CN104774265A - Neutralizing monoclonal antibody 11F1 against human nerve growth factor and hybridoma cell strain thereof - Google Patents
Neutralizing monoclonal antibody 11F1 against human nerve growth factor and hybridoma cell strain thereof Download PDFInfo
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- CN104774265A CN104774265A CN201510073966.2A CN201510073966A CN104774265A CN 104774265 A CN104774265 A CN 104774265A CN 201510073966 A CN201510073966 A CN 201510073966A CN 104774265 A CN104774265 A CN 104774265A
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Abstract
The invention provides a neutralizing monoclonal antibody 11F1 against the human nerve growth factor and a hybridoma cell strain thereof, belonging to the field of immunology. The neutralizing monoclonal antibody against the human nerve growth factor is secreted by a hybridoma cell with an accession number of CGMCC No. 8773, can undergo neutralization reaction with the human nerve growth factor and has the following classification subtypes: heavy-chain IgG1 and light-chain Kappa. The monoclonal antibody has specific neutralizing activity to the human nerve growth factor and can be used as an antagonist against the human nerve growth factor.
Description
Technical field
The present invention relates to field of immunology, relate to monoclonal antibody and the hybridoma cell strain thereof of anti human nerve somatomedin particularly.
Background technology
Nerve growth factor (NGF) is a kind of nerve growth regulatory factor having neurotrophic and promote enation double biological function, and it all has important regulating and controlling effect to the expression of the growth of maincenter and peripheral nerve unit, differentiation, growth, regeneration and functional performance.
Research shows, nerve growth factor (NGF) is a kind of mediator in the generation of allergy and the generation of allodynia with keying action.A large amount of preclinical study shows: the interaction stoping NGF and its acceptor, gets final product alleviating pain.NGF monoclonal antibody and NGF neutralize, and block the interaction of the acceptor in NGF and its Sensory neurone thus reach lenitive effect.(anti-nerve growth factor monoclonal antibody Tanezumab. pharmacy progress .2010,34 (1): 526.).There is report display to adopt Anti-NGF Antibody antagonist prevent or treat postoperative pain, comprise from operation or the pain from cutting or traumatic wound.Also have report display anti-ngf antibodies to be used for the treatment of various disease, comprise asthma, sacroiliitis and psoriatic (D.L. Xie Er pauses. anti-ngf antibodies is used for the treatment of various disease. Chinese Patent Application No.: 02814992.0).But, at present to the report of Anti-NGF Antibody be mostly research polyclonal antibody (Zhao little Lin, by the east that shakes. the preparation of Anti-NGF Antibody and application thereof. Chinese Journal of Neuroscience .2004,20 (2): 171-173.).And the poor specificity of polyclonal antibody, high for the background that develops the color during immunohistochemical methods, quality is wayward, is difficult to promote in clinical application.Monoclonal antibody has high specificity, advantage that susceptibility is high, is widely used, can uses in the multiple method such as immunohistochemical methods, ELISA in clinical detection.Uncommon to the research of nerve growth factor monoclonal antibody both at home and abroad.Now having been reported the anti-rhNGF monoclonal antibody obtained is NGF (antigen: thalline 1:5) intrasplenic injection by adopting the Salmonellas thalline adsorption and purification after acid treatment, splenocyte and myeloma cell screen and obtain hybridoma after PEG merges, and preliminary identification and analysis (Su Jin has been carried out to the anti-rhNGF monoclonal antibody obtained, Zhang Yali, You Changxuan, Deng. the preparation of anti-human NGF monoclonal's antibody and preliminary evaluation. cell and molecular immunology magazine .2002, 18 (2): 168), but the Neutralization effect not resisting rhNGF monoclonal antibody is identified.Therefore, also need to obtain a kind of high specificity, anti-human NGF monoclonal's antibody that Neutralization effect is high at present, for the alleviation of Clinical Pain and the treatment of disease.
Summary of the invention
According to the demand in above-mentioned field, the invention provides a kind of neutralizing monoclonal antibody and hybridoma cell strain thereof of anti human nerve somatomedin.
The technical scheme of request protection of the present invention is as follows:
The neutralizing monoclonal antibody 11F1 of anti human nerve somatomedin, by preserving number secreted by the hybridoma of CGMCC No.8773, described neutralizing monoclonal antibody and growth factor of human nerve have neutralization reaction effect, and its classification hypotype is: heavy chain IgG1, light chain Kappa.
The hybridoma cell strain of secretion growth factor of human nerve neutralizing monoclonal antibody 11F1, its preserving number is CGMCC No.8773.
Based on the research of this area for the neutralizing antibody of anti human nerve somatomedin, also below the above-mentioned antibody of request protection: pharmaceutical applications:
A kind of analgesic medicine, is characterized in that: its effective component comprises described neutralizing monoclonal antibody 11F1.
A kind of antianaphylaxis medicament, is characterized in that: its effective component comprises described neutralizing monoclonal antibody 11F1.
A kind of anti-antasthmatic medicament, is characterized in that: its effective component comprises described neutralizing monoclonal antibody 11F1.One is used for the treatment of psoriatic medicament, it is characterized in that: its effective component comprises described neutralizing monoclonal antibody 11F1.
Identify a method for the Neutralization effect of anti-nerve growth factor antibody, step is as follows:
(1) with maintain basigamy antibody samples solution, on the basis of pre-dilution concentration 40 μ g/ml, carry out 2 times of serial dilutions, totally 9 extent of dilution, testing sample solution is transferred in 96 orifice plates, the multiple hole of each concentration 3, every hole 50 μ l, alternative select 3 holes add not containing the substratum of antibody as negative control, every hole 100 μ l;
Described 9 extent of dilution are respectively: 10 μ g/ml, 5 μ g/ml, 2.5 μ g/ml, 1.25 μ g/ml, 0.625 μ g/ml, 0.3125 μ g/ml, 0.15625 μ g/ml, 0.078125 μ g/ml, 0.039063 μ g/ml;
(2) the another NGF protein solution with maintain basigamy 200ng/ml, be added in 96 orifice plates containing anti-ngf antibodies sample solution, every hole 50 μ l, negative control hole does not add NGF substratum, alternative is selected 3 holes and is added positive control solution 100ng/ml NGF, then 96 orifice plates are positioned over 37 DEG C of incubators and hatch 1h by every hole 100 μ l;
(3) amount of taking fully TF-1 cell culture, collected by centrifugation TF-1 cell, PBS washs 2 times, and the cell of collected by centrifugation hangs with maintain basic weight, is made into every 1ml containing 5 × 10
4the cell suspension of individual cell, is inoculated in the hole containing NGF albumen and anti-ngf antibodies in 96 porocyte culture plates, negative control hole and Positive control wells, every hole 100 μ l; 96 orifice plates are placed in 37 DEG C, and 5% carbonic acid gas is cultivated; After 72h, every hole adds MTS solution 20 μ l, and in 37 DEG C, 5% carbonic acid gas cultivates 3 hours;
(4) from incubator, take out 96 orifice plates, put into microplate reader, measure absorbancy in wavelength 490nm place, record measurement result;
(5) by the absorbancy of goods to be measured, according to formula: with inhibiting rate (%)=[(positive hole OD490nm mean value-negative hole OD490nm mean value)-(antibody hole OD490nm mean value-negative hole OD490nm mean value)]/(positive hole OD490nm mean value-negative hole OD490nm mean value) in anti-ngf antibodies hole, calculate in every hole and inhibiting rate, in and inhibiting rate higher, then the Neutralization effect of anti-ngf antibodies is stronger.
Experiment proves, the neutralizing monoclonal antibody of anti human nerve somatomedin provided by the invention can with growth factor of human nerve specific binding, there is neutralization reaction activity, therefore can as the antagonist of hNGF.Compared with existing anti human nerve somatomedin polyclonal antibody, monoclonal antibody high specificity provided by the invention, cross reaction can be avoided, and monoclonal antibody provided by the invention have passed through the qualification of Neutralization effect, its height of tiring, avidity is strong, specificity good, with growth factor of human nerve, obvious neutralization reaction can occur.
The present invention also asks the hybridoma cell line of the monoclonal antibody protecting secretion anti human nerve somatomedin, and its preserving number is respectively CGMCC No.8773.
On the other hand, the present invention also provides a kind of method identifying the Neutralization effect of anti-nerve growth factor antibody, and described method is TF-1 cell proliferation method.The present invention adopts TF-1 cell proliferation method to detect the Neutralization effect of anti-ngf antibodies first, the method can be accurate, detect the Neutralization effect of anti-ngf antibodies efficiently, its principle is: the TF-1 cell that NGF relies on can promote TF-1 cell proliferation under NGF albumen existent condition, but after with the addition of certain density anti-ngf antibodies, there is neutralizing effect in anti-ngf antibodies and NGF, thus suppress the good growth of TF-1 cell, then by adding the colour developing of MTS mixed solvent cell, in microplate reader, OD490nm place surveys absorbancy, calculate in every hole and inhibiting rate, in and inhibiting rate higher, the Neutralization effect of anti-ngf antibodies is also stronger.
Biological deposits information:
Biomaterial title: 12C11
Classification And Nomenclature: mouse hybridoma cell
Preservation date: on January 16th, 2014
Preserving number: CGMCC No.8772
Preservation mechanism: China Committee for Culture Collection of Microorganisms's common micro-organisms center
Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica
Biomaterial title: 11F1
Classification And Nomenclature: mouse hybridoma cell
Preservation date: on January 16th, 2014
Preserving number: CGMCC No.8773
Preservation mechanism: China Committee for Culture Collection of Microorganisms's common micro-organisms center
Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica
Accompanying drawing explanation
Fig. 1. immunized mice serum titer detected result
Fig. 2. the SDS-PAGE qualification of the monoclonal antibody-purified effect of anti-hNGF
Fig. 3. the Western Blot result of anti-hNGF monoclonal antibody protein
Fig. 4. with Inhibition test result in anti-hNGF monoclonal antibody
Embodiment
Be described in more detail the present invention below by specific embodiment, it should be noted that, following examples only for explaining and illustrating, instead of limit the present invention by any way.
Experiment material:
BALB/c mouse: purchased from Department Of Medicine, Peking University's Experimental Animal Center.
RhNGF: recombinant human nerve growth factor, (pleasure is big, Peng Lujia, history authority to express also purifying by applicant unit, Deng. the high expression of recombinant human nerve growth factor and evaluated biological activity research. Chinese Pharmaceutical Affairs .2014,28 (6): 601-606.).
The TF-1-A2 cell that NGF relies on: by our company of applicant unit tame TF-1 cell that rear cloneization screens (Liu Hezhong, happy big, Huo Lihong, etc. subcloned cells strain TF-1-A2 and preparation method thereof and purposes. Chinese invention patent grant number: ZL 201210119401.X.).
(Soviet Union's peptide is raw, Beijing SHUTAISHEN pharmaceutcal corporation, Ltd, lot number: 201211148) for injection mouse nerve growth factor
The sheep anti-mouse igg of AP mark: purchased from SIGMA company (Cat.No.:A3562).
The not specified experiment reagent of the present invention is this area conventional reagent, and can obtain by being purchased or adopting this area ordinary method preparation, specification is for testing pure level.
The preparation of the anti-hNGF monoclonal antibody of embodiment 1.
1, BALB/c mouse immunity
Prepared by rhNGF: expressed voluntarily by applicant, purifying and prepare (happy big, Peng Lujia, history authority, etc. the high expression of recombinant human nerve growth factor and evaluated biological activity research. Chinese Pharmaceutical Affairs .2014,28 (6): 601-606.).
Choose 5 female BAl BIc/c mouse, mixed by rhNGF antigen with Freund's complete adjuvant (sigma company), subcutaneous injection after emulsification completely, the immunizing dose of every mouse is 80 μ g immunity.After first immunisation, 2 weeks and 3 weeks, interval, by rhNGF antigen with repeats immune twice after not formula Freund's incomplete adjuvant (sigma company) carries out emulsification, mouse tail gets blood, detect tiring (result as shown in Figure 1) of serum specific antibody with indirect ELISA method, wherein 2# Mouse titers can reach 1:10
4, respectively test for fusion is carried out to 1#, 2#, 3#, 4# and 5# mouse.Merge first 3 days, abdominal cavity booster immunization 1 time.
2, the preparation of hybridoma
The spleen getting immune BALB/c mouse in super clean bench under gnotobasis is developed into splenocyte suspension, washs 2 times, collect 1 × 10 with RPMI1640 substratum
8splenocyte and 2 × 10
7-5 × 10
7myeloma cell SP2/0 is mixed in a 50ml fusion pipe, adds RPMI1640 substratum to 30ml, fully mixes.Centrifugal 10 minutes of 1000r/min, by supernatant exhaustion as far as possible.Palm touches at the bottom of fusion pipe, make sedimentation cell evenly loose.Added 50% PEG2000 (PH 8.0) 1ml being preheated to 37 DEG C at about 1 minute with 1ml suction pipe, limit edged rotates gently, and naked eyes have particle to occur as seen, slowly adds RPMI1640 substratum to 20ml.Centrifugal 6 minutes of 1000r/min, supernatant discarded.Within first 10 days, cultivate in HAT selective medium, afterwards until first time cloning selects HT culture medium culturing before completing.The positive rate of 2 weeks rear indirect enzyme-linked immunosorbent assay (ELISA method) detection fusion cells, select the hole that positive value is higher, through limiting dilution, the positive hybridoma cell detected is cloned, after continuous cloning makes for 3 times the positive rate in positive colony hole reach twice 100%, select the high hole of value and turn hole and enlarged culturing frozen.Obtain the hybridoma cell strain of the anti-hNGF monoclonal antibody of stably excreting, it is numbered 11F1 and 12C11, send preservation, and preserving number is respectively: CGMCC No.8772 and CGMCC No.8773.
3, the preparation of anti-hNGF monoclonal antibody ascites, titer of ascites measure and antibody purification
Induce method in employing body and prepare monoclonal antibody in a large number.Get the healthy Balb/c female mice in 6-8 age in week, abdominal injection paraffin (0.5ml/ only).After 1 week, hybridoma centrifuge washing, adjusts cell count to 1 × 10 with PBS
6individual/ml, every injected in mice 0.5ml.After 5 ~ 7d, after mouse web portion expands, gather ascites and titer of ascites is detected.Ascites collected after centrifugation supernatant, with Protein A Sepharose antibody purification.In-70 DEG C of preservations after packing.
Embodiment 2. anti-hNGF monoclonal antibody subgroup identification
Adopt Rapid ELISA Mouse Antibody Isotyping Kit (Cat.No.:37503) of Pierce company to identify class and the subclass of monoclonal antibody, operate according to test kit specification sheets.
The qualification result of table 1 anti-hNGF monoclonal antibody hypotype
Embodiment 3. anti-hNGF monoclonal antibody affinity constant measures
Adopt the method for capture antibody, detect avidity and the binding kinetics of anti-hNGF monoclonal antibody with GE company biomolecular interaction analysis instrument Biacore 3000.
Table 2 Biacore detects the avidity of anti-hNGF monoclonal antibody and rhNGF antigen
The specificity identification of the anti-hNGF monoclonal antibody of embodiment 4.
Western Blot is adopted to identify the specificity of monoclonal antibody.The purification effect SDS-PAGE of anti-hNGF monoclonal antibody protein identifies (Fig. 2), and show by the result of Gel-blot-pro analytical electrophoresis band, antibody purity is all not less than 90%.Carry out SDS-PAGE electrophoresis to rhNGF and mNGF, after electric transfer printing, hatch with the monoclonal antibody (1 μ g/ml) of preparation, the sheep anti-mouse igg (1: 5000 dilution) of AP mark resists as two, operates by the general step of Western Blot.The Western Blot experimental result of anti-hNGF monoclonal antibody protein as shown in Figure 3, because homology between the NGF of people and mouse NGF is up to 89.1%, and have between data reference and mouse NGF and there is immunological cross-reaction, therefore have also been obtained identical result in Western qualification process.There is faint cross reaction in anti-hNGF monoclonal antibody and mNGF.
Embodiment 5. anti-hNGF monoclonal antibody Neutralization effect detects
The TF-1 cell relied on according to NGF can rely on the principle of NGF propagation, and this experiment is with in the monoclonal antibody of anti-hNGF and the biologic activity of rhNGF albumen, thus suppression TF-1 cell proliferation.
Specific experiment step is as follows:
1) with maintain basigamy system anti-hNGF monoclonal antibody sample solution, on the basis of pre-dilution concentration 40 μ g/ml, carry out 2 times of serial dilutions, totally 9 extent of dilution, detected sample solution is transferred in 96 orifice plates, the multiple hole of each concentration 3, every hole 50 μ l, alternative is selected 3 holes and is added negative control solution (maintain base), every hole 100 μ l.
2) another with maintain basigamy rhNGF protein solution, concentration 200ng/ml, is transferred in 96 orifice plates containing anti-hNGF monoclonal antibody sample solution by rhNGF protein solution, 3 multiple every hole 50, hole μ l.Negative control hole does not add the nutrient solution containing rhNGF albumen.Alternative is selected 3 blank well and is added positive control solution rhNGF protein solution 100ng/ml, every hole 100 μ l.After above two step end of operations, 96 orifice plates are positioned over 37 DEG C of incubators and hatch 1h.
3) amount of taking fully TF-1-A2 cell culture, collected by centrifugation TF-1-A2 cell, resuspended 2 times of PBS, is resuspended in maintain basigamy and becomes every 1ml containing 5 × 10 after centrifugal collecting cell
4the cell suspension of individual cell, is inoculated in the hole containing anti-hNGF monoclonal antibody and rhNGF albumen in 96 porocyte culture plates, negative control hole and Positive control wells, every hole 100 μ l.Be placed in 37 degree, 5% carbonic acid gas is cultivated.After 72h, every hole adds MTS solution 20 μ l, and in 37 degree, 5% carbonic acid gas cultivates 3 hours.From incubator, take out 96 orifice plates, put into microplate reader, measure absorbancy in wavelength 490nm place, record measurement result.According to formula: in anti-hNGF monoclonal anti body opening and inhibiting rate (%)=(50ngNGF OD490nm-negative hole value)-(this antibody hole OD490nm-negative hole value)/(50ngNGF OD490nm-negative hole value) calculate in and inhibiting rate.
Result as shown in Figure 4, along with the increase of anti-hNGF MAb concentration, in anti-hNGF monoclonal antibody and inhibiting rate also constantly increase, when antibody reaches finite concentration, in and inhibiting rate there is plateau.As shown in table 3, in 11F1 and 12C11 and inhibiting rate up to 100%, show that anti-hNGF monoclonal antibody and rhNGF albumen have obvious neutralization reaction effect.
Table 3 anti-hNGF monoclonal antibody every hole inhibiting rate (%)
Claims (7)
1. the neutralizing monoclonal antibody 11F1 of anti human nerve somatomedin, by preserving number secreted by the hybridoma of CGMCC No.8773, described neutralizing monoclonal antibody and growth factor of human nerve have neutralization reaction effect, and its classification hypotype is: heavy chain IgG1, light chain Kappa.
2. secrete the hybridoma cell strain of growth factor of human nerve neutralizing monoclonal antibody 11F1, its preserving number is CGMCCNo.8773.
3. an analgesic medicine, is characterized in that: its effective component comprises neutralizing monoclonal antibody 11F1 according to claim 1.
4. an antianaphylaxis medicament, is characterized in that: its effective component comprises neutralizing monoclonal antibody 11F1 according to claim 1.
5. an anti-antasthmatic medicament, is characterized in that: its effective component comprises neutralizing monoclonal antibody 11F1 according to claim 1.
6. be used for the treatment of a psoriatic medicament, it is characterized in that: its effective component comprises neutralizing monoclonal antibody 11F1 according to claim 1.
7. identify a method for the Neutralization effect of anti-nerve growth factor antibody, step is as follows:
(1) with maintain basigamy antibody samples solution, on the basis of pre-dilution concentration 40 μ g/ml, carry out 2 times of serial dilutions, totally 9 extent of dilution, testing sample solution is transferred in 96 orifice plates, the multiple hole of each concentration 3, every hole 50 μ l, alternative select 3 holes add not containing the substratum of antibody as negative control, every hole 100 μ l;
Described 9 extent of dilution are respectively: 10 μ g/ml, 5 μ g/ml, 2.5 μ g/ml, 1.25 μ g/ml, 0.625 μ g/ml, 0.3125 μ g/ml, 0.15625 μ g/ml, 0.078125 μ g/ml, 0.039063 μ g/ml;
(2) the another NGF protein solution with maintain basigamy 200ng/ml, be added in 96 orifice plates containing anti-ngf antibodies sample solution, every hole 50 μ l, negative control hole does not add NGF substratum, alternative is selected 3 holes and is added positive control solution 100ng/ml NGF, then 96 orifice plates are positioned over 37 DEG C of incubators and hatch 1h by every hole 100 μ l;
(3) amount of taking fully TF-1 cell culture, collected by centrifugation TF-1 cell, PBS washs 2 times, and the cell of collected by centrifugation hangs with maintain basic weight, is made into every 1ml containing 5 × 10
4the cell suspension of individual cell, is inoculated in the hole containing NGF albumen and anti-ngf antibodies in 96 porocyte culture plates, negative control hole and Positive control wells, every hole 100 μ l; 96 orifice plates are placed in 37 DEG C, and 5% carbonic acid gas is cultivated; After 72h, every hole adds MTS solution 20 μ l, and in 37 DEG C, 5% carbonic acid gas cultivates 3 hours;
(4) from incubator, take out 96 orifice plates, put into microplate reader, measure absorbancy in wavelength 490nm place, record measurement result;
(5) by the absorbancy of goods to be measured, according to formula: with inhibiting rate (%)=[(positive hole OD490nm mean value-negative hole OD490nm mean value)-(antibody hole OD490nm mean value-negative hole OD490nm mean value)]/(positive hole OD490nm mean value-negative hole OD490nm mean value) in anti-ngf antibodies hole, calculate in every hole and inhibiting rate, in and inhibiting rate higher, then the Neutralization effect of anti-ngf antibodies is stronger.
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| WO2021238886A1 (en) * | 2020-05-27 | 2021-12-02 | Staidson (Beijing) Biopharmaceuticals Co., Ltd. | Antibodies specifically recognizing nerve growth factor and uses thereof |
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| CN101827609A (en) * | 2007-08-10 | 2010-09-08 | 里珍纳龙药品有限公司 | High affinity human antibodies to human nerve growth factor |
| CN102660505A (en) * | 2012-04-20 | 2012-09-12 | 北京华安科创生物技术有限公司 | Subcloned cell strain TF-1-A2, preparation method thereof and application |
| CN103764677A (en) * | 2011-05-06 | 2014-04-30 | Nvip私人有限公司 | Anti-nerve growth factor antibodies and methods of preparing and using the same |
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| CN1849138A (en) * | 2003-07-15 | 2006-10-18 | 安姆根有限公司 | Human anti-NGF neutralizing antibodies as selective ngf pathway inhibitors |
| CN1790024A (en) * | 2004-12-16 | 2006-06-21 | 浙江永宁制药厂 | Protein content determination method for nerve growth factor and application thereof |
| CN101827609A (en) * | 2007-08-10 | 2010-09-08 | 里珍纳龙药品有限公司 | High affinity human antibodies to human nerve growth factor |
| CN103764677A (en) * | 2011-05-06 | 2014-04-30 | Nvip私人有限公司 | Anti-nerve growth factor antibodies and methods of preparing and using the same |
| CN102660505A (en) * | 2012-04-20 | 2012-09-12 | 北京华安科创生物技术有限公司 | Subcloned cell strain TF-1-A2, preparation method thereof and application |
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| WO2021238886A1 (en) * | 2020-05-27 | 2021-12-02 | Staidson (Beijing) Biopharmaceuticals Co., Ltd. | Antibodies specifically recognizing nerve growth factor and uses thereof |
| TWI796700B (en) * | 2020-05-27 | 2023-03-21 | 大陸商舒泰神（北京）生物製藥股份有限公司 | Antibodies specifically recognizing nerve growth factor and uses thereof |
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