The neutralizing monoclonal antibody 11F1 and its hybridoma of anti human nerve growth factor are thinBorn of the same parents' strainTechnical field
The present invention relates to field of immunology, more particularly to the monoclonal antibody and its hybridoma of anti human nerve growth factorCell line.
Background technology
Nerve growth factor (NGF) is one kind god for having the function of neurotrophic and promoting enation double biologicalThrough cell growth regulator, its development, differentiation, growth, regeneration and expression of functional characteristic to maincenter and peripheral neuronsIt is respectively provided with important regulating and controlling effect.
Research shows that nerve growth factor (NGF) is a kind of in the generation of hypersensitivity and the generation of allodyniaMediator with key effect.A large amount of preclinical studies show：Prevent the interaction of NGF and its acceptor, you can relieve pain.NGF monoclonal antibodies are neutralized with NGF, block the interaction of NGF and the acceptor in its sensory neuron so as to reach alleviation painThe effect of pain.(anti-nerve growth factor monoclonal antibody Tanezumab. pharmacy progress .2010,34 (1):526.).Have been reported thatDisplay is prevented using Anti-NGF Antibody antagonist or is treated postoperative pain, including from operation or from cuttingOr the pain of traumatic wound.Also have been reported that display anti-ngf antibodies are used to treat various diseases, including asthma, arthritis and silver(Xie Er anti-ngf antibodies of D.L. are used to treat various disease Chinese Patent Application No. bits disease：02814992.0).However, meshThe preceding report to Anti-NGF Antibody is research polyclonal antibody (Zhao little Lin, by the eastern Anti-NGF Antibodies that shake mostlyPrepare and its using China Journal of Neuroscience .2004,20 (2):171-173.).And the poor specificity of polyclonal antibody, useThe background that develops the color when immunohistochemistry is high, and quality is difficult to control, it is difficult to is promoted in clinical practice.Monoclonal antibody has specificityBy force, the advantages of sensitiveness is high, is widely used in clinical detection, can be used in a variety of methods such as immunohistochemistry, ELISA.The research to nerve growth factor monoclonal antibody is not common both at home and abroad.The anti-rhNGF monoclonal antibodies having had been reported that are logicalCross the NGF (antigens using the salmonella thalline adsorption and purification after acid treatment：Thalline 1：5) intrasplenic injection, splenocyte and marrowOncocyte screens after PEG is merged and obtains hybridoma, and the anti-rhNGF monoclonal antibodies to obtaining have carried out preliminary mirror(Su Jin, Zhang Yali, You Changxuan, wait preparation and Preliminary Identification cells and the molecular immune of anti-human NGF monoclonal's antibody to setting analysisLearn magazine .2002,18 (2):168), but without the neutralization activity of confrontation rhNGF monoclonal antibodies identified.Therefore, at presentAlso need to obtain the high anti-human NGF monoclonal's antibody of a kind of high specificity, neutralization activity, alleviation and disease for Clinical PainTreatment.
The content of the invention
According to the demand in above-mentioned field, the present invention provide a kind of anti human nerve growth factor neutralizing monoclonal antibody andIts hybridoma cell strain.
The claimed technical solution of the present invention is as follows：
The neutralizing monoclonal antibody 11F1 of anti human nerve growth factor, by the hybridization that preserving number is CGMCC No.8773Secreted by oncocyte, the neutralizing monoclonal antibody has neutralization reaction effect with growth factor of human nerve, its hypotype of classifying is：Heavy chain IgG1, light chain Kappa.
The hybridoma cell strain of growth factor of human nerve neutralizing monoclonal antibody 11F1 is secreted, its preserving number is CGMCCNo.8773。
This area of research based on to(for) the neutralizing antibody of anti human nerve growth factor, be also claimed above-mentioned antibody withUnder：Pharmaceutical applications：
A kind of analgesic medicine, it is characterised in that：Its effective component includes the neutralizing monoclonal antibody 11F1.
A kind of antiallergy medicament, it is characterised in that：Its effective component includes the neutralizing monoclonal antibody 11F1.
One kind resists antasthmatic medicament, it is characterised in that：Its effective component includes the neutralizing monoclonal antibody11F1.A kind of medicament for being used to treat psoriasis, it is characterised in that：Its effective component includes the neutralizing monoclonal antibody11F1。
A kind of method for the neutralization activity for identifying anti-nerve growth factor antibody, step are as follows：
(1) with maintaining culture medium to prepare antibody samples solution, on the basis of 40 μ g/ml of pre-dilution concentration, carrying out 2 times isRow dilution, totally 9 dilution factors, testing sample solution is transferred in 96 orifice plates, each 3 multiple holes of concentration, per 50 μ l of hole, is selected elseSelect 3 holes and add the culture medium without antibody as negative control, per 100 μ l of hole；
9 dilution factors are respectively：10μg/ml、5μg/ml、2.5μg/ml、1.25μg/ml、0.625μg/ml、0.3125μg/ml、0.15625μg/ml、0.078125μg/ml、0.039063μg/ml；
(2) the another NGF protein solutions for maintaining culture medium to prepare 200ng/ml, add it to containing anti-ngf antibodies sampleIn 96 orifice plates of product solution, per 50 μ l of hole, negative control hole does not add NGF culture mediums, alternative to select 3 holes to add positive controls molten96 orifice plates per 100 μ l of hole, are then positioned over 37 DEG C of incubators and are incubated 1h by liquid 100ng/ml NGF；
(3) amount of taking fully TF-1 cell cultures, are collected by centrifugation TF-1 cells, and PBS is washed 2 times, and the cell being collected by centrifugation is usedMaintain culture medium to be resuspended, be made into every 1ml containing 5 × 104The cell suspension of a cell, is inoculated in 96 porocyte culture plates and containsIn the hole of NGF albumen and anti-ngf antibodies, negative control hole and Positive control wells, per 100 μ l of hole；96 orifice plates are placed in 37 DEG C,5% carbon dioxide culture；After 72h, 20 μ l of MTS solution are added per hole, in 37 DEG C, when 5% carbon dioxide culture 3 is small；
(4) 96 orifice plates are taken out from incubator, are put into microplate reader, absorbance, record measure knot are measured at wavelength 490nmFruit；
(5) by the absorbance of product to be measured, according to formula：The neutralization inhibiting rate (%) in anti-ngf antibodies hole=[(positiveHole OD490nm average values-negative hole OD490nm average values)-(antibody hole OD490nm average values-negative hole OD490nm is averagedValue)]/(positive hole OD490nm average values-negative hole OD490nm average values), the neutralization inhibiting rate per hole is calculated, neutralizes and suppressesRate is higher, then the neutralization activity of anti-ngf antibodies is stronger.
It is demonstrated experimentally that the neutralizing monoclonal antibody of anti human nerve growth factor provided by the invention can be with people's nerve growthFactor-specific combines, and has neutralization reaction activity, therefore can be as the antagonist of hNGF.Grown with existing anti human nerveFactor polyclonal antibody is compared, and monoclonal antibody high specificity provided by the invention, can avoid cross reaction, and provided by the inventionMonoclonal antibody have passed through the identification of neutralization activity, its potency is high, affinity is strong, specificity is good, can occur with growth factor of human nerveObvious neutralization reaction.
The hybridoma cell line of the monoclonal antibody of secretion anti human nerve growth factor, its preservation is also claimed in the present inventionNumber be respectively CGMCC No.8773.
On the other hand, the present invention also provides a kind of method for the neutralization activity for identifying anti-nerve growth factor antibody, instituteIt is TF-1 cell proliferation methods to state method.The present invention, should first using the neutralization activity of TF-1 cell proliferation methods detection anti-ngf antibodiesMethod can accurately and efficiently detect the neutralization activity of anti-ngf antibodies, its principle is：The TF-1 cells that NGF is relied on are in NGFIt can promote TF-1 cell Proliferations under the conditions of albumen is existing, but after it with the addition of certain density anti-ngf antibodies, anti-NGFWith NGF neutralization occurs for antibody, so as to suppress the good growth of TF-1 cells, then by adding MTS mixed solvent cellsColour developing, surveys absorbance in microplate reader at OD490nm, calculate the neutralization inhibiting rate per hole, neutralization inhibiting rate is higher, and anti-NGF resistsThe neutralization activity of body 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：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, 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：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica
Brief description of the drawings
Fig. 1 immunized mice serum titer testing results
The SDS-PAGE identifications of the monoclonal antibody-purified effects of the anti-hNGF of Fig. 2
The Western Blot results of the anti-hNGF monoclonal antibody proteins of Fig. 3
In the anti-hNGF monoclonal antibodies of Fig. 4 and inhibition assay result
Embodiment
The present invention is described in more detail, it is necessary to which explanation is below by specific embodiment, following embodiments are onlyFor explanation and illustration, rather than it limit the invention in any way.
Experiment material：
BALB/c mouse：Purchased from Department Of Medicine, Peking University's Experimental Animal Center.
rhNGF：Recombinant human nerve growth factor, expressed by applicant's unit and purify (happy big, Peng Lujia, history authority,Deng the high efficient expression and biological evaluation research Chinese Pharmaceutical Affairs .2014 of recombinant human nerve growth factors, 28 (6)：601-606.)。
The TF-1-A2 cells that NGF is relied on：By TF-1 cell (Liu of applicant's unit our company domestication rear cloneization screeningIn lotus, find pleasure in big, Huo Lihong, waits subcloned cells strains TF-1-A2 and preparation method thereof and purposes Chinese invention patent grant numbers：ZL 2012 1 0119401.X.)。
Injection mouse nerve growth factor (Soviet Union's peptide life, Beijing SHUTAISHEN pharmaceutcal corporation, Ltd, lot number：201211148)
The sheep anti-mouse igg of AP marks：Purchased from SIGMA companies (Cat.No.:A3562).
The not specified experiment reagent of the present invention is this area conventional reagent, can be by commercially available or using this areaConventional method is prepared, and specification is the pure level of experiment.
The preparation of the anti-hNGF monoclonal antibodies of embodiment 1.
1st, BALB/c mouse is immunized
It is prepared by rhNGF：Voluntarily express, purify and prepare that (happy big, Peng Lujia, history authority, waits recombined humans refreshing by applicantHigh efficient expression and biological evaluation research Chinese Pharmaceutical Affairses .2014 through growth factor, 28 (6)：601-606.).
5 female BAl BIcs/c mouse are chosen, rhNGF antigens are mixed with Freund's complete adjuvant (sigma companies), have been emulsifiedIt is subcutaneously injected after complete, the immunizing dose of every mouse is immunized for 80 μ g.After first immunisation, it is spaced 2 weeks and 3 weeks, by rhNGF antigensRepeat to be immunized twice after being emulsified with not formula Freund's incomplete adjuvant (sigma companies), mouse tail takes blood, with indirect ELISA sideMethod detects the potency (the results are shown in Figure 1) of serum specific antibody, and wherein 2# Mouse titers are up to 1：104, respectively to 1#,2#, 3#, 4# and 5# mouse carry out test for fusion.3 days before fusion, abdominal cavity booster immunization 1 time.
2nd, the preparation of hybridoma
Take the spleen of immune BALB/c mouse to be developed into splenocyte suspension under gnotobasis in super-clean bench, trained with RPMI1640Support base to wash 2 times, collect 1 × 108Splenocyte and 2 × 107-5×107Myeloma cell SP2/0 is mixed in a 50ml fusion pipeIn, RPMI1640 culture mediums are added to 30ml, are fully mixed.1000r/min is centrifuged 10 minutes, and supernatant is exhausted as far as possible.In handPalm touches bottom of fusion pipe, makes sedimentation cell loosely uniform.Added with 1ml suction pipes at 1 minute or so be preheated to 37 DEG C 50%PEG2000 (PH 8.0) 1ml, side edged gently rotate, and naked eyes are visible particle appearance, is slowly added to RPMI1640 culture mediums extremely20ml.1000r/min is centrifuged 6 minutes, supernatant discarding.Cultivated in HAT selective mediums within first 10 days, afterwards until theCloning selects HT medium cultures before completing.It is thin with indirect enzyme-linked immunosorbent assay (ELISA method) detection fusion after 2 weeksThe positive rate of born of the same parents, selects the higher hole of positive value, the positive hybridoma cell detected is cloned through limiting dilution, continuouslyCloning makes for 3 times the positive rate in positive colony hole select the high hole turn hole of value up to after twice 100% and expand culture and freeze.The hybridoma cell strain of the anti-hNGF monoclonal antibodies of stably excreting is obtained, its numbering is 11F1 and 12C11, send preservation, preservationNumber be respectively：CGMCC No.8772 and CGMCC No.8773.
3rd, the preparation of anti-hNGF monoclonal antibodies ascites, titer of ascites measure and antibody purification
Using inducing method largely prepares monoclonal antibody in vivo.Take the healthy Balb/c female mices of 6-8 week old, abdominal cavity notePenetrate paraffin (0.5ml/ is only).After 1 week, hybridoma centrifuge washing, cell number is adjusted to 1 × 10 with PBS6A/ml is every smallMouse injects 0.5ml.After 5~7d, after mouse web portion expands, gather ascites and titer of ascites is detected.After ascites centrifugationSupernatant is collected, with Protein A Sepharose antibody purifications.Packing is after -70 DEG C of preservations.
The anti-hNGF monoclonal antibodies subgroup identification of embodiment 2.
Using the Rapid ELISA Mouse Antibody Isotyping Kit (Cat.No. of Pierce companies:37503) class and subclass of monoclonal antibody are identified, is operated according to kit specification.
The qualification result of the anti-hNGF monoclonal antibodies hypotype of table 1
The anti-hNGF monoclonal antibodies affinity constant measure of embodiment 3.
Using the method for capture antibody, detected with GE companies biomolecular interaction analysis instrument Biacore 3000 anti-The affinity and binding kinetics of hNGF monoclonal antibodies.
2 Biacore of table detects anti-hNGF monoclonal antibodies and the affinity of rhNGF antigens
The specificity identification of the anti-hNGF monoclonal antibodies of embodiment 4.
The specificity of monoclonal antibody is identified using Western Blot.The purifying effect of anti-hNGF monoclonal antibody proteinsFruit identifies (Fig. 2) with SDS-PAGE, with Gel-blot-pro analytical electrophoresis bands the result shows that, antibody purity is not less than90%.SDS-PAGE electrophoresis is carried out to rhNGF and mNGF, after electricity transfer, is incubated with the monoclonal antibody (1 μ g/ml) of preparation, AP marksThe sheep anti-mouse igg (1: 5000 dilution) of note is used as secondary antibody, is operated by the general step of Western Blot.Anti- hNGF monoclonals resistThe Western Blot experimental results of body protein as shown in figure 3, due between the NGF and mouse NGF of people homology be up to89.1%, and there are data to show between people and mouse NGF there are immunological cross-reaction, therefore in the Western qualification processes alsoIdentical result is arrived.There are faint cross reaction with mNGF for anti-hNGF monoclonal antibodies.
The anti-hNGF monoclonal antibodies neutralization activity detection of embodiment 5.
The principle of NGF propagation, the monoclonal antibody of the anti-hNGF of this experiment can be relied on according to the NGF TF-1 cells relied onThe biological activity of rhNGF albumen is neutralized, so as to suppress TF-1 cell Proliferations.
Specific experiment step is as follows：
1) with maintaining culture medium to prepare anti-hNGF monoclonal antibodies sample solution, on the basis of 40 μ g/ml of pre-dilution concentrationOn, carry out 2 times and be serially diluted, totally 9 dilution factors, detected sample solution is transferred in 96 orifice plates, and each concentration 3 is multipleHole, it is alternative to select 3 holes addition negative control solutions (maintenance culture medium) per 50 μ l of hole, per 100 μ l of hole.
2) another with maintaining culture medium to prepare rhNGF protein solutions, concentration 200ng/ml, rhNGF protein solutions are transferred toIn 96 orifice plates containing anti-hNGF monoclonal antibodies sample solution, 3 multiple holes are per 50 μ l of hole.Negative control hole do not add containingThe nutrient solution of rhNGF albumen.It is alternative to select 3 blank wells addition positive control solution rhNGF protein solution 100ng/ml, per hole100μl.After two step end of operation of the above, 96 orifice plates are positioned over 37 DEG C of incubators and are incubated 1h.
3) amount of taking fully TF-1-A2 cell cultures, are collected by centrifugation TF-1-A2 cells, and PBS is resuspended 2 times, and cell is collected by centrifugationAfter be resuspended in and maintain culture medium to be made into every 1ml containing 5 × 104The cell suspension of a cell, is inoculated in 96 porocyte culture plates and containsThere are hole, negative control hole and the Positive control wells of anti-hNGF monoclonal antibodies and rhNGF albumen, per 100 μ l of hole.37 degree are placed in,5% carbon dioxide culture.After 72h, 20 μ l of MTS solution are added per hole, in 37 degree, when 5% carbon dioxide culture 3 is small.From culture96 orifice plates are taken out in case, are put into microplate reader, absorbance is measured at wavelength 490nm, record measurement result.According to formula：It is anti-The neutralization inhibiting rate (%) in hNGF monoclonal antibodies hole=(50ngNGF OD490nm- negative holes value)-(antibody holeOD490nm- negative holes value)/(50ngNGF OD490nm- negative holes value) calculating neutralization inhibiting rate.
The results are shown in Figure 4, with the increase of anti-hNGF MAb concentrations, the neutralization suppression of anti-hNGF monoclonal antibodiesRate processed is also continuously increased, and when antibody reaches a certain concentration, neutralizes inhibiting rate and plateau occurs.As shown in table 3,11F1 andThe neutralization inhibiting rate of 12C11 is up to 100%, shows that anti-hNGF monoclonal antibodies and rhNGF albumen have obvious neutralization reactionEffect.
The anti-hNGF monoclonal antibodies of table 3 are per hole inhibiting rate (%)