CN102827279A - 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 ofly highly tire, 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, expresses because of its instantaneity in the cns growth course and is cloned, and is used as the specific sign of NSC always.Along with the very fast development of molecular biology and gene therapy technology, adopt neural stem cells transplantation treatment Spinal injury (SCI) to be acknowledged as up-to-date, one of the most promising treat-ment.Because both at home and abroad scientific research institution is placed on vitro culture, amplification, the transplanting aspect of NSC with main energy, therefore, it is particularly important to set up the NSC detection architecture.Anti-people Nestin monoclonal antibody is that human nerve stem cell is identified the most special method, meets the molecular immunology principle fully, and still, height is tired, the anti human nerve nidogen monoclonal antibody of high specific is not seen report at present as yet.
Summary of the invention
The object of the present invention is to provide a kind ofly highly tire, 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 multiple tissue of cytogamy, subclone and many cases and cell; The acquisition height is tired, high specific anti human nerve nidogen monoclonal antibody; Height, specificity are good because this monoclonal antibody is tired; Can directly apply to basic medical research, or process multiple external diagnosis reagent case and carry out evaluation of NSC and the therapeutic evaluation behind the neural stem cells transplantation.
Anti human nerve nidogen monoclonal antibody according to the invention, its weight chain variable region amino acid sequence are shown in SEQ ID NO.1, and its light chain variable region amino acid sequence is shown in SEQ ID NO.2; Or its variable region of heavy chain by the aminoacid sequence shown in the SEQ ID NO.1 through replacement, disappearance or add one or several amino acid derived aminoacid sequence and have 95% consistence at least with SEQ ID NO.1, variable region of light chain by the aminoacid sequence shown in the SEQ ID NO.2 through replacement, disappearance or add one or several amino acid derived aminoacid sequence and have at least 95% consistence and said monoclonal antibody to have the activity that specificity combines Nestin with SEQ ID NO.2.
As preferably, said monoclonal antibody comprises verivate, function equivalent and the homologue of single-chain antibody, double-stranded antibody, chimeric antibody, humanized antibody and above-mentioned antibody, also comprises antibody fragment and any polypeptide that contains the antigen binding domains.
" antibody " according to the invention should be interpreted as and contain any specificity binding factor with required specific binding domains.Thereby the function equivalent and the homologue of homologous antibody fragment, verivate, humanized antibody and antibody with it contained in this term, also comprises any polypeptide that contains the antigen binding domains, no matter is natural or synthetic the generation.The instance of antibody is Tegeline hypotype (like IgG, IgE, IgM, IgD and IgA) and hypotype subclass thereof; Also can be fragment such as Fab, scFv, Fv, dAb, the Fd that comprises the antigen binding domains; With double-stranded antibody (diabodies).Mosaic molecule or equivalent fusion to another polypeptide, that comprise the antigen binding domains are also included within wherein.The cloning and expression of chimeric antibody is described in EP.A-0120694 and EP.A.0125023.
Monoclonal antibody according to the invention can be; For example; Monovalent or verivate, function equivalent and the homologue of single-chain antibody, double-stranded 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 through many modes, and available DNA recombinant technology produces other antibody or the chimeric molecule that keeps original antibodies specific.This technology can comprise that constant region or constant region that the DNA with the immune globulin variable region of encoding antibody or complementarity-determining region (CDRs) introduces different Tegelines 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 the antibody that produces.
Being used for the also available hybridoma method of monoclonal antibody of the present invention makes; 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 obtaining 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 the suitable expression vector.At last, under the condition that is fit to antibody expression of the present invention, the host cell of culture transformation gained, those skilled in the art use the conventional separation and purification means purifying of knowing and obtain monoclonal antibody of the present invention then.
As stated, the present invention also provides reagent, test kit or the chip that is used for embodiment of the present invention antibody.Reagent, test kit or chip comprise at least following one or more: according to the antibody that above method is processed, the Nucleotide of this antibody of encoding, or comprise eukaryotic cell, prokaryotic cell prokaryocyte and the virus and the optional buffered soln of this antibody.
The present invention also specifically discloses said anti human nerve nidogen MONOCLONAL ANTIBODIES SPECIFIC FOR method, and it comprises following process step:
(1) animal immune: 1ml is fully emulsified with highly purified people's neural nest protein fragments (LASTPIP PTPQAPSPAV); Immune and used myeloma cell's homologous BALB/c healthy mice; High purity people's neural nest albumen after every abdominal injection emulsification is measured its antiserum(antisera) with 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 that changes behind the liquid is adjusted into cell suspension, the mouse boosting cell of separating immune is also processed cell suspension;
(3) cytogamy: will have greater activity Sp2/0 myeloma cell and splenocyte suspension by the 1:10 mixed; Add 45%PEG (molecular weight: 4000) in 30 seconds gradually; Static 90 seconds; Cell is merged each other, in the mixed cell suspension of two kinds of cells, drip nutrient solution, select substratum to carry out cell cultures with HAT;
(4) screening hybridization tumour cell: cell cultures to the to be merged is in the time of seven days; Draw the culture supernatant that occurs clone cell bunch in the hole of 96 well culture plates and detect anti-body contg with the enzyme-linked immunosorbent assay method; Carry out three subclone screenings through limiting dilution; Secretion situation according to antibody filters out the antibody-secreting hole that height is tired, and with cell enlarged culturing in the hole, carries out antigen specific immune histological chemistry in-site detecting then; Select height to tire, the cell strain of high specific is enlarged culturing and frozen again;
(5) monoclonal antibody specificity screening: choosing detects the supernatant of antibody titer greater than the positive hole of 1:10000 through enzyme-linked immunosorbent assay, carries out the immunohistochemical methods specificity screening with the outer culturing cell strain of people's embryo NSC of vitro culture, multiple adult's healthy tissues, tumor tissues, people's embryonic tissue and human body.
(6) monoclonal antibody purification storage: select BALB/c mouse or its parental generation mouse for use; Earlier with pristane or the capable mouse peritoneal injection of whiteruss; After hybridoma is inoculated in the mouse peritoneal; The ascites of mouse is collected in one week back, uses AKTA-FPLC protein purification appearance that mouse IgG monoclonal antibody ascites solution is collected when the A280nm, and 1.44 (absorbance unit) concentration is 1mg/ml.
The present invention also provides described monoclonal antibody to be used for identifying the purposes of the reagent of NSC in preparation.
Another object of the present invention provides a kind of reagent, test kit or chip, comprises described monoclonal antibody.
Institute of the present invention acquisition of technology progress is: tire height, specificity of the anti human nerve nest monoclonal antibody that is obtained is very good; The direct multiple tracer of mark, by present advanced person's detecting instrument as: flow cytometer, chemiluminescence detector, magnetic separator etc. carry out the sun choosing of NSC.
The present invention cultivates high purity people neural nest albumen and thereby a large amount of histocyte original position specificity screenings obtains highly to tire, the anti-people Nesting monoclonal antibody of high specific through immune animal, cytogamy, cloning, and this antibody can directly apply to clinical detection and fundamental research.Monoclonal antibody has great using value in biology and medical research field, be part important in the affinity chromatography, is antibody main in the immunohistochemical methods, is the novel agent in the immunity inspection, is the guiding weapon of biotherapy.As the detection reagent of NSC, 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, has reduced possible cross reaction, makes the test-results confidence level bigger.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 ofly highly tire, 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 that all similarly replace and change apparent to those skilled in the art, they all are regarded as and are included in the present invention.Product of the present invention and application are described through preferred embodiment; The related personnel obviously can change or suitably change and combination methods and applications as herein described in not breaking away from content of the present invention, spirit and scope, realizes and use technology of the present invention.
Before further setting forth the present invention, we are necessary to recognize that the specific embodiment that the present invention is not limited to describe that is to say, on specific form, possibly exist variation.Also having what a bit need to remind is that because scope of the present invention receives the restriction of additional claims, therefore, the term that this paper uses is just in order to describe the purpose of particular, rather than in order to limit the object of the invention.
Term " antibody " and " Tegeline " can exchange use in this article.These terms are term well-known to those skilled in the art, specifically are meant the protein that is made up of antigenic one or more polypeptide of ability specific combination.A kind of form of antibody has constituted the basic structural unit of antibody.This form is a tetramer, and it is made up of two pairs of identical antibody chains, and each is to all having 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 then is responsible for the effector functions of antibody.
Present known immunoglobulin polypeptides comprises κ and lambda light chain, and alpha, gamma (IgG
1, IgG
2, IgG
3, IgG
4), δ, ε and μ heavy chain or their other type equivalence thing.The Tegeline of total length " light chain " (approximately 25kDa or about 214 amino acid) comprises one by NH
2About 110 amino acids formed variable regions on the-end, and κ or λ constant region on COOH-end.The Tegeline of total length " heavy chain " (approximately 50kDa or about 446 amino acid) comprises a variable region (about 116 amino acid) equally, and one of CH, for example γ (about 330 amino acid).
Term " antibody " and " Tegeline " comprise the antibody or the Tegeline of any phenogen; Or maintenance and antigen-specific bonded antibody fragment; Include but not limited to Fab; Fv, scFv and Fd fragment, chimeric antibody, humanized antibody, single-chain antibody and the antigen-binding portion thereof and the proteinic fused protein of non-antibody that comprise antibody.Antibody can be labeled and detect, for example, and can be through ri, can produce the enzyme that can detect thing, fluorescence protein, vitamin H or the like and carry out mark and to be detected.Antibody can also be incorporated into solid phase carrier, includes but not limited to polystyrene plate or bead or the like.This term also comprises Fab ', Fv, F (ab ')
2And/or other can with antigen-specific bonded antibody fragment and monoclonal antibody.
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 with single stranded form (for example, Huston etc., Proc.Natl.Acad.Sci.U.S.A., 1988; 85,5879 with Bird etc., Science, 1988; 242,423, be incorporated herein by reference) exist.(be also referred to as " complementary determining region " or CDR) form, these hypervariable regions by framework region (FR) at interval by three hypervariable regions for the heavy chain of Tegeline 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 ordering of all antibody aminoacid sequences that discuss in this place is all with reference to the Kabat system.Light chain that same species are different and heavy chain framework region sequence are conservative relatively.The framework region of antibody is used 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 making up, and particularly utilizes genetic engineering modified antibody variable region that belongs to different plant species and constant region gene.For example, can the variable region fragment of mouse monoclonal antibody gene be connected to people's antibody constant region fragment such as γ 1 and γ 3.For example treating with chimeric antibody is a kind of chimeric protein; Its origin comes from rabbit antibody variable region fragment or antigen binding domain fragment and people's antibody constant region or effect district and combines (like the anti-Tac chimeric antibody by the cell preparation of A.T.C.C. preservation registration number CRL 9688); Certainly, the gene source of chimeric antibody also can use 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 combine antigen or the not influence basically of 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, more than the inner amino acid of each combination can replace each other.
" 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 with sequence alignment, be that the basis defines by existing structure knowledge.The evaluation of framework region and CDR residue and numbering are pressed Chothia and other people said (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 possibly 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 " is meant when two or more aminoacid sequences are compared, and is positioned at the amino-acid residue of same position (just they correspond to each other).The method of antibody sequence comparison and numbering is at Chothia, and on seeing, Kabat sees upward and in other to obtain elaboration.Those of ordinary skills are known (referring to like Kabat 1991Sequences of Proteins of Immunological Interest; DHHS; Washington; DC), can in one or two amino acid of antibody, make one, two or three breach sometimes and/or insert 1,2,3 or 4 residue or about at the most 15 residues (particularly in L3 and H3CDR), compare thereby accomplish once.
" instead position " refers to a specific position of antibody, and it can be replaced and can not make that the combination of antibody is active significantly to be reduced by different amino acid.How the method for evaluation instead position can be substituted in below with them will be carried out more detailed description.The instead position also can be called " variation tolerance position ".
In order to make those skilled in the art understand technical scheme of the present invention better, the present invention is done further detailed description below in conjunction with specific embodiment.
Embodiment 1: the preparation of monoclonal antibody according to the invention
Height according to the invention is tired, high specific anti human nerve nidogen MONOCLONAL ANTIBODIES SPECIFIC FOR, comprises following process step:
(1) animal immune: with highly purified people's neural nest protein fragments (LASTPIP PTPQAPSPAV; The autonomous design composition sequence) 1ml is fully emulsified; Immune and used myeloma cell's homologous BALB/c healthy mice; High purity people's neural nest albumen after every abdominal injection emulsification is measured its antiserum(antisera) with 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 that changes behind the liquid is adjusted into cell suspension, the mouse boosting cell of separating immune is also processed cell suspension;
(3) cytogamy: will have greater activity Sp2/0 myeloma cell and splenocyte suspension by the 1:10 mixed; Adding PEG merges cell each other; In the mixed cell suspension of two kinds of cells, drip nutrient solution, select substratum to carry out cell cultures with HAT;
(4) screening hybridoma: cell cultures to the to be merged is in the time of seven days; Draw the culture supernatant that occurs clone cell bunch in the hole of 96 well culture plates and detect anti-body contg, carry out three subclones screenings, filter out the antibody-secreting hole that height is tired according to the secretion situation of antibody through limiting dilution with the enzyme-linked immunosorbent assay method; With cell enlarged culturing in the hole; Carry out antigen specific immune histological chemistry in-site detecting then, select height to tire, the cell strain of high specific is enlarged culturing and frozen again;
(5) monoclonal antibody specificity screening: choosing detects the supernatant of antibody titer greater than the positive hole of 1:10000 through enzyme-linked immunosorbent assay, carries out the immunohistochemical methods specificity screening with the outer culturing cell strain of multiple adult's healthy tissues, tumor tissues, people's embryonic tissue and human body.
(6) monoclonal antibody purification storage: select BALB/c mouse or its parental generation mouse for use; Earlier with pristane or the capable mouse peritoneal injection of whiteruss; After hybridoma is inoculated in the mouse peritoneal; The ascites of mouse is collected in one week back, uses AKTA protein purification appearance FPLC that mouse IgG monoclonal antibody ascites solution is collected when the A280nm, and 1.44 (absorbance unit) concentration is 1mg/ml.
Step (1) with height tire, high specific anti human nerve nidogen 1ml adds the complete freund adjuvant of equivalent and through fully emulsified, with used myeloma cell's homologous BALB/c healthy mice, mouse age is in 9 weeks, every abdominal injection 0.2ml, the injection of 2 weeks is once at interval; Measure antiserum(antisera).
Adopt the enzyme-linked immunosorbent assay method to measure antiserum(antisera) after step (2) finishes, this method is made up of following operation steps:
A, encapsulate:
Carbonate buffer solution with 50mmol/L, pH=9 dilutes the high purity people's neural nest albumen 1:500 in the step (1), encapsulates 96 hole polyethylene boards, and vacuum is drained, and it is subsequent use to seal 4 ℃ of preservations.
B, sealing:
Every hole adds pH and is 7.4 phosphate buffered saline buffer 200 μ l washing, includes 1% lowlenthal serum;
C, application of sample:
Every hole adds immune for the third time back 3 days mouse periphery serum 50 μ l (1:5000 dilution), and every plate is established a normal control, positive control and blank (phosphate buffered saline buffer), washing;
D, the every hole 100 μ l of adding 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 judge: P/N=measures sample O.D average/negative serum O.D average, and P/N >=2.1 are positive.
Polyethylene board specification among the step a be 200 μ l/ holes, vacuum to drain temperature be 4 ℃, washing is adopted 0.05% Tween-20 phosphate buffered saline buffer washing 3 times.
Processing parameter among the 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 among the step c are the immune for the third time back 3 days mouse periphery serum 50 μ l after every hole adds the 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 the 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 among the step e are that room temperature, time are 10 minutes, use the stop buffer termination reaction then, read OD value through ELIASA at wavelength 450nm place.
Get non-immune BALB/c healthy mice peritoneal macrophage in the step (3), sod 96 well culture plates, the Sp2/0 cell that changed behind the liquid 15 hours is adjusted into 9 * 10
5/ ml cell suspension, the mouse boosting cell of separating immune.
Have the mixed of the ratio of highly active Sp2/0 myeloma cell and splenocyte in the step (4), add PEG cell is merged each other by 1:10, in the mixed cell suspension of two kinds of cells, the 1st minute dropping 4.5ml nutrient solution; At interval 2 minutes Dropwise 5 ml nutrient solutions add nutrient solution 50ml then, and selecting substratum with HAT is that 1 cells/well is carried out cell cultures by 36% hole.
Be when cell cultures is at the bottom of cover 10% hole in the step (5); Draw culture supernatant and detect anti-body contg with the enzyme-linked immunosorbent assay method; Secretion situation according to antibody filters out the antibody-secreting hole that height is tired; With the capable again cloning of cell in the hole, carry out the immunohistochemistry of antigen-specific then and measure, the high secretion of choosing specific cell strain enlarged culturing or frozen.
Adult's healthy tissues and the people's embryonic tissue selected for use in the step (6) comprise main organs and nervous tissue; Tumor tissues comprises common and multiple tumor tissues; Be lower than 5% with the positive expression of above various tissues and cell, with the expression that is positive of the isolating NSC from the embryo more than 98%.
Select BALB/c mouse or its parental generation mouse in the step (6) for use, earlier with pristane or the capable mouse peritoneal injection of whiteruss, after the week with 5 * 10
5Hybridoma is inoculated in the mouse peritoneal and goes; After one week of inoculation, there is tangible ascites to produce; Every mouse can be collected the ascites of 15ml, uses AKTA protein purification appearance 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 each item index according to the invention detects
The hybridoma cell strain excretory monoclonal antibody of embodiment 1 screening is carried out sequential analysis, and its variable region of heavy chain is following:
QLQESGGGAVQPGRSLRLSCAASGFTFSTNLMNWVRQAPGKGLEWVIAISTYGYSY KYADAAVKGRFTISRDNSKNTIYLQMNSLRAEDTAVYYCARGDYADEYMLDLWGNG TVTVSSL (shown in SEQ ID No.1)
Its variable region of light chain is following:
DLNISQSPGSISVSIGEDATLSCRASQSISSYIAWYQQKPGQAPRLLIYSASTKVT GVPERFSGSASGTDFTLTITRLQPVDFAAYPCQQTSQFPPTFGYGTKVSIKRS (shown in SEQ ID No.2).
Embodiment 3: simultaneous test
With reference to the prior art application number is that 200610012998.2 specification sheets prepares anti human nerve nidogen monoclonal antibody as control group, compares with the anti human nerve nidogen monoclonal antibody of the embodiment of the invention 1 preparation, and the result is following.
The enzyme-linked immunosorbent assay of embodiment 1 each step tired detect, its each item index is following:
1, the present invention is the positive colony of said monoclonal antibody embodiment 1 preparation, and enzyme-linked immunosorbent assay is tired and is 1:64000; The control group enzyme-linked immunosorbent assay is tired and is 1:50000;
2, carry out the specific expressed experiment in positive colony hole with immunohistochemical method:,, specific as follows with the expression that is negative of other histocyte with the human nerve stem cell expression that is positive:
The outer primary cultured cell of human body:
The outer culturing cell of human body system:
Annotate: negative (-) positive (+)
Adult's healthy tissues:
People's embryonic tissue:
Above presentation of results; It is all positive that anti human nerve nest monoclonal antibody according to the invention is carried out the immunohistochemical methods expressed in situ through the NSC of many cases vitro culture; Carry out immunohistochemical methods expressed in situ positive rate with the tissue of multiple many cases number and cell and be lower than 2%; Explain 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 only is a preferred implementation of the present invention; Should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention; Can also make some improvement and retouching, these improvement and retouching also should be regarded as protection scope of the present invention.
Claims (6)
1. anti human nerve nidogen monoclonal antibody is characterized in that, its weight chain variable region amino acid sequence is shown in SEQ ID NO.1, and its light chain variable region amino acid sequence is shown in SEQ ID NO.2; Or its variable region of heavy chain by the aminoacid sequence shown in the SEQ ID NO.1 through replacement, disappearance or add one or several amino acid derived aminoacid sequence and have 95% consistence at least with SEQ ID NO.1, variable region of light chain by the aminoacid sequence shown in the SEQ ID NO.2 through replacement, disappearance or add one or several amino acid derived aminoacid sequence and have at least 95% consistence and said monoclonal antibody to have the activity that specificity combines Nestin with SEQ ID NO.2.
2. monoclonal antibody according to claim 1; It is characterized in that; Said monoclonal antibody comprises verivate, function equivalent and the homologue of single-chain antibody, double-stranded antibody, chimeric antibody, humanized antibody and above-mentioned antibody, also comprises antibody fragment and any polypeptide that contains the antigen binding domains.
3. each described monoclonal antibody of claim 1 to 2 is used for identifying the purposes of the reagent of NSC in preparation.
4. a reagent comprises each described monoclonal antibody of claim 1 to 2.
5. a test kit comprises each described monoclonal antibody of claim 1 to 2.
6. a chip comprises each described monoclonal antibody of claim 1 to 2.
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| WO2014143886A2 (en) * | 2013-03-15 | 2014-09-18 | Edimer Pharmaceuticals, Inc. | Anti-ectodysplasin antibodies |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1903879A (en) * | 2006-08-01 | 2007-01-31 | 李彬 | Preparation method of high liter, high specificity anti human nerve nest protein monoclone antibody |
| US20100086998A1 (en) * | 1999-02-12 | 2010-04-08 | Buck David W | Enriched Central Nervous System Stem Cell and Progenitor Cell Populations, and Methods For Identifying, Isolating and Enriching For Such Populations |
| CN102127150A (en) * | 2010-01-19 | 2011-07-20 | 中国农业科学院北京畜牧兽医研究所 | Nestin portion polypeptide and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100086998A1 (en) * | 1999-02-12 | 2010-04-08 | Buck David W | Enriched Central Nervous System Stem Cell and Progenitor Cell Populations, and Methods For Identifying, Isolating and Enriching For Such Populations |
| CN1903879A (en) * | 2006-08-01 | 2007-01-31 | 李彬 | Preparation method of high liter, high specificity anti human nerve nest protein monoclone antibody |
| CN102127150A (en) * | 2010-01-19 | 2011-07-20 | 中国农业科学院北京畜牧兽医研究所 | Nestin portion polypeptide and application thereof |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014143886A2 (en) * | 2013-03-15 | 2014-09-18 | Edimer Pharmaceuticals, Inc. | Anti-ectodysplasin antibodies |
| WO2014143886A3 (en) * | 2013-03-15 | 2014-11-20 | Edimer Pharmaceuticals, Inc. | Anti-ectodysplasin antibodies |
| US9695245B2 (en) | 2013-03-15 | 2017-07-04 | Edimer Pharmaceuticals, Inc | Anti-ectodysplasin antibodies |
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