis factors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/745—Assays involving non-enzymic blood coagulation factors
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Abstract
The present invention relates to biological technical field, more particularly to a kind of monoclonal antibody.Monoclonal antibody 3G3 combinations FVII relies on the presence of calcium ion.3G3 antibody subtypes are identified using the antibody subtype identification test strips of Roche companies, it is IgG1, Kappa types as a result to confirm 3G3 monoclonal antibodies hypotype.The Sepharose 4B being coupled using monoclonal antibody 3G3 can successful purification recombinant human blood coagulation factor VII, destination protein molecular weight is shown as 60kDa, and expected consistent.Determine the affinity costant of 3G3 monoclonal antibodies.As a result show that monoclonal antibody 3G3 and clotting factor FVII that the present invention is provided affinity are preferable.
Description
Technical field
The present invention relates to biological technical field, more particularly to a kind of monoclonal antibody.
Background technology
Coagulation cascade reaction is the key link of hemostasis.When coagulation pathway existing defects, the bleeding time
(bleeding time) extends, amount of bleeding increase.Clotting factor is the protein molecule for participating in coagulation process, under physiological conditions,
Most clotting factor exist with inactive zymogen forms, when tissue damage, and these clotting factor are turned by enzymolysis activation in succession
Active protease is turned to, final conversion of prothrombin is fibrin ferment, be changed into soluble fibrinogen insoluble
Fibrin agglomerate.
Proconvertin belongs to the clotting factor of vitamin K dependence, is synthesized by liver, its main function is and tissue factor
Compound is formed, so as to start exogenous cruor pathway.
Hemophilia A and the hereditary hemorrhagic disease that hemophilia B is X-linkage, respectively with blood coagulation factor VIII
(FVIII) it is characterized with IX (FIX) shortages.This kind of patient is faced with the danger bled profusely after wound with hematostaxis.Generation
Boundary's health organization statistics shows that the incidence of disease of hemophilia A is about 1/5000 newborn boy baby, the incidence of disease about 1/20000 of hemophilia B
Newborn boy baby.Haemophiliachemophiliac traditional treatment mode is clotting factor supplementary therapy, i.e. venoclysis blood plasma source or genetic recombination
FVIII or FIX, it is abnormal to correct clotting factor, reduces hemorrhagic tendency.But, the severe Hemophilia A patients that there are about 25% produce
FVIII mortifiers (antibody), the severe hemophilia B patient that there are about 4% produces FIX mortifiers, treats clotting factor supplementary therapy
Effect reduction is even invalid.Many patients die from uncontrollable bleeding；Because caused by bleeding the problem of severe joint damage still
It is so extremely serious；How to stop blooding when being performed the operation to the patient for having mortifier, especially one special problem.FVIIa can lead to
Cross alternative pathway and bypass coagulation pathway dependent on FVIII and FIX, activation FX produces FXa, promotes substantial amounts of conversion of prothrombin
Into fibrin ferment, formed " fibrin ferment outburst ".Restructuring FVIIa is used to treat the congenital blood friend that there are FVIII or FIX mortifiers
The spontaneous or surgical bleeding of patient, congenital factor VII lacks patient.
Human blood coagulation factor VII antibody can be used for recombinant blood coagulation factor VII affinitive layer purification and detection.However, general
Antibody-mediated affinity chromatography need to be de- using pickling, and one of resulting influence is to change the Sample Buffer in purifying process
Liquid, complicates purifying process；In addition, low pH very likely influences the activity of sample.And the antibody that calcium ion is relied on can be in original
Have and calcium ion chelator EDTA is added in sample buffer separate antigen-antibody, it is to avoid the de- above-mentioned unfavorable shadow brought of pickling
Ring.Therefore, in the urgent need to the anti-human coagulation factor VII monoclonal antibodies of independent development calcium ion dependence.
The content of the invention
In view of this, the present invention provides a kind of monoclonal antibody.The monoclonal antibody can be tied with proconvertin specificity
Close, with preferable affinity.
In order to realize foregoing invention purpose, the present invention provides following technical scheme：
The monoclonal antibody of the specific binding proconvertin relied on the invention provides a kind of calcium ion, by preservation
Numbering is CCTCC No：C2012158 hybridoma cell strain is produced.
The preparation method of monoclonal antibody of the present invention is as follows：With the recombined human activated clotting factor VII of commercialization
(NovoSeven) emulsification is sufficiently mixed after the subcutaneous multi-point injection of Balb/c mouse backs, 4 with isometric Freund's complete adjuvant
NovoSeven and the subcutaneous multi-point injection booster immunization one of mouse back after isometric incomplete Freund's adjuvant mixing and emulsifying are taken after week
It is secondary, the immune impact of NovoSeven mouse tail vein injections is taken after 8 weeks, spleen is taken out after 4 days, isolates spleen cell, with mouse
Myeloma cell SP2/0 is with 10:1 cell number ratio mixing, centrifugation is carried out after addition fusion agent PEG1500 in cell precipitation
Fusion, is transferred in 96 orifice plates and is screened with the selective medium containing 1%HAT, change liquid every 2 days double later, takes within 10-14 days thin
Born of the same parents' culture supernatant carries out ELISA detection screenings.
The anti-FVII antibody relied on for screening calcium ion, NovoSeven (1 μ g/ml) coated elisa plate is previously added
Containing/the TBS without 10mM EDTA, cells and supernatant is then added, 37 DEG C are incubated 1 hour, TBST (0.1%tween-20)
The sheep anti-mouse igg secondary antibody of horseradish peroxidase-labeled is added after washing 5 times, 37 DEG C are incubated 1 hour, TBST (0.1%tween-
20) tmb substrate colour developing, 1N sulfuric acid terminating reactions, ELIASA reading OD450 are added after washing 5 times.Obtain one plant without
EDTA supernatant combination strong positive and add the hybridoma 3G3 (table 1, Fig. 1) that 10mM EDTA significantly inhibit antibody binding.
As a result show that 3G3 antibody bindings FVII relies on the presence of calcium ion；And another plant of hybridoma 3D7 is in the presence of EDTA
Remain to be combined with FVII, illustrate 3D7 combinations FVII independent of calcium ion.And then limiting dilution is used to 3G3 hybridomas
Method carries out monoclonal, will freeze conservation after 3G3 cell expansion cultures.
The purifying of monoclonal antibody：5-7 days before injection hybridoma, Pristane0.5ml/ is injected intraperitoneally in advance
Only, 3G3 cells are taken to be injected intraperitoneally, 10-14 days pneumoretroperitoneum donor source mouse ascites were placed for 4 DEG C after 37 DEG C of placement half an hour
Night.Centrifugation in second day removes blood clot, is added in ascites after the phosphate buffer of 3 times of volumes, centrifugation, supernatant membrane filtration
Purified with protein G posts.
The identification of monoclonal antibody：3G3 antibody subtypes are reflected using the antibody subtype identification test strips of Roche companies
Fixed, it is IgG1, Kappa types (Fig. 2) as a result to confirm 3G3 monoclonal antibodies hypotype.
Present invention also offers a kind of hybridoma cell strain, its deposit number is CCTCC No：C2012158.
Present invention also offers described monoclonal antibody answering in the instrument for purifying blood coagulation factor Ⅴ II is prepared
With.In some specific embodiments of the present invention, the purifying is nucleophilic chromatographic purifying.
Present invention also offers a kind of tools for purification, including described monoclonal antibody.Some in the present invention are specific real
Apply in scheme, the purifying is nucleophilic chromatographic purifying.
Prepared present invention also offers described monoclonal antibody for detecting in the detection instrument of proconvertin
Using.In some specific embodiments of the present invention, the detection instrument is kit, chip or test paper.
In addition, present invention also offers a kind of detection instrument, including described monoclonal antibody.
In some specific embodiments of the present invention, the detection instrument is kit, chip or test paper.
The invention provides a kind of hybridoma cell strain, (deposit number is CCTCC No：C2012158), it is and thus miscellaneous
The monoclonal antibody 3G3 for handing over tumor cell strain to produce.Prepared present invention also offers monoclonal antibody for the purifying blood coagulation factor
Application in VII instrument and its preparing the application in being used to detect the detection instrument of proconvertin, contain the Dan Ke
The detection instrument of grand antibody.The monoclonal antibody that the present invention is provided
3G3 combinations FVII relies on the presence of calcium ion.
3G3 antibody subtypes are identified using the antibody subtype identification test strips of Roche companies, as a result confirm that 3G3 is mono-
Clonal antibody hypotype is IgG1, Kappa types.
The monoclonal antibody immobilization provided by the present invention is used for purifying blood coagulation factor Ⅴ II, as a result shows, using Dan Ke
The Sepharose 4B of grand antibody 3G3 coupling can successful purification recombinant human blood coagulation factor VII, destination protein molecular weight is shown as
60kDa, with being expected unanimously.
It is coated on the recombinant blood coagulation factor FVII (NovoSeven) of commercialization on bio-sensing chip, uses Biacore
The affinity costant of X100 protein-interacting system measurement 3G3 monoclonal antibodies.As a result the affinity costant for showing 3G3 antibody is
1.3×10-9M.Show that the affinity for the monoclonal antibody 3G3 and clotting factor FVII that the present invention is provided is very good.
When the monoclonal antibody that the present invention is provided is used for purifying blood coagulation factor FVII, the monoclonal relied on due to calcium ion
Antibody 3G3 can add calcium ion chelator EDTA in original sample buffer separates antigen-antibody, it is to avoid sour elution band
The adverse effect come.
Biological deposits explanation
Hybridoma cell strain Anti-F7-3G3：Classification And Nomenclature：Hybridoma cell strain Anti-F7-3G3, in November, 2012
China typical culture collection center (CCTCC) is deposited within 8th, collection address is that Hubei Province Wuchang city Luo Jia Shan Wuhan is big
In school, deposit number is CCTCC No.C2012158.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
There is the accompanying drawing used required in technology description to be briefly described.
Fig. 1 shows the anti-human FVII antibody that Elisa screening calcium ions are relied on；
Fig. 2 shows that 3G3 antibody subtypes are identified；
Fig. 3 shows the recombinant blood coagulation factor VII of coomassie brilliant blue staining identification affinitive layer purification after SDS-PAGE electrophoresis；
NR:Non reducing conditions；R：Reducing condition；
Fig. 4 shows the affinity costant of 3G3 monoclonal antibodies；Wherein, line 1 shows 150nM；Line 2 shows 75nM；Line 3 shows 37.5nM；Line
4 show 18.75nM；Line 5 shows 9.375nM.
Embodiment
The invention discloses a kind of monoclonal antibody, those skilled in the art can use for reference present disclosure, be suitably modified work
Skill parameter is realized.In particular, all similar replacements and change be aobvious and easy for a person skilled in the art
See, they are considered as being included in the present invention.The method of the present invention and application are described by preferred embodiment,
Related personnel substantially can not departing from present invention, method described herein and application are modified in spirit and scope or
Suitably change with combining, to realize and apply the technology of the present invention.
Raw materials used and reagent can be purchased by market in each specific embodiment for the monoclonal antibody that the present invention is provided
.
With reference to embodiment, the present invention is expanded on further：
The animal immune of embodiment 1 and hybridoma screening
With recombined human activated clotting factor VII (NovoSeven) the 100 μ g and isometric Freund's complete adjuvant of commercialization
Emulsification is sufficiently mixed after the subcutaneous multi-point injection of Balb/c mouse backs, 100 μ gNovoSeven and isometric Freund are taken after 4 weeks
The subcutaneous multi-point injection booster immunization of mouse back once, takes 100 μ gNovoSeven small after Freund's incomplete adjuvant mixing and emulsifying after 8 weeks
The immune impact of tail vein injection, takes out spleen after 4 days, isolates spleen cell, with murine myeloma cell SP2/0 with 10:1
Cell number ratio mixing, centrifugation after in cell precipitation add 1ml fusion agents PEG1500 merged, be transferred in 96 orifice plates
Screened with the selective medium containing 1%HAT, change liquid every 2 days double later, take within 10-14 days cells and supernatant to carry out
ELISA detection screenings.The anti-FVII antibody relied on for screening calcium ion, NovoSeven (1 μ g/ml) coated elisa plate, in advance
50 μ l are first added containing/the TBS without 10mM EDTA, 50 μ l cells and supernatants are then added, 37 DEG C are incubated 1 hour, TBST
(0.1%tween-20) adds the sheep anti-mouse igg secondary antibody of horseradish peroxidase-labeled after washing 5 times, 37 DEG C are incubated 1 hour,
TBST (0.1%tween-20) adds tmb substrate colour developing, 1N sulfuric acid terminating reactions, ELIASA reading OD450 after washing 5 times.Obtain
Obtain one plant and add the hybridoma that 10mM EDTA significantly inhibit antibody binding in the supernatant combination strong positive without EDTA
3G3 (table 1, Fig. 1), as a result shows that 3G3 antibody bindings FVII relies on the presence of calcium ion；And another plant of hybridoma 3D7 is in EDTA
In the presence of remain to be combined with FVII, illustrate 3D7 combinations FVII independent of calcium ion.And then have to the use of 3G3 hybridomas
Limit dilution method and carry out monoclonal, conservation will be frozen after 3G3 cell expansion cultures.
The EDTA of table 1 suppresses 3G3 antibody binding proconvertins
| Group | OD450 |
| 3G3 | 2.144 |
| 3D7 | 2.238 |
| 3G3+EDTA | 0.173 |
| 3D7+EDTA | 2.231 |
The purifying of the anti-human coagulation factor VII monoclonal antibodies of embodiment 2
5-7 days before injection hybridoma, intraperitoneal injection Pristane 0.5ml/ only, take 5 × 10 in advance63G3 cells
Intraperitoneal injection, 10-14 days pneumoretroperitoneum donor source mouse ascites, 4 DEG C stand overnight after 37 DEG C of placement half an hour.Centrifuge within second day
Blood clot is removed, the phosphate buffer of 3 times of volumes is added in ascites, 13000rpm is centrifuged 30 minutes, and supernatant is with 0.4 μm of filter membrane
Purified after filtering with protein G posts.
The antibody subtype of embodiment 3 is identified
3G3 antibody subtypes are identified using the antibody subtype identification test strips of Roche companies, as a result confirm that 3G3 is mono-
Clonal antibody hypotype is IgG1, Kappa types (Fig. 2).
The antibody immobilization of embodiment 4 is used for purifying blood coagulation factor Ⅴ II
3G3 monoclonal antibodies are coupled the preparation of Sepharose 4B gels：Weigh 0.268g cyanogen bromide-activateds
Sepharose 4B dry glues, add the 1mM salt acid-swellable of 10ml precoolings 30 minutes, and centrifuged deposit adds the 1mM of 15ml precoolings
Salt acid elution, centrifugation, gel washed once with coupling buffer, add 6mg 3G3 monoclonal antibodies, and room temperature gently mixes reaction
2 hours, free 3G3 antibody was washed in centrifugation with coupling buffer, is then closed with 0.1MTris-Hcl (pH8.0), room temperature reaction
1 hour.Successively with low pH lavation buffer solutions (0.1M sodium acetates, 0.5M sodium chloride, pH3.5) and high pH lavation buffer solutions (0.1M
Sodium acid carbonate, 0.5M sodium chloride, pH8.3) washing, post is filled after cyclic washing 4 times.4 DEG C of preservations.
Affinitive layer purification recombinant blood coagulation factor VII:Recombinant blood coagulation factor VII Chinese hamster ovary celI is transfected, 24 hours after transfection
The serum free medium CD-CHO (Invitrogen companies) containing 10 μ g/ml vitamin K1s is changed, after continuously cultivating 7 days, in collection
Clearly, high speed centrifugation removes insoluble granule, and 0.22 μm of filtering is added in 3G3-Sepharose 4B gel columns, washing is removed not
With reference to impurity protein, destination protein elutes with the TBS buffer solutions of the EDTA containing 10mM, collects eluted product, often pipe collects 2ml,
5 are collected altogether to manage.Eluted product adds isometric reduced form sample-loading buffer, boils and is cooled down after 5 minutes, loading, through 10%SDS-
PAGE electrophoresis, coomassie brilliant blue staining decolourizes, and observes clotting factor purification effect.As a result：It can be seen that application 3G3 monoclonal antibodies
The Sepharose 4B of coupling can successful purification recombinant human blood coagulation factor VII, destination protein molecular weight is shown as 60kDa, and pre-
Phase is consistent (Fig. 3).
The monoclonal antibody 3G3 affinity for the FVII that the calcium ion of embodiment 5 is relied on is determined
It is coated on the recombinant blood coagulation factor FVII (NovoSeven) of commercialization on bio-sensing chip, uses Biacore
The affinity costant of X100 protein-interacting system measurement 3G3 monoclonal antibodies.
Experiment material：
CM5 chips：GE, article No. BR100012；
Running buffer：10mM HEPES, 150mMNaCl, 5mMEDTANa2,0.05%P20, pH7.4；
Amino coupled kit：GE, article No.：2060499；
Sample：3G3 monoclonal antibodies and each 1 of recombinant human blood coagulation factor VII (NovoSeven).
Experimental method：
1st, it is coupled
Optimize coupling condition, slope according to isoelectric point and according to BiacoreX100control soft protocol
Optimum choice sodium acetate is used as coupling dilution buffer.NovoSeven samples are diluted with this buffer solution to being coupled to after 25 μ g/ml
On CM5 chips.The default horizontal 1500RU of coupling.
2nd, KD is tested
Dilute 3G3 monoclonal antibodies with pH7.4 Running buffer, dilute a series of concentration to 0nM, 9.375nM,
18.75nM、37.5nM、75nM、150nM.Setting binding time is 180s, and Dissociation time 15min, regeneration buffer is used
50mMGly-HCl(pH1.7).Examination with computer is carried out according to BiacoreX100control soft protocol.
3rd, data processing
Analysis software is carried using biacore X100 to be analyzed.Data are understood as follows：
Ka：Binding constant, reaction bonded speed degree, numerical value is bigger, and combination is faster.
Kd：Dissociation constant, reaction dissociation speed degree, the smaller dissociation of numerical value is slower.
KD：KD=Kd/Ka, affinity constant, reacting antigen antibody binding firmness, numerical value is smaller, and affinity is stronger.
Experimental result：
1st, it is coupled
According to the isoelectric point characteristic of antigen and slope is combined, final choice pH4.5 sodium acetates are used as coupling dilution buffer
Liquid.Coupling level is 1500RU.
2nd, affinity result：See Fig. 4.As a result the affinity costant for showing 3G3 antibody is 1.3 × 10-9M。
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of monoclonal antibody for the specific binding proconvertin that calcium ion is relied on, is CCTCC No by deposit number：
C2012158 hybridoma cell strain is produced.
2. a kind of hybridoma cell strain, its deposit number is CCTCC No：C2012158.
3. application of the monoclonal antibody as claimed in claim 1 in the instrument for purifying blood coagulation factor Ⅴ II is prepared.
4. application according to claim 3, it is characterised in that the purifying is nucleophilic chromatographic purifying.
5. a kind of tools for purification, it is characterised in that including monoclonal antibody as claimed in claim 1.
6. tools for purification according to claim 5, it is characterised in that the purifying is nucleophilic chromatographic purifying.
7. monoclonal antibody as claimed in claim 1 is preparing the application in being used to detect the detection instrument of proconvertin.
8. application according to claim 7, it is characterised in that the detection instrument is kit, chip or test paper.
9. a kind of detection instrument, it is characterised in that including monoclonal antibody as claimed in claim 1.
10. detection instrument according to claim 9, it is characterised in that it is kit, chip or test paper.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110314414A (en) * | 2019-07-23 | 2019-10-11 | 宝锐生物科技泰州有限公司 | Immune affinity chromatographic column filler, immune affinity chromatographic column, the isolation and purification method of factor XIII and its application |
| CN116375859A (en) * | 2023-06-05 | 2023-07-04 | 北京大学第一医院 | anti-vWF/PF 4 protein monoclonal antibody and application thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110314414A (en) * | 2019-07-23 | 2019-10-11 | 宝锐生物科技泰州有限公司 | Immune affinity chromatographic column filler, immune affinity chromatographic column, the isolation and purification method of factor XIII and its application |
| CN110314414B (en) * | 2019-07-23 | 2021-10-19 | 宝锐生物科技泰州有限公司 | Separation and purification method of blood coagulation factor XIII and application thereof |
| CN116375859A (en) * | 2023-06-05 | 2023-07-04 | 北京大学第一医院 | anti-vWF/PF 4 protein monoclonal antibody and application thereof |
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| CN107118277B (en) | 2020-12-04 |
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