CN109942707A - A kind of monoclonal antibody of anti-human NT-proBNP polypeptide - Google Patents
A kind of monoclonal antibody of anti-human NT-proBNP polypeptide Download PDFInfo
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Abstract
The invention discloses a kind of monoclonal antibody of anti-human NT-proBNP polypeptide, including selecting NT-proBNP antigen determinant polypeptide segment aa1 and aa2, preparing NT-proBNP antigen determinant polypeptide segment aa1 and aa2, the monoclonal antibody that forms coupling protein aa1-KLH-aa2, use coupling protein aa1-KLH-aa2 as antigen-immunized animal and prepare the anti-human NT-proBNP polypeptide.Coupling protein prepared by production process of the invention has good protein surface activity and antigenicity, convenient for filtering out the good monoclonal antibody of specificity, coupling protein in the present invention can increase the quantity of antigenic determinant, to increase the type of monoclonal antibody generation, and a possibility that improving the pairing of later period monoclonal antibody, preparation process of the invention is simple, affinity of antibody is high.The present invention has a vast market foreground.
Description
Technical field
The present invention relates to antibody engineering technical fields, and in particular to a kind of monoclonal antibody of anti-human NT-proBNP polypeptide.
Background technique
With the raising of living condition and medical condition, social senilization's phenomenon is more and more significant, heart failure patient number
Increase year by year.Heart failure (Heart Failure) is the clinic of a kind of sign generated by heart dysfunction and symptom
Syndrome.Although some drugs can delay, sb.'s illness took a turn for the worse, unless carrying out heart transplant, otherwise heart failure is can not to cure
's.Heart failure early diagnosis is the most critical issue that current health care faces, more early to start to treat, and is more had to patient
Benefit.It is difficult to (especially in heart failure early stage) however, heart failure to make a definite diagnosis.The classical symptom of heart failure, such as fatigue, breathing are anxious
Rush, ankle swelling etc. do not have specificity, and the slight patient of some state of an illness does not also show classical symptom.Primary medical care machine
Structure relies only on clinical criteria as diagnosis basis, and the false positive rate of diagnosis of heart failure may be up to 50%.Current diagnosis heart failure is most
Commonly using effective method mainly has echocardiogram, radionuclide imaging and heart nuclear magnetic resonance check；But daily practical diagnosis
When left ventricular function failure, above-mentioned detection methods are that can trust completely, and have good reproducibility without one.Cause
This, is clinically badly in need of a kind of objective, simple, scientific fast diagnosis method, improves the diagnosis of patients with heart failure.Research shows that brain
Sodium peptide (Brain natriuretic peptide, BNP) and amino-terminal brain natriuretic peptide precursor (N-terminal pro-brain
Natriuretic peptide, NT-proBNP) concentration raising for heart failure diagnosis have very high sensibility,
And the energy evaluation of cardiac function extent of damage, it is the very effective biomarker of diagnosis of heart failure.
BNP is a kind of peptide hormone, is mainly secreted by ventricle, has sharp sodium, expansion blood vessel, inhibits endothelium tight
The effect of Zhang Su-aldosterone system.This substance of studies have shown that has the diagnosis of chronic heart failure and cardiac dysfunction
There is good directive significance.BNP is formed by way of proteolysis by preceding brain natriuretic peptide first (preproBNP).
PreproBNP is the polypeptide chain synthesized in cardiac muscle cell, includes 134 amino acid residues, when 1-26 signal peptides are cut
Fall the brain natriuretic peptide (proBNP) that rear just formation includes 108 amino acid residues.Pro-BNP has been cracked into when ventricle is stimulated
Active BNP (32 amino acid) and inactive NT-proBNP (76 amino acid).NT-proBNP and BNP equal proportion is released
It puts, the two has similar clinical application in terms of the diagnosis of disease of cardiovascular system, treatment, monitoring and prognosis.
The biological half-life of NT-proBNP is 60-120 minutes, biological half-life of 20 minutes compared to BNP will long several times,
And the elevation amplitude of NT-proBNP is noticeably greater than BNP when heart failure, thus be more conducive to detect.In addition, NT-proBNP exists
It is highly stable in serum and blood plasma, without pre-processing to sample, transmission and the processing side of Conventional blood sample can be used
Method.Under normal circumstances, NT-proBNP content is almost without making a variation in the daytime, thus can sample detection at any time；But also do not suffered from
The influence of person's position and moving situation.Blood plasma NT-proBNP level is aggravated with degree of heart failure and is increased.The adult blood of the right side of fifty
450 pg/mL of NT-proBNP concentration is starched, the sensibility and specificity for diagnosing acute heart failure is respectively 93% and 95%；50 years old with
On 900 pg/mL of human plasma, the sensibility and specificity for diagnosing heart failure is respectively 91% and 80%.
By comparing with New York Heart association (New York heart association, NYHA) cardiac functional grading, NT-
ProBNP more can really reflect the variation of heart function.NT-proBNP level is related to the severity of heart failure, horizontal higher
Lesion is more serious, and prognosis is also poorer.Meanwhile NT-proBNP is conducive in early stage or the slight stage discovery heart failure of lesion.
It is increasing with heart failure case, carry out and reinforce NT-proBNP detection for diagnosing and preventing and treating mental and physical efforts
Failure has very important significance.Correlative study and report, WO93/24531 had been carried out for the identification of NT-proBNP
(US5,786,163) polyclonal antibody and the detection side that can combine NT-proBNP47-64 amino acids are disclosed
Method,
Hunt, P.J. etc. disclose the measuring method for 1-13 amino acids.In addition there are be directed to other different aminoacids
The measuring method of section, but these methods all exist less reproducible, sample pretreating method is complicated, characterization or/and clinically relevant
The problems such as property is poor.Therefore, it is necessary to more effective antibody and method for detecting specificity to solve the above problems.
Summary of the invention
The purpose of the present invention is overcoming the drawbacks described above of the prior art, provide it is a kind of it is reproducible, sample pretreatment is simple,
It characterizes or/and clinical correlation is preferable, effect is good, the monoclonal antibody of anti-human NT-proBNP polypeptide at low cost.
To realize the above-mentioned technical purpose, the present invention provides a kind of monoclonal antibodies of anti-human NT-proBNP polypeptide, special
Sign is that the monoclonal antibody of the anti-human NT-proBNP polypeptide is prepared by the inclusion of the method for following steps:
1) selects NT-proBNP antigen determinant polypeptide segment aa1 and aa2, and the aa1 is shown in SEQ ID NO.1
Sequence, that is, ETSGLQEQRNHL, the aa2 are sequence i.e. IRGHRKMVLYTLRAPR shown in SEQ ID NO.2；
2) preparation step 1) described in NT-proBNP antigen determinant polypeptide segment aa1 and aa2；
3) polypeptide fragment aa1, aa2 and KLH carrier protein that obtains step 2) connects, and forms coupling protein
aa1-KLH-aa2；
4) the coupling protein aa1-KLH-aa2 that uses step 3) to obtain is prepared described anti-human as antigen-immunized animal
The monoclonal antibody of NT-proBNP polypeptide；
Wherein, the step 4) method particularly includes: Balb/c mouse is immunized with the coupling protein aa1-KLH-aa2,
Obtain it is immune after mouse boosting cell and merged with SP2/0 cell, will cell strain intraperitoneal injection that fusion obtains be immunized it is small
Mouse, collect purifying after ascites be prepared anti-human NT-proBNP polypeptide monoclonal it is anti-.
Beneficial effects of the present invention include at least: 1. coupling protein prepared by production process of the invention has good
Protein surface activity and antigenicity, convenient for filtering out the good monoclonal antibody of specificity；2. the coupling protein energy in the present invention
Increase the quantity of antigenic determinant, to increase the type of monoclonal antibody generation, and improves the pairing of later period monoclonal antibody
Possibility；3. preparation process of the invention is simple, affinity of antibody is high.
Detailed description of the invention
Fig. 1 is that antibody prepared by the present invention and industry generally acknowledge certain preferable import brand agent effective comparison diagram.
Specific embodiment
Now in conjunction with the embodiment and attached drawing technical solution that the present invention will be described in detail.It should be understood that following embodiment and attached drawing are only
For illustrating the present invention rather than limiting the scope of the invention.Without departing from the spirit and scope of the present invention, this hair
Bright to will also have various changes and improvements, these changes and improvements are within the scope of the present invention.
Unless otherwise specified, the conventional means that technological means used in embodiment is well known to those skilled in the art.
Experimental material used in following embodiment have HMP resin (P- hydroxymethyl phenoxy methyl poly vinyl),
FmocAA (amino acid of 9- fluorenylmethoxycarbonyl carbonyl acyl group protection), MeoH (methanol), DCM (methylene chloride), DMAP (dimethylamino
Pyridine), Piperidine (piperidines), DCC (dicyclohexylcarbodiimide), HOBT (hydroxybenzotriazole), NMP (N-methyl pyrrole
Pyrrolidone), EDT (1,2- dithioglycol), TFA (trifluoroacetic acid), Thioanisole (thioanisole), Phenol,
Crystalline (crystalline phenol), Acetonitrile (acetonitrile), paraffin, KLH carrier protein, EDC, NT-proBNP standard
Product；
Major experimental instrument used in following embodiment has polypeptide automatic synthesizer, Rotary Evaporators, high performance liquid chromatography
Instrument, freeze drier, refrigerated centrifuge, carbon dioxide incubator, sample injector, thermostat water bath, fall at protein G is affine layer
Set microscope, Biohazard Safety Equipment, microplate reader, Sephadex G-25 chromatographic column, PH instrument, assay balance, ultra low temperature freezer, liquid nitrogen
Tank, generic centrifuge, chromatograph Waters 600, people's NT-proBNP enzyme linked immunological kit.
Embodiment 1
1) selects NT-proBNP antigen determinant polypeptide segment aa1 and aa2:
The albumen knot of early period is carried out to people's NT-proBNP full length amino acid sequence using DNAstar (Genetool) software
Structure and Characterization of antigenic epitopes choose two polypeptide fragments aa1 and aa2, the aa1 is SEQ ID NO.1 institute according to optimal conditions
The sequence shown, the aa2 are sequences shown in SEQ ID NO.2.Sequence SEQ ID NO.1, sequence SEQ ID NO.2 are as follows:
Sequence SEQ ID NO.1:ETSGLQEQRNHL
Sequence SEQ ID NO.2:IRGHRKMVLYTLRAPR
2) prepares NT-proBNP antigen determinant polypeptide segment aa1 and aa2:
HMP resin 100mg is weighed, in the reaction chamber of polypeptide automatic synthesizer, by synthesizer automatically by specific amino
Acid links up in a different order, and Conjugate ratio is greater than 99%.Reaction step is as follows:
1. the activation (HOBt/DCC method) of amino acid: 0.15mol Fmoc-Gln and 0.15mol HOBt is taken, with appropriate DMF
Dissolution；0.15molDCC separately is taken, is slowly added under stirring, is stirred to react in room temperature environment 30 minutes, is activated after filtering
Amino acid solution afterwards, it is spare；
2. on connection amino acid to resin: amino acid solution and resin carrier after making activation under the action of coupling reagent
(Wang resin) carries out esterification, and the reaction time is 100 minutes, obtains peptide resin 1；
3. sloughing the Fomc protecting group of amino acid: selecting PIP/DMF (piperidines/N,N-dimethylformamide) mixed solution de-
Except Fomc protecting group, piperidines concentration of volume percent is 15% in mixed solution, remaining is DMF.Elution time is 20 minutes.It goes
The dosage of protecting group reagent is every 0.05 mol peptide resin 1200mL.
4. activation of amino acid (HOBt/DCC method): taking 0.15mol amino acid and 0.15mol HOBt, dissolved with appropriate DMF；
0.15molDCC separately is taken, is slowly added into, is stirred to react in room temperature environment 30 minutes, the ammonia after being activated after filtering under stirring
Base acid solution, it is spare；
5. coupling: the amino acid solution after activation being added in the peptide resin 1 of Fmoc, coupling reaction 100 minutes, instead
It answers terminal to be subject to ninhydrin method to detect, filtration washing obtains peptide resin 2；
6. repeating (3)~(5) until synthesis terminates；
7. peptide resin: peptide chain is cut with TFA (trifluoroacetic acid), uses EDT, thioanisole, H2O as scavenger,
In room temperature reaction 2 hours, cutting reagent is removed, is extracted with ether, obtains the crude product of NT-proB00NP polypeptide.
8. peptide purification
It is as follows using high-efficient liquid phase chromatogram purification method:
C810 × 100mm chromatographic column and chromatograph is selected to carry out purification work；Mobile phase A is that concentration of volume percent is
0.1% TFA, B are that the concentration of volume percent that the acetonitrile for being 60% with concentration of volume percent is solvent preparation is 0.1%
TFA；Detection wavelength is set as 214nm；Flow velocity is set as 4ml/min；Gradient is set as 20%-60%B；Elution time is set
It is set to 30min.
It is as follows using HPLC (high performance liquid chromatography) analysis method:
C184.6 × 150mm chromatographic column and chromatograph is selected to be analyzed；Mobile phase A is that concentration of volume percent is
0.1% TFA, B are that the concentration of volume percent that the acetonitrile for being 60% with concentration of volume percent is solvent preparation is 0.1%
TFA；Detection wavelength is set as 214nm；Flow velocity is set as 1ml/min；Gradient is set as 0%-60%B；Elution time is set
It is set to 30 min；Two kinds of polypeptide fragments analyze 95% or more purity as the result is shown.
3) connects polypeptide fragment aa1, aa2 and KLH carrier protein, forms coupling protein aa1-KLH-aa2:
The resulting NT-proBNP antigen determinant polypeptide segment aa1 of step 2) is connect with aa2 with KLH carrier protein, shape
At coupling protein aa1-KLH-aa2.Steps are as follows for albumen coupling:
1. weighing the KLH with required connecting peptides equal quality, it is dissolved in PH7.2 PBS buffer solution, 8 mg/ of final concentration
ml；
2. solution is dialysed 7 hours for 6 DEG C in PH7.2 PBS buffer solution, a buffer is changed in centre；
3. again dialyses solution 2 hours at room temperature in connection buffer, then go in clean container；
4. NT-proBNP antigen determinant polypeptide segment aa1 and aa2 to be connected with distilled water are dissolved to 10mg/ml；
5. 1:1 in mass ratio mixes NT-proBNP antigen determinant polypeptide segment aa1 with aa2 with KLH solution, formed
Mixed liquor；
6. 1.0mgEDC is added in every 1ml mixed liquor, react at room temperature 2 hours.
7. mixture is dialysed 7 hours for 6 DEG C in PH7.2 PBS buffer solution, buffer three times is changed therebetween.
8. solution is taken out, concentration is calculated, packing freezes；
4) the coupling protein aa1-KLH-aa2 that uses step 3) to obtain is prepared described special as antigen-immunized animal
The monoclonal antibody of property anti-human NT-proBNP polypeptide:
1. animal immune: by coupling protein and Freund's adjuvant mixed in equal amounts 3 Balb/c mouse of fully emulsified rear injection, often
25 μ g of mouse is immunized 3 times, interval time 15 days altogether.
2. cell fusion, selectivity culture, screening: first 3 days of fusion, mouse peritoneal are injected without 25 μ g of adjuvant coupling protein,
The spleen cell for taking out immune mouse, by 2.0 × 107A SP2/0 myeloma cell and 2.0 × 108It is a through step (1) be immunized
The splenocyte of Balb/c mouse mixes, centrifugation, abandons supernatant, and slight oscillatory mixes, and in 37 DEG C of water-baths, 1ml is added dropwise in 90 seconds
Then 20ml DMEM culture medium is added dropwise in the PEG-1500 aqueous solution that volumetric concentration is 50%, supernatant is abandoned in centrifugation, then washes once,
Centrifugation abandons supernatant, obtains hybridoma.Hybridoma is selected in 96 porocyte culture plates using HAT Selective agar medium,
Total fusion rate > 95% is detected under mirror.The supernatant in monoclonal cell hole is selected to be detected under mirror, the hole of picking OD450 > 0.8
Inner cell is subcloned, the final cell clone obtained to NT-proBNP reacting positive rate > 99%, anti-human as secreting
The hybridoma cell strain of NT-proBNP monoclonal antibody.
3. cell clone: being cloned to the positive cell strain of acquisition, using limiting dilution assay, clone 3 times, finally obtain
8 plants of anti-human NT-proBNP monoclonal antibody hybridoma cell line of high-titer are generated, culture is expanded, places and is frozen in liquid nitrogen container.
4. prepared by ascites: 6-8 week old female Balb/c mouse takes hybridoma with 2 × 106 with after Treating Cuttings with Paraffin Wax 10 days
A cell/be only injected intraperitoneally.The ascites for being rich in antibody is obtained after 10 days from mouse peritoneal, measures titer of ascites > 105.
5. Purification: using protein G affinity chromatography.Protein G affinity column is prepared first, uses PBS
It after balancing pillar, takes ascites to cross column, then cleans pillar with PBS, until OD value is close to zero, with the glycinate of 50 nmol/L
Acid salt solution elution, collects eluent, measures the OD value of each collecting pipe, retain the eluent of peak region, eluent is after dialysing
It collects.It is the monoclonal antibody of purifying through SDS-PAGE electroresis appraisal, purity is 98.5% or more.
Embodiment 2
1) selects NT-proBNP antigen determinant polypeptide segment aa1 and aa2, and method is the same as embodiment 1；
2) prepares NT-proBNP antigen determinant polypeptide segment aa1 and aa2:
HMP resin 100mg is weighed, in the reaction chamber of polypeptide automatic synthesizer, by synthesizer automatically by specific amino
Acid links up in a different order, and Conjugate ratio is greater than 99%.Reaction step is as follows:
1. the activation (HOBt/DCC method) of amino acid: 0.15mol Fmoc-Ile and 0.15mol HOBt is taken, with appropriate DMF
Dissolution；0.15molDCC separately is taken, is slowly added under stirring, is stirred to react in room temperature environment 50 minutes, is activated after filtering
Amino acid solution afterwards, it is spare；
2. on connection amino acid to resin: amino acid solution and resin carrier after making activation under the action of coupling reagent
(Wang resin) carries out esterification, and the reaction time is 250 minutes, obtains peptide resin 1；
3. sloughing the Fomc protecting group of amino acid: selecting PIP/DMF (piperidines/N,N-dimethylformamide) mixed solution de-
Except Fomc protecting group, piperidines concentration of volume percent is 25% in mixed solution, remaining is DMF.Elution time is 50 minutes.It goes
The dosage of protecting group reagent is every 0.05 mol peptide resin 1800mL.
4. activation of amino acid (HOBt/DCC method): taking 0.15mol amino acid and 0.15mol HOBt, dissolved with appropriate DMF；
0.15molDCC separately is taken, is slowly added into, is stirred to react in room temperature environment 50 minutes, the ammonia after being activated after filtering under stirring
Base acid solution, it is spare；
5. coupling: the amino acid solution after activation being added in the peptide resin 1 of Fmoc, coupling reaction 250 minutes, instead
It answers terminal to be subject to ninhydrin method to detect, filtration washing obtains peptide resin 2；
6. repeating (3)~(5) until synthesis terminates.
7. peptide resin: peptide chain is cut with TFA (trifluoroacetic acid), uses EDT, thioanisole, H2O as scavenger,
In room temperature reaction 4 hours, cutting reagent is removed, is extracted with ether, obtains the crude product of NT-proB00NP polypeptide.
8. peptide purification
High-efficient liquid phase chromatogram purification method, HPLC analytical method are same as Example 1.
3) connects polypeptide fragment aa1, aa2 and KLH carrier protein, forms coupling protein aa1-KLH-aa2:
The resulting NT-proBNP antigen determinant polypeptide segment aa1 of step 2) is connect with aa2 with KLH carrier protein, shape
At coupling protein aa1-KLH-aa2.Steps are as follows for albumen coupling:
1. weighing the KLH with required connecting peptides equal quality, it is dissolved in PH7.2 PBS buffer solution, 8 mg/ of final concentration
ml；
2. solution is dialysed 9 hours for 6 DEG C in PH7.2 PBS buffer solution, a buffer is changed in centre；
3. solution is dialysed 2 hours at room temperature in connection buffer again, then go in clean container；
4. NT-proBNP antigen determinant polypeptide segment aa1 and aa2 to be connected with distilled water are dissolved to 10mg/ml；
5. 1:1 in mass ratio mixes NT-proBNP antigen determinant polypeptide segment aa1 with aa2 with KLH solution, formed
Mixed liquor；
6. 1.0mgEDC is added in every 1ml mixed liquor, react at room temperature 4 hours.
7. mixture is dialysed 9 hours for 6 DEG C in PH7.2 PBS buffer solution, buffer three times is changed therebetween.
8. solution is taken out, concentration is calculated, packing freezes；
4) uses coupling protein aa1-KLH-aa2 as antigen-immunized animal and prepares the specific anti-human NT-proBNP and is more
The monoclonal antibody of peptide:
1. animal immune: by coupling protein and Freund's adjuvant mixed in equal amounts 3 Balb/c mouse of fully emulsified rear injection, often
25 μ g of mouse is immunized 3 times, interval time 15 days altogether.
2. cell fusion, selectivity culture, screening: first 3 days of fusion, mouse peritoneal are injected without 25 μ g of adjuvant coupling protein,
The spleen cell for taking out immune mouse, by 2.0 × 107A SP2/0 myeloma cell and 2.0 × 108It is a through step (1) be immunized
The splenocyte of Balb/c mouse mixes, centrifugation, abandons supernatant, and slight oscillatory mixes, and in 37 DEG C of water-baths, 1ml is added dropwise in 90 seconds
Then 20ml DMEM culture medium is added dropwise in the PEG-1500 aqueous solution that volumetric concentration is 50%, supernatant is abandoned in centrifugation, then washes once,
Centrifugation abandons supernatant, obtains hybridoma.Hybridoma is selected in 96 porocyte culture plates using HAT Selective agar medium,
Total fusion rate > 95% is detected under mirror.The supernatant in monoclonal cell hole is selected to be detected under mirror, the hole of picking OD450 > 0.8
Inner cell is subcloned, the final cell clone obtained to NT-proBNP reacting positive rate > 99%, anti-human as secreting
The hybridoma cell strain of NT-proBNP monoclonal antibody.
3. cell clone: being cloned to the positive cell strain of acquisition, using limiting dilution assay, clone 3 times, finally obtain
8 plants of anti-human NT-proBNP monoclonal antibody hybridoma cell line of high-titer are generated, culture is expanded, places and is frozen in liquid nitrogen container.
4. prepared by ascites: 6-8 week old female Balb/c mouse takes hybridoma with 2 × 10 with after Treating Cuttings with Paraffin Wax 10 days6
A cell/be only injected intraperitoneally.The ascites for being rich in antibody is obtained after 10 days from mouse peritoneal, measures titer of ascites > 105.
5. Purification: using protein G affinity chromatography.Protein G affinity column is prepared first, uses PBS
It after balancing pillar, takes ascites to cross column, then cleans pillar with PBS, until OD value is close to zero, with the glycinate of 50 nmol/L
Acid salt solution elution, collects eluent, measures the OD value of each collecting pipe, retain the eluent of peak region, eluent is after dialysing
It collects.It is the monoclonal antibody of purifying through SDS-PAGE electroresis appraisal, purity is 98.5% or more.
Embodiment 3
1) selects NT-proBNP antigen determinant polypeptide segment aa1 and aa2, and method is same as Example 1；
2) prepares NT-proBNP antigen determinant polypeptide segment aa1 and aa2；
HMP resin 100mg is weighed, in the reaction chamber of polypeptide automatic synthesizer, by synthesizer automatically by specific amino
Acid links up in a different order, and Conjugate ratio is greater than 99%.Reaction step is as follows:
1. the activation (HOBt/DCC method) of amino acid: 0.15mol Fmoc-Gln and 0.15mol HOBt is taken, with appropriate DMF
Dissolution；0.15molDCC separately is taken, is slowly added under stirring, is stirred to react in room temperature environment 40 minutes, is activated after filtering
Amino acid solution afterwards, it is spare；
2. on connection amino acid to resin: amino acid solution and resin carrier after making activation under the action of coupling reagent
(Wang resin) carries out esterification, and the reaction time is 200 minutes, obtains peptide resin 1；
3. sloughing the Fomc protecting group of amino acid: selecting PIP/DMF (piperidines/N,N-dimethylformamide) mixed solution de-
Except Fomc protecting group, piperidines concentration of volume percent is 20% in mixed solution, remaining is DMF.Elution time is 30 minutes.It goes
The dosage of protecting group reagent is every 0.05 mol peptide resin 1500ml.
4. activation of amino acid (HOBt/DCC method): taking 0.15mol amino acid and 0.15mol HOBt, dissolved with appropriate DMF；
0.15molDCC separately is taken, is slowly added into, is stirred to react in room temperature environment 40 minutes, the ammonia after being activated after filtering under stirring
Base acid solution, it is spare；
5. coupling: the amino acid solution after activation being added in the peptide resin 1 of Fmoc, coupling reaction 200 minutes, instead
It answers terminal to be subject to ninhydrin method to detect, filtration washing obtains peptide resin 2；
6. repeating (3)~(5) until synthesis terminates.
7. peptide resin: peptide chain is cut with TFA (trifluoroacetic acid), uses EDT, thioanisole, H2O as scavenger,
In room temperature reaction 3 hours, cutting reagent is removed, is extracted with ether, obtains the crude product of NT-proB00NP polypeptide.
8. peptide purification: high-efficient liquid phase chromatogram purification method, HPLC analytical method are same as Example 1；
3) connects polypeptide fragment aa1, aa2 and KLH carrier protein, forms coupling protein aa1-KLH-aa2:
The resulting NT-proBNP antigen determinant polypeptide segment aa1 of step 2) is connect with aa2 with KLH carrier protein, shape
At coupling protein aa1-KLH-aa2.Steps are as follows for albumen coupling:
1. weighing the KLH with required connecting peptides equal quality, it is dissolved in PH7.2 PBS buffer solution, 8 mg/ of final concentration
ml；
2. solution is dialysed 8 hours for 6 DEG C in PH7.2 PBS buffer solution, a buffer is changed in centre；
3. solution is dialysed 2 hours at room temperature in connection buffer again, then go in clean container；
4. NT-proBNP antigen determinant polypeptide segment aa1 and aa2 to be connected with distilled water are dissolved to 10mg/ml；
5. 1:1 in mass ratio mixes NT-proBNP antigen determinant polypeptide segment aa1 with aa2 with KLH solution, formed
Mixed liquor；
6. 1.0mgEDC is added in every 1ml mixed liquor, react at room temperature 3 hours.
7. mixture is dialysed 8 hours for 6 DEG C in PH7.2 PBS buffer solution, buffer three times is changed therebetween.
8. solution is taken out, concentration is calculated, packing freezes；
4) uses coupling protein aa1-KLH-aa2 as antigen-immunized animal and prepares the specific anti-human NT-proBNP and is more
The monoclonal antibody of peptide；
1. animal immune: by coupling protein and Freund's adjuvant mixed in equal amounts 3 Balb/c mouse of fully emulsified rear injection, often
25 μ g of mouse is immunized 3 times, interval time 15 days altogether.
2. cell fusion, selectivity culture, screening: first 3 days of fusion, mouse peritoneal are injected without 25 μ g of adjuvant coupling protein,
The spleen cell for taking out immune mouse, by 2.0 × 107A SP2/0 myeloma cell and 2.0 × 108It is a through step (1) be immunized
The splenocyte of Balb/c mouse mixes, centrifugation, abandons supernatant, and slight oscillatory mixes, and in 37 DEG C of water-baths, 1ml is added dropwise in 90 seconds
Then 20ml DMEM culture medium is added dropwise in the PEG-1500 aqueous solution that volumetric concentration is 50%, supernatant is abandoned in centrifugation, then washes once,
Centrifugation abandons supernatant, obtains hybridoma.Hybridoma is selected in 96 porocyte culture plates using HAT Selective agar medium,
Total fusion rate > 95% is detected under mirror.The supernatant in monoclonal cell hole is selected to be detected under mirror, the hole of picking OD450 > 0.8
Inner cell is subcloned, the final cell clone obtained to NT-proBNP reacting positive rate > 99%, anti-human as secreting
The hybridoma cell strain of NT-proBNP monoclonal antibody.
3. cell clone: being cloned to the positive cell strain of acquisition, using limiting dilution assay, clone 3 times, finally obtain
8 plants of anti-human NT-proBNP monoclonal antibody hybridoma cell line of high-titer are generated, culture is expanded, places and is frozen in liquid nitrogen container.
4. prepared by ascites: 6-8 week old female Balb/c mouse takes hybridoma with 2 × 10 with after Treating Cuttings with Paraffin Wax 10 days6
A cell/be only injected intraperitoneally.The ascites for being rich in antibody is obtained after 10 days from mouse peritoneal, measures titer of ascites > 105
(105)。
5. Purification: using protein G affinity chromatography.Protein G affinity column is prepared first, uses PBS
It after balancing pillar, takes ascites to cross column, then cleans pillar with PBS, until OD value is close to zero, with the glycinate of 50 nmol/L
Acid salt solution elution, collects eluent, measures the OD value of each collecting pipe, retain the eluent of peak region, eluent is after dialysing
It collects.It is the monoclonal antibody of purifying through SDS-PAGE electroresis appraisal, purity is 98.5% or more.
Embodiment 4
Detection model of the monoclonal antibody of the anti-human NT-proBNP polypeptide prepared using embodiment 1 in chemiluminescence platform
It encloses for 5~35000pg/mL, the antibody and industry generally acknowledge certain preferable import brand reagent 220 parts of clinical serums of testing number simultaneously
Sample, testing result show that antibody prepared by the present invention and the brand correlation are R2=0.9593 (Fig. 1).
Sequence table
<110>calm and peaceful auspicious biotechnology (Wuhan) Co., Ltd is begged
<120>a kind of monoclonal antibody of anti-human NT-proBNP polypeptide
<141> 2019-04-10
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 12
<212> PRT
<213>homo sapiens (Homo sapiens)
<400> 1
Glu Thr Ser Gly Leu Gln Glu Gln Arg Asn His Leu
1 5 10
<210> 2
<211> 16
<212> PRT
<213>homo sapiens (Homo sapiens)
<400> 2
Ile Arg Gly His Arg Lys Met Val Leu Tyr Thr Leu Arg Ala Pro Arg
1 5 10 15
Claims (1)
1. a kind of monoclonal antibody of anti-human NT-proBNP polypeptide, it is characterised in that the monoclonal of the anti-human NT-proBNP polypeptide
Antibody is prepared by the inclusion of the method for following steps:
1) selects NT-proBNP antigen determinant polypeptide segment aa1 and aa2, and the aa1 is sequence shown in SEQ ID NO.1
That is ETSGLQEQRNHL, the aa2 are sequence i.e. IRGHRKMVLYTLRAPR shown in SEQ ID NO.2；
2) preparation step 1) described in NT-proBNP antigen determinant polypeptide segment aa1 and aa2；
3) polypeptide fragment aa1, aa2 and KLH carrier protein that obtains step 2) connects, and forms coupling protein aa1-
KLH-aa2；
4) the coupling protein aa1-KLH-aa2 that uses step 3) to obtain prepares the anti-human NT- as antigen-immunized animal
The monoclonal antibody of proBNP polypeptide；
Wherein, the step 4) method particularly includes: Balb/c mouse is immunized with the coupling protein aa1-KLH-aa2, obtains
Mouse boosting cell after immune is simultaneously merged with SP2/0 cell, and immune mouse is injected intraperitoneally in the cell strain that fusion is obtained, and receives
The monoclonal that anti-human NT-proBNP polypeptide is prepared in purifying after collection ascites resists.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112592398A (en) * | 2021-02-07 | 2021-04-02 | 天津奇云诺德生物医学有限公司 | BNP antigenic determinant polypeptide and application thereof |
| CN112707964A (en) * | 2019-10-25 | 2021-04-27 | 东莞市朋志生物科技有限公司 | Recombinant antibody for resisting N-terminal brain natriuretic peptide precursor |
| CN112745390A (en) * | 2019-10-29 | 2021-05-04 | 东莞市朋志生物科技有限公司 | Binding protein containing NT-proBNP antigen binding structural domain |
| CN112759639A (en) * | 2020-12-31 | 2021-05-07 | 重庆中元汇吉生物技术有限公司 | N-terminal brain natriuretic peptide precursor polypeptide, antibody and kit |
| CN113248590A (en) * | 2021-06-24 | 2021-08-13 | 天津奇云诺德生物医学有限公司 | NT-proBNP protein antigenic determinant polypeptide and application thereof |
| US11609230B2 (en) | 2019-08-13 | 2023-03-21 | Gentian As | Highly sensitive particle enhanced assay for the quantification of NT-proBNP |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040096439A1 (en) * | 2002-11-18 | 2004-05-20 | Debold Adolfo J. | Monoclonal antibodies against N-Terminus proBNP |
| CN103087191A (en) * | 2012-12-26 | 2013-05-08 | 天健生物制药（天津）有限公司 | Monoclonal antibody of anti-human NT-proBNP polypeptide and application thereof |
| CN103304665A (en) * | 2013-05-21 | 2013-09-18 | 上海贝西生物科技有限公司 | NT-proBNP (Amino terminal pro-brain natriuretic peptide precursor) resistant monoclonal antibody and application thereof |
| CN106916225A (en) * | 2017-03-23 | 2017-07-04 | 暨南大学 | A kind of monoclonal antibody for detecting N-terminal plasma pro-brain natriuretic peptide levels and its hybridoma cell strain and application |
-
2019
- 2019-04-17 CN CN201910307614.7A patent/CN109942707A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040096439A1 (en) * | 2002-11-18 | 2004-05-20 | Debold Adolfo J. | Monoclonal antibodies against N-Terminus proBNP |
| CN103087191A (en) * | 2012-12-26 | 2013-05-08 | 天健生物制药（天津）有限公司 | Monoclonal antibody of anti-human NT-proBNP polypeptide and application thereof |
| CN103304665A (en) * | 2013-05-21 | 2013-09-18 | 上海贝西生物科技有限公司 | NT-proBNP (Amino terminal pro-brain natriuretic peptide precursor) resistant monoclonal antibody and application thereof |
| CN106916225A (en) * | 2017-03-23 | 2017-07-04 | 暨南大学 | A kind of monoclonal antibody for detecting N-terminal plasma pro-brain natriuretic peptide levels and its hybridoma cell strain and application |
Non-Patent Citations (3)
| Title |
|---|
| MICHAEL D. WILKINS等: "Quantum dot enabled lateral flow immunoassay for detection of cardiac biomarker NT-proBNP", 《SENSING AND BIO-SENSING RESEARCH》 * |
| NO: "UniProtKB - P16860 (ANFB_HUMAN)", 《UNIPROT》 * |
| 刘鹏展等: "NT-proBNP特异性单克隆抗体的制备及鉴定", 《生物技术通报》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11609230B2 (en) | 2019-08-13 | 2023-03-21 | Gentian As | Highly sensitive particle enhanced assay for the quantification of NT-proBNP |
| CN112707964A (en) * | 2019-10-25 | 2021-04-27 | 东莞市朋志生物科技有限公司 | Recombinant antibody for resisting N-terminal brain natriuretic peptide precursor |
| CN112707964B (en) * | 2019-10-25 | 2022-11-08 | 东莞市朋志生物科技有限公司 | Recombinant antibody for resisting N-terminal brain natriuretic peptide precursor |
| CN112745390A (en) * | 2019-10-29 | 2021-05-04 | 东莞市朋志生物科技有限公司 | Binding protein containing NT-proBNP antigen binding structural domain |
| CN112759639A (en) * | 2020-12-31 | 2021-05-07 | 重庆中元汇吉生物技术有限公司 | N-terminal brain natriuretic peptide precursor polypeptide, antibody and kit |
| CN112592398A (en) * | 2021-02-07 | 2021-04-02 | 天津奇云诺德生物医学有限公司 | BNP antigenic determinant polypeptide and application thereof |
| CN113248590A (en) * | 2021-06-24 | 2021-08-13 | 天津奇云诺德生物医学有限公司 | NT-proBNP protein antigenic determinant polypeptide and application thereof |
| CN113248590B (en) * | 2021-06-24 | 2021-09-10 | 天津奇云诺德生物医学有限公司 | NT-proBNP protein antigenic determinant polypeptide and application thereof |
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Application publication date: 20190628 |