CN102827279B - Anti-neuroepithelial stem cell protein monoclonal antibody with high titer and high specificity and application thereof - Google Patents
Anti-neuroepithelial stem cell protein monoclonal antibody with high titer and high specificity and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of bioengineering, and discloses an anti-neuroepithelial stem cell protein monoclonal antibody with high titer and high specificity and application of the anti-neuroepithelial stem cell protein monoclonal antibody. According to the monoclonal antibody with high titer and high specificity, a heavy chain variable region amino acid sequence is shown in SEQ ID NO.1; and a light chain variable region amino acid sequence is shown in SEQ ID NO.2, so that the titer and the specificity of the monoclonal antibody are high. Because of uniformity of a monoclonal antibody and oneness of biological activity, the quality of an antigen antibody reaction result is conveniently controlled, the standardization and the normalization are facilitated, and the monoclonal antibody can be directly applied to clinical detection and basic research and has a high application value in the fields of biology and medical research.
Description
Technical field
The present invention relates to technical field of bioengineering, particularly a kind of high-titer, high specific anti human nerve nidogen monoclonal antibody and application thereof.
Background technology
People's neural nest albumen is called for short Nestin, is a moderate fiber skelemin, belongs to VI class intermediate filament protein, and the instantaneity because of it in the development of central nervous system process is expressed and is cloned, and is used as the specific sign of neural stem cell always.Along with the very fast development of molecular biology and gene therapy technology, adopt Neural Stem Cells ' Transplantation Spinal injury (SCI) to be acknowledged as up-to-date, one of the most promising methods for the treatment of.Because domestic and international scientific research institution is placed on main energy vitro culture, amplification, the transplanting aspect of neural stem cell, therefore, set up the neural stem cell detection system particularly important.Anti-human Nestin monoclonal antibody is identified the most special method to human nerve stem cell, meets the molecular immunology principle fully, and still, the anti human nerve nidogen monoclonal antibody of high-titer, high specific has not yet to see report.
Summary of the invention
The object of the present invention is to provide a kind of high-titer, high specific anti human nerve nidogen monoclonal antibody and application thereof.
Overall technology design of the present invention is: the specificity screening technology of utilizing cytogamy, subclone and many cases Various Tissues and cell, obtain high-titer, high specific anti human nerve nidogen monoclonal antibody, because tire height, specificity of this monoclonal antibody is good, can directly apply to basic medical research, or make multiple external diagnosis reagent case and carry out the evaluation of neural stem cell and the therapeutic evaluation after neural stem cells transplantation.
Anti human nerve nidogen monoclonal antibody of the present invention, its weight chain variable region amino acid sequence is as shown in SEQ ID NO.1, and its light chain variable region amino acid sequence is as shown in SEQ ID NO.2; Or its variable region of heavy chain by the aminoacid sequence shown in SEQ ID NO.1 through replacing, disappearance or add one or several amino acid derived aminoacid sequence with SEQ ID NO.1 and have 95% consistence at least, variable region of light chain by the aminoacid sequence shown in SEQ ID NO.2 through replacing, disappearance or add one or several amino acid derived aminoacid sequence with SEQ ID NO.2 and have at least 95% consistence and described monoclonal antibody to there is the activity of specific binding Nestin.
As preferably, described monoclonal antibody comprises derivative, function equivalent and the homologue of single-chain antibody, double-chain antibody, chimeric antibody, humanized antibody and above-mentioned antibody, also comprises antibody fragment and any polypeptide that contains the antigen binding domains.
" antibody " of the present invention should be interpreted as containing any specific binding factor with required specific binding domains.Thereby function equivalent and the homologue of the antibody fragment of homology, derivative, humanized antibody and antibody with it contained in this term, also comprises any polypeptide that contains the antigen binding domains, no matter be natural or synthetic the generation.The example of antibody is immunoglobulin (Ig) hypotype (as IgG, IgE, IgM, IgD and IgA) and hypotype subclass thereof; Can be also comprise the antigen binding domains fragment as Fab, scFv, Fv, dAb, Fd; And double-chain antibody (diabodies).Fusion to another polypeptide, mosaic molecule or equivalent that comprise the antigen binding domains is also included within wherein.The cloning and expression of chimeric antibody is described in EP.A-0120694 and EP.A.0125023.
Monoclonal antibody of the present invention can be, for example, derivative, function equivalent and homologue unit price or single-chain antibody, double-chain antibody, chimeric antibody, humanized antibody and above-mentioned antibody, also comprise antibody fragment and any polypeptide that contains the antigen binding domains.
Antibody can be modified by many modes, can produce other antibody or the chimeric molecule that retains original antibodies specific with the DNA recombinant technology.This technology can comprise that constant region or constant region that the DNA of the immune globulin variable region of encoding antibody or complementarity-determining region (CDRs) is introduced to different immunoglobulin (Ig)s add framework region.Referring to, EP.A.184187, GB 2188638A or .EP.A.239400.Can also carry out genetic mutation or other change to other cell of hybridoma or generation antibody, this can change or not change the binding specificity of produced antibody.
For monoclonal antibody of the present invention also available hybridoma method, make, because the DNA sequence dna of code book invention humanized antibody can be used conventional means well known to those skilled in the art, as obtained according to aminoacid sequence synthetic disclosed by the invention or with the amplification of PCR method, thereby also available recombinant DNA method, available the whole bag of tricks well known in the art is connected into this sequence in suitable expression vector.Finally, under the condition that is applicable to antibody expression of the present invention, cultivate the host cell that transforms gained, the conventional separation and purification means purifying that then those skilled in the art's application is known obtains monoclonal antibody of the present invention.
As mentioned above, the present invention also provides reagent, test kit or the chip for implementing antibody of the present invention.Reagent, test kit or chip at least comprise following one or more: the antibody of making according to above method, the Nucleotide of this antibody of encoding, or the eukaryotic cell that comprises this antibody, prokaryotic cell prokaryocyte and virus and optional buffered soln.
The present invention also specifically discloses the preparation method of described anti human nerve nidogen monoclonal antibody, and it comprises following processing step:
(1) animal immune: highly purified people's neural nest protein fragments (LASTPIP PTPQAPSPAV) 1ml is fully emulsified, the BALB/c healthy mice of myeloma cell's homology immune and used, high purity people's neural nest albumen after every abdominal injection emulsification, measure its antiserum(antisera) by the enzyme-linked immunosorbent assay method;
(2) separating Morr. cell: get non-immune BALB/c healthy mice peritoneal macrophage, sod 96 well culture plates, the Sp2/0 cell changed after liquid is adjusted into to cell suspension, the mouse boosting cell of separating immune is also made cell suspension;
(3) cytogamy: will there is greater activity Sp2/0 myeloma cell and mix in the 1:10 ratio with splenocyte suspension, add gradually the 45%PEG(molecular weight in 30 seconds: 4000), static 90 seconds, cell is merged each other, drip nutrient solution in the mixed cell suspension of two kinds of cells, with HAT, select substratum to carry out cell cultures;
(4) screening hybridization tumour cell: wait the cell cultures to the merged in the time of seven days, draw in the hole of 96 well culture plates the culture supernatant that clone cell bunch occurs and detect anti-body contg by the enzyme-linked immunosorbent assay method, carry out three subclone screenings through limiting dilution, filter out the antibody-secreting hole of high-titer according to the secretion situation of antibody, by cell enlarged culturing in hole, then carry out antigen specific immune histological chemistry in-site detecting, select high-titer, the cell strain of high specific is enlarged culturing frozen again;
(5) monoclonal antibody specificity screening: choosing detects through enzyme-linked immunosorbent assay the supernatant that antibody titer is greater than the positive hole of 1:10000, with the outer culturing cell strain of people's embryo neural stem cell of vitro culture, multiple adult normal tissue, tumor tissues, human embryo tissue and human body, carries out the immunohistochemical methods specificity screening.
(6) monoclonal antibody purification storage: select BALB/c mouse or its parental generation mouse, first with pristane or the capable mouse peritoneal injection of whiteruss, after hybridoma is inoculated in mouse peritoneal, collect the ascites of mouse after one week, use AKTA-FPLC protein purification instrument that mouse IgG monoclonal antibody ascites solution is collected when the A280nm, the 1.44(absorbance unit) concentration is 1mg/ml.
The present invention also provides the purposes of described monoclonal antibody in the reagent for the preparation of identifying neural stem cell.
Another object of the present invention is to provide a kind of reagent, test kit or chip, comprises described monoclonal antibody.
The invention technological progress is: the anti human nerve nest antibody titer obtained is high, specificity is very good, the direct multiple tracer of mark, by current advanced person's detecting instrument as: flow cytometer, chemiluminescence detector, magnetic separator etc. carry out the sun choosing of neural stem cell.
Thereby the present invention obtains high purity people neural nest albumen the anti-human Nesting monoclonal antibody of high-titer, high specific through immune animal, cytogamy, cloning cultivation and a large amount of histocyte original position specificity screenings, this antibody can directly apply to clinical detection and fundamental research.Monoclonal antibody has great using value in biology and medical research field, is part important in affinity chromatography, is antibody main in immunohistochemical methods, is the novel agent in immunity inspection, is the guiding weapon of biotherapy.As the detection reagent of neural stem cell, anti human nerve nidogen monoclonal antibody can be given full play to its advantage.The high specificity of monoclonal antibody, can improve the specificity of antigen antibody reaction greatly, reduced possible cross reaction, makes the test-results confidence level larger.The homogeneity of monoclonal antibody and biological activity unicity make the antigen antibody reaction result be convenient to quality control, are beneficial to stdn and standardization.
Embodiment
The invention discloses a kind of high-titer, high specific anti human nerve nidogen monoclonal antibody and application thereof, those skilled in the art can use for reference this paper content, suitably improve processing parameter and realize.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the artly, they all are deemed to be included in the present invention.Product of the present invention and application are described by preferred embodiment, the related personnel obviously can be changed methods and applications as herein described or suitably change and combination within not breaking away from content of the present invention, spirit and scope, realizes and apply the technology of the present invention.
Before further setting forth the present invention, we are necessary to recognize, the present invention is not limited to the specific embodiment of description, that is to say, on specific form, may exist variation.Also have and a bit to need to remind, because scope of the present invention is subject to the restriction of additional claims, therefore, term used herein is just in order to describe the purpose of particular, rather than in order to limit purpose of the present invention.
Term " antibody " and " immunoglobulin (Ig) " in this article can Alternates.The term that these terms are well known to the skilled person, specifically refer to by can specific combination the protein that forms of one or more polypeptide of antigen.A kind of form of antibody has formed the basic structural unit of antibody.This form is tetramer, and it consists of two pairs of identical antibody chains, every a pair of have a light chain and a heavy chain.In every antagonist chain, the variable region gang of light chain and heavy chain is responsible for conjugated antigen jointly, and constant region is responsible for the effector functions of antibody.
Known immunoglobulin polypeptides comprises κ and lambda light chain at present, and alpha, gamma (IgG
1, IgG
2, IgG
3, IgG
4), δ, ε and μ heavy chain or their other type equivalence thing.The immunoglobulin (Ig) of total length " light chain " (approximately 25kDa or about 214 amino acid) comprises one by NH
2about 110 amino acids formed variable regions on-end, and κ or λ constant region on COOH-end.The immunoglobulin (Ig) of total length " heavy chain " (approximately 50kDa or about 446 amino acid), comprise a variable region (about 116 amino acid) equally, and one of CH, for example γ (about 330 amino acid).
Term " antibody " and " immunoglobulin (Ig) " comprise antibody or the immunoglobulin (Ig) of any phenogen, or the maintenance antibody fragment of being combined with antigen-specific, include but not limited to Fab, Fv, the fused protein of scFv and Fd fragment, chimeric antibody, humanized antibody, single-chain antibody and the antigen-binding portion thereof that comprises antibody and non-antibody protein.Antibody can be labeled and detect, for example, and can be by radio isotope, can produce the enzyme that can detect thing, fluorescence protein, vitamin H etc. and carry out mark detected.Antibody can also be incorporated into solid phase carrier, includes but not limited to polystyrene plate or bead etc.This term also comprises Fab ', Fv, F (ab ')
2and/or other antibody fragment and the monoclonal antibody that can be combined with antigen-specific.
Antibody can also exist in a variety of forms, for example comprises Fv, Fab and (Fab')
2, and difunctional hybrid antibody (document for example, Lanzavecchia etc., Eur.J.Immunol., 1987; 17,105) and for example, with single stranded form (, Huston etc., Proc.Natl.Acad.Sci.U.S.A., 1988; 85,5879 and Bird etc., Science, 1988; 242,423, be incorporated herein by reference) exist.(also referred to as " complementary determining region " or CDR), form, these hypervariable regions are by framework region (FR) interval by three hypervariable regions for the heavy chain of immunoglobulin (Ig) or variable region of light chain.The scope of framework region and complementary determining region is by explication (referring to " Sequences of Proteins of Immunological Interest, " E.Kabat etc., U.S.Department of Health and Human Services, 1991).The sequence of all antibody aminoacid sequences that discuss in this place is all with reference to the Kabat system.The different light chain of same species is relative conservative with heavy chain framework region sequence.The framework region of antibody is for location and calibration CDR.CDR mainly is responsible for the epi-position of conjugated antigen.
Chimeric antibody is its heavy chain and the antibody of light chain gene through building, and particularly utilizes the genetic engineering modified antibody variable region that belongs to different plant species and constant region gene.For example, the variable region fragment of mouse monoclonal antibody gene can be connected to people's antibody constant region fragment as γ 1 and γ 3.For example treatment is a kind of chimeric protein with chimeric antibody, its origin comes from rabbit antibody variable region fragment or antigen binding domain fragment and people's antibody constant region or effect district in conjunction with (the anti-Tac chimeric antibody prepared as the cell by A.T.C.C. preservation registration number CRL 9688), certainly, the gene source of chimeric antibody also can be used other mammalian species.
Be appreciated that the humanized antibody that the present invention designs and produces may substitute some conservative amino acid, these amino acid there is no impact to antigen combination or other functions of antibody.In other words, gly and ala; Val, ile and leu; Asp and glu; Asn and gln; Ser and thr; Lys and arg; Phe and tyr, abovely respectively combine inner amino acid and can mutually replace.
" variable region " of heavy chain of antibody or light chain is the ripe zone of N end of this chain.All Ranges, CDR and residue numbering all take sequence alignment, by existing structure knowledge, as basis, defined.The evaluation of framework region and CDR residue and numbering are pressed Chothia and other people described (Chothia, Structural determinants in the sequences of immunoglobulin variable domain.J Mol Biol.1998; 278,457).
VH is the variable region of heavy chain of antibody.VL is the variable region of light chain of antibody, and it may have κ and λ isotype.K-1 antibody has κ-1 isotype and K-2 antibody has κ-2 isotype, and V λ is variable lambda light chain.
" corresponding amino acid ", refer to when two or more aminoacid sequence comparison, is positioned at the amino-acid residue of same position (namely they correspond to each other).The method of antibody sequence comparison and numbering is at Chothia, and on seeing, Kabat, be shown in above and in other and obtain elaboration.Those of ordinary skills are known (referring to as Kabat 1991Sequences of Proteins of Immunological Interest, DHHS, Washington, DC), sometimes can in one or two amino acid of antibody, manufacture one, two or three breach and/or insert 1,2,3 or 4 residue or about 15 residues (particularly in L3 and H3CDR) at the most, thereby complete once comparison.
But " the position of substitution ", refer to a specific position of antibody, it can not made by different aminoacid replacement significantly reducing in conjunction with active of antibody.But how the method for identifying the position of substitution can be substituted in below and will be further described in more detail with them.But the position of substitution also can be called " variation tolerance position ".
In order to make those skilled in the art understand better technical scheme of the present invention, below in conjunction with specific embodiment, the present invention is described in further detail.
Embodiment 1: the preparation of monoclonal antibody of the present invention
The preparation of high-titer of the present invention, high specific anti human nerve nidogen monoclonal antibody comprises following processing step:
(1) animal immune: by highly purified people's neural nest protein fragments (LASTPIP PTPQAPSPAV, the autonomous design composition sequence) 1ml is fully emulsified, the BALB/c healthy mice of myeloma cell's homology immune and used, high purity people's neural nest albumen after every abdominal injection emulsification, measure its antiserum(antisera) by the enzyme-linked immunosorbent assay method;
(2) separating Morr. cell: get non-immune BALB/c healthy mice peritoneal macrophage, sod 96 well culture plates, the Sp2/0 cell changed after liquid is adjusted into to cell suspension, the mouse boosting cell of separating immune is also made cell suspension;
(3) cytogamy: will there is greater activity Sp2/0 myeloma cell and mix in the 1:10 ratio with splenocyte suspension, add PEG that cell is merged each other, drip nutrient solution in the mixed cell suspension of two kinds of cells, with HAT, select substratum to carry out cell cultures;
(4) screening hybridoma: wait the cell cultures to the merged in the time of seven days, draw in the hole of 96 well culture plates the culture supernatant that clone cell bunch occurs and detect anti-body contg by the enzyme-linked immunosorbent assay method, carry out three subclone screenings through limiting dilution, filter out the antibody-secreting hole of high-titer according to the secretion situation of antibody, by cell enlarged culturing in hole, then carry out antigen specific immune histological chemistry in-site detecting, select high-titer, the cell strain of high specific is enlarged culturing frozen again;
(5) monoclonal antibody specificity screening: choosing detects through enzyme-linked immunosorbent assay the supernatant that antibody titer is greater than the positive hole of 1:10000, with the outer culturing cell strain of multiple adult normal tissue, tumor tissues, human embryo tissue and human body, carries out the immunohistochemical methods specificity screening.
(6) monoclonal antibody purification storage: select BALB/c mouse or its parental generation mouse, first with pristane or the capable mouse peritoneal injection of whiteruss, after hybridoma is inoculated in mouse peritoneal, collect the ascites of mouse after one week, use AKTA protein purification instrument FPLC that mouse IgG monoclonal antibody ascites solution is collected when the A280nm, the 1.44(absorbance unit) concentration is 1mg/ml.
Step (1) adds the complete freund adjuvant of equivalent by high-titer, high specific anti human nerve nidogen 1ml and through fully emulsified, and with the BALB/c healthy mice of myeloma cell's homology used, mouse age is at 9 weeks, every abdominal injection 0.2ml, and interval injection in 2 weeks is once; Measure antiserum(antisera).
Step (2) adopts the enzyme-linked immunosorbent assay method to measure antiserum(antisera) after carrying out, and the method is comprised of following operation steps:
A, coated:
Carbonate buffer solution with 50mmol/L, pH=9 dilutes the high purity people's neural nest albumen 1:500 in step (1), coated 96 hole polyethylene boards, and vacuum is drained, and seals 4 ℃ and saves backup.
B, sealing:
Every hole adds the phosphate buffered saline buffer 200 μ l that pH is 7.4 to wash, include 1% lowlenthal serum;
C, application of sample:
Every hole adds the clear 50 μ l(1:5000 dilutions of the mouse peripheral blood of immune latter 3 days for the third time), every plate is established a normal control, positive control and blank (phosphate buffered saline buffer), washing;
D, add the every hole 100 μ l of ELIAS secondary antibody, washing;
E, colour developing:
Every hole adds substrate 100 μ l;
F, colorimetric:
With the blank zeroing, the 405nm wavelength is measured optical density(OD) (O.D);
G, result judgement: P/N=measure sample O.D average/negative serum O.D average, and P/N >=2.1 are positive.
Polyethylene board specification in step a be 200 μ l/ holes, vacuum to drain temperature be 4 ℃, washing adopts 0.05% Tween-20 phosphate buffered saline buffer washing 3 times.
Processing parameter in step b is that every hole adds pH=7.4, contains 1% lowlenthal serum phosphate buffered saline buffer 200 μ l, and temperature is 37 ℃, 1 hour time, washs 3 times.
Processing condition in step c are the clear 50 μ l of the mouse peripheral blood of immune latter 3 days for the third time after every hole adds 1:5000 dilution, and every plate is established a normal control, positive control and blank (phosphate buffered saline buffer), and temperature is that 37 ℃, time are 1 hour, washs 3 times.
Processing condition in steps d add the every hole 100 μ l of ELIAS secondary antibody, and temperature is that 37 ℃, time are 1 hour, washs 3 times.
Processing condition in step e are that room temperature, time are 10 minutes, then use the stop buffer termination reaction, through microplate reader, at wavelength 450nm place, read optical density value.
Get non-immune BALB/c healthy mice peritoneal macrophage in step (3), sod 96 well culture plates, the Sp2/0 cell changed after liquid 15 hours is adjusted into to 9 * 10
5/ ml cell suspension, the mouse boosting cell of separating immune.
There is highly active Sp2/0 myeloma cell in step (4) and mix in the ratio of 1:10 with the ratio of splenocyte, add PEG that cell is merged each other, in the mixed cell suspension of two kinds of cells, within the 1st minute, drip the 4.5ml nutrient solution; Interval 2 minutes drips the 5ml nutrient solution, then adds nutrient solution 50ml, and the HAT of take selects substratum to carry out cell cultures by 36% hole as 1 cells/well.
In step (5), be by cell cultures when covering at the bottom of 10% hole, draw culture supernatant and detect anti-body contg by the enzyme-linked immunosorbent assay method, filter out the antibody-secreting hole of high-titer according to the secretion situation of antibody, by the capable cloning again of cell in hole, then carry out the Immunohistochemistry of antigen-specific, select hypersecretion specific cell strain enlarged culturing or frozen.
Adult normal tissue and the human embryo tissue in step (6), selected comprise main organs and nervous tissue, tumor tissues comprises common and multiple tumor tissues, with the positive expression of above various tissues and cell lower than 5%, with the neural stem cell positive expression separated from the embryo more than 98%.
Select BALB/c mouse or its parental generation mouse in step (6), first with pristane or the injection of the capable mouse peritoneal of whiteruss, after one week by 5 * 10
5hybridoma is inoculated in mouse peritoneal and goes, in inoculation, after one week, there is obvious ascites to produce, every mouse can be collected the ascites of 15ml, uses AKTA protein purification instrument FPLC that mouse IgG monoclonal antibody ascites solution is collected when the A280nm, and absorbance unit=1.44 o'clock concentration is 1mg/ml.
Embodiment 2: monoclonal antibody indices of the present invention detects
The monoclonal antibody of the hybridoma cell strain secretion of embodiment 1 screening is carried out to sequential analysis, and its variable region of heavy chain is as follows:
QLQESGGGAVQPGRSLRLSCAASGFTFSTNLMNWVRQAPGKGLEWVIAISTYGYSY KYADAAVKGRFTISRDNSKNTIYLQMNSLRAEDTAVYYCARGDYADEYMLDLWGNG TVTVSSL(is as shown in SEQ ID No.1)
Its variable region of light chain is as follows:
DLNISQSPGSISVSIGEDATLSCRASQSISSYIAWYQQKPGQAPRLLIYSASTKVT GVPERFSGSASGTDFTLTITRLQPVDFAAYPCQQTSQFPPTFGYGTKVSIKRS(is as shown in SEQ ID No.2).
Embodiment 3: simultaneous test
The specification sheets that is 200610012998.2 with reference to the prior art application number prepares anti human nerve nidogen monoclonal antibody as a control group, with the anti human nerve nidogen monoclonal antibody of the embodiment of the present invention 1 preparation, compares, and result is as follows.
The enzyme-linked immunosorbent assay of embodiment 1 each step is tired and detected, and its indices is as follows:
1, the present invention is the positive colony of described monoclonal antibody embodiment 1 preparation, and enzyme-linked immunosorbent assay is tired as 1:64000; The control group enzyme-linked immunosorbent assay is tired as 1:50000;
2, carry out the specific expressed experiment in positive colony hole with immunohistochemical method: with the human nerve stem cell positive expression, with the expression that is negative of other histocyte, specific as follows:
The outer primary cultured cell of human body:
The outer culturing cell of human body system:
Annotate: negative (-) positive (+)
The adult normal tissue:
Human embryo tissue:
These results suggest that, it is all positive that anti human nerve nest monoclonal antibody of the present invention is carried out the immunohistochemical methods expressed in situ through the neural stem cell of many cases vitro culture, carry out immunohistochemical methods expressed in situ positive rate lower than 2% with tissue and the cell of multiple many cases number, illustrate that this monoclonal antibody specificity is higher than control group (5%), be applicable to clinical evaluation and the therapeutic evaluation of carrying out neural stem cells transplantation.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (5)
1. anti human nerve nidogen monoclonal antibody, is characterized in that, its weight chain variable region amino acid sequence is as shown in SEQ ID NO.1, and its light chain variable region amino acid sequence is as shown in SEQ ID NO.2.
2. the purposes of monoclonal antibody claimed in claim 1 in the reagent for the preparation of identifying neural stem cell.
3. a reagent, comprise monoclonal antibody claimed in claim 1.
4. a test kit, comprise monoclonal antibody claimed in claim 1.
5. a chip, comprise monoclonal antibody claimed in claim 1.
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