CN102863529A - VEGF (vascular endothelial growth factor) monoclonal antibody and fracture healing evaluation antibody chip with same - Google Patents
VEGF (vascular endothelial growth factor) monoclonal antibody and fracture healing evaluation antibody chip with same Download PDFInfo
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Abstract
The invention relates to the technical field of bioengineering and discloses a VEGF (vascular endothelial growth factor) monoclonal antibody and a fracture healing evaluation antibody chip with the same. The amino acid sequence of a heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO.1, and the amino acid sequence of a light chain variable region of the monoclonal antibody is shown as SEQ ID NO.2. Homogeneity and biological activity unicity of the monoclonal antibody enables antigen antibody reaction results to be convenient for quality control and beneficial to standardization and normalization. The chip with the monoclonal antibody can be directly applied to clinical therapeutic evaluation on fracture healing, solves the technical problems of time and labor consumption, poor result repeatability and the like of the prior art, has extremely high application value in the field of clinical treatment and has wide application prospect.
Description
Technical field
The present invention relates to biological technical field, particularly antibody chip is estimated in a kind of VEGF monoclonal antibody and the union of fracture that comprises this monoclonal antibody.
Background technology
Vasculogenesis is significant in fracture healing process.New vascularization may be relevant with factors, such as fibroblast growth factor, Delicious peptide, transforming growth factor.The platelet-derived long factor, PGE, vascular endothelial growth factor, tumour necrosis factor, insulingrowthfactorⅰ etc.
Vascular endothelial growth factor (VEGF) Human Umbilical Vein Endothelial Cells has its propagation of promotion and vasculogenesis, increases vascular permeability, and is particularly important.VEGF by with vascular endothelial cell on receptors bind, have the short vascular endothelial proliferation of powerful, specificity and angiogenic action, be the final passage of short revascularization, the angiogenesispromoting effect of other factors is that all or part of effect by VEGF is achieved.Using VEGF increases the fracture end volume of blood flow within a certain period of time than control group; Anti-VEGF can cause the fracture end volume of blood flow obviously to reduce, and illustrates that variation may affect union of fracture to VEGF on the fracture end volume of blood flow, and is significant.
There is the cytokine that promotes bone marrow stroma stem cell mitotic division and propagation in the craniocerebral injury body fluid.As if wherein Delicious peptide (BMP-2) is the successfully epochmaking factor of healing of fracturing, yet Delicious peptide is not to be one of craniocerebral injury union of fracture accelerator; Craniocerebral injury Prostatropin (bFGF) positive expression occurs in advance, and (VEGF) has obvious time and concentration relationship with vascular endothelial growth factor, by the expression promotion skeletonization of vascular endothelial growth factor; Insulingrowthfactorⅰ (IGF-I) is the main growth that promotes cartilage in union of fracture; Property neural (BDNF) nutritional factor in brain source all has promoter action at the union of fracture stages; The platelet derived growth factor serum-concentration is starkly lower than other groups, mechanism of action may be to finish by (IGF-II) rising of content of serum insulin growth factor II and the gathering of local platelet derived growth factor, the time that the time that insulin-like growth factor Ⅱ (IGF-II) serum-concentration raises and platelet derived growth factor (PDGF) reduce all concentrates on first two weeks, consistent on time, synergy may be arranged between the two; Epidermal growth factor (EGF) mainly is by the secretion of inflammatory cell Monocytes/Macrophages, acts on simultaneously repair cell inoblast, endotheliocyte, epidermic cell etc., has the function that promotes cell proliferation.But the anti-human VEGF monoclonal antibody of high-titer, high specific and union of fracture are estimated antibody chip and are had not yet to see report.
Summary of the invention
The object of the present invention is to provide a kind of VEGF monoclonal antibody and comprise chip and the application of this antibody.
Overall technology design of the present invention is: utilize the synthetic VEGF polypeptide (sequence is IFQEYPDEIE YIFKPS) of autonomous design to carry out the specificity screening technology through immune animal, cytogamy, subclone and enzyme-linked immunosorbent assay, obtain high-titer, the anti-human VEGF monoclonal antibody of high specific, height, specificity are good because this monoclonal antibody is tired, with other 7 kinds with union of fracture the antibody chip of relevant monoclonal antibody point system, complement C3 antibody and examined product in conjunction with the CY3 mark have been set up complete detection system, estimate the union of fracture effect for detection of reaching.This antibody chip has highly sensitive, and the advantages such as specificity is good, simple to operate, high-throughput can be used for clinical and monitoring and Quality Control health check-up serum.
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 contains any polypeptide of antigen binding domains.
" antibody " of the present invention should be interpreted as containing any specific binding factor with required specific binding domains.Thereby with it function equivalent and the homologue of the antibody fragment of homology, derivative, humanized antibody and antibody contained in this term, also comprises any polypeptide that contains the antigen binding domains, no matter is natural or synthetic the generation.The example of antibody is immunoglobulin (Ig) hypotype (such as IgG, IgE, IgM, IgD and IgA) and hypotype subclass thereof; Also can be to comprise the fragment of antigen binding domains such as Fab, scFv, Fv, dAb, Fd; And double-chain antibody (diabodies).Fusion to mosaic molecule or equivalent another polypeptide, 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 keeps original antibodies specific with the DNA recombinant technology.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 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 the antibody that produces.
Make for monoclonal antibody of the present invention also available hybridoma method, 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, cultivate the host cell that transforms gained, then those skilled in the art use the conventional separation and purification means purifying of knowing and obtain monoclonal antibody of the present invention.
As mentioned above, the present invention also provides reagent, test kit or the chip that is used for implementing antibody of the present invention.Reagent, test kit or chip comprise at least following one or more: according to the antibody that above method is made, the Nucleotide of this antibody of encoding, or comprise eukaryotic cell, prokaryotic cell prokaryocyte and virus and the optional buffered soln of this antibody.
As preferably, antibody chip of the present invention also comprises the antibody of the following antigen of specific binding: IGF-1, IGF-2, BMP-2, EGF, PDGF, bFGF, VEGF, BDNF.
The present invention also provides the test kit that comprises described antibody chip, also comprises the C3 complement antibody of beacon molecule marker, and described beacon molecule is preferably CY3.
In embodiment, the present invention also provides the preparation method of VEGF antibody, comprises following processing step:
(1) cytogamy: the splenocyte that VEGF polypeptide (sequence the is IFQEYPDEIE YIFKPS) immune animal that autonomous design is synthetic is got its sensitization mixes in the 10-100:1 ratio with Sp2/0 myeloma cell's suspension, adding polyoxyethylene glycol (U.S. Sigma company product) merges cell each other, in the mixed cell suspension of two kinds of cells, drip nutrient solution, carry out cell cultures with HAT selective medium (U.S. sea cloning companies produces);
(2) screening hybridoma: in the time of cell cultures to the 5-10 to be merged 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, filter out the antibody-secreting hole of high-titer according to the secretion situation of antibody, with cell enlarged culturing in the hole, then carry out antigen specific immune histological chemistry in-site detecting, select high-titer, the cell strain of high specific is enlarged culturing and frozen again;
(3) monoclonal antibody specificity screening: choosing detects antibody titer greater than the supernatant in the positive hole of 1:5000 through enzyme-linked immunosorbent assay, respectively IGF-1, IGF-2, BMP-2, EGF, PDGF, bFGF, VEGF, BDNF antigen is carried out specificity and the screening of tiring.
(4) monoclonal antibody purification storage: specificity is good and tire the nutrient solution sucking-off of clone hole high, use AKTA-FPLC protein purification instrument (manufacturing of AM General company) that IgG monoclonal antibody culture supernatant solution is collected the 1.44(absorbance unit when the A280nm) concentration is 1-10mg/ml.The monoclonal antibody of the VEGF hybridoma cell strain secretion of screening carries out sequential analysis, and its variable region of heavy chain is as follows:
QVQLV QSGAE VKKPG ASVKV SCKAS GGTFS SYAIS WVRQA PGQGL EWMGG FDPED GETIY AQKFQ GRVTM TEDTS TDTAY MELSS LRSED TAVYY CATGR SMVRG VIIPF NGMDV WGQGT TVTVS SY(is shown in SEQ ID No.1)
Its variable region of light chain is as follows:
DIR MTQSP SSLSA SVGDR VTITC RASQSISSYL NWYQQ KPGKA PKLLI YAASS LQSGV PSRFS GSGSG TDFTL TISSL QPEDFATYYC QQSYSTPLTF GGGTK VEIKP(is shown in SEQ ID No.2)
(5) CY3 traget antibody: with the fluorescein-labelled complement C3 of CY3 antibody.
(6) monoclonal antibody microarray point sample: 96 points of a chip point, put on chip two points of each monoclonal antibody, the content 0.1-1ng/ml of each point through limiting dilution with other 7 kinds of monoclonal antibodies with union of fracture.
Processing parameter in concrete technology step of the present invention and each step is:
Have highly active Sp2/0 myeloma cell in the step (1) and mix in the ratio of 1:10-100 with the ratio of splenocyte, add PEG cell is merged each other, in the mixed cell suspension of two kinds of cells, the 1st minute dropping 4.5ml nutrient solution; Interval 2 minutes drips the 5ml nutrient solution, then adds nutrient solution 50ml, selects substratum to carry out cell cultures by 36% hole as 1 cells/well take HAT.
In the step (2) be with cell cultures when covering at the bottom of 0%～20% hole, draw culture supernatant and detect anti-body contg with the enzyme-linked immunosorbent assay method, filter out the antibody-secreting hole of high-titer according to the secretion situation of antibody, with more capable cloning of cell in the hole, select hypersecretion specific cell strain enlarged culturing or frozen.
Adopt in the step (3) enzyme-linked immunosorbent assay to detect antibody titer greater than the supernatant in the positive hole of 1:5000, the mark antigen relevant with 8 kinds of union of fracture carries out specificity and the screening of tiring respectively.
Specificity is good and tire the nutrient solution sucking-off of clone hole high in the step (4), use AKTA-FPLC protein purification instrument (manufacturing of AM General company) that IgG monoclonal antibody culture supernatant solution is collected the 1.44(absorbance unit when the A280nm) concentration is 1-10mg/ml.
What select in the step (5) is that CY3 is to the mark of complement C3 antibody
The design of step (6) Plays product point sample matrix: every kind of antibody repeats 2-5 point, chooses 6-18 different antibody concentration gradient, and 6-18 matrix adopts SmartArrayerTM micro-array chip point sample system to carry out point sample.
The invention technological progress is: the anti-human VEGF monoclonal antibody that obtains is respectively through AKTA protein purification instrument FPLC purifying, its height of tiring, specificity are very good, put on chip through limiting dilution with other 7 kinds of monoclonal antibodies relevant with union of fracture, with the complement C3 antibody of mark with detect sample and hybridize, detect by present advanced person's chip detection instrument.This technology and traditional detection method relatively have fast, the characteristics of simple operation, high throughput testing; And can carry out quickly and accurately qualitative detection.
Thereby the method disclosed in the present is the VEGF polypeptide that autonomous design is synthetic obtains high-titer, high specific through immune animal, cytogamy, cloning cultivation and a large amount of histocyte original position specificity screenings anti-human VEGF monoclonal antibody, and this antibody and 7 kinds of antibody chips made from the monoclonal antibody of union of fracture mark of correlation thing 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.Its 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 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.
This patent is studied the union of fracture factor of influence in conjunction with up-to-date biochip technology first, broken away from present immunohistochemical methods, the drawback that the research methods such as enzyme-linked immunosorbent assay are difficult to compare because of systematic error, can carry out simultaneously high flux screening to dozens or even hundreds of kind of biotic factor simultaneously, find out rapidly and accurately synergistic mechanism between the biotic factor that promotes union of fracture and these biotic factors, instruct clinical medication treatment, and the union of fracture curative effect is estimated.This studies another new breakthrough is exactly to put up a bridge by complement C3, and strict double antibodies sandwich method before having broken away from, the multiple union of fracture factor only need a kind of two anti-(antibody of anticomplement C3) just can solve.So not only greatly reduce the technical costs of experiment, and make operation more easy, quick.
Embodiment
The invention discloses a kind of VEGF monoclonal antibody and comprise chip and the application of this antibody, 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 similarly replace and change apparent to those skilled in the art, and 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 change or suitably change and combination methods and applications as herein described within not breaking away from content of the present invention, spirit and scope, realizes and use 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, may exist variation on specific form.Also having what a bit need to remind is that because the restriction of claims that scope of the present invention is added, 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 consists of of one or more polypeptide of antigen.A kind of form of antibody has consisted of the basic structural unit of antibody.This form is tetramer, and it is made 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 then 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 the-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) comprises 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, scFv and Fd fragment, chimeric antibody, humanized antibody, single-chain antibody and comprise the antigen-binding portion thereof of antibody and the fused protein of 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 and 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 with single stranded form (for example, 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.(be 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 ordering 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 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 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 such 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 (such as 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 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, the inner amino acid of above each combination can replace mutually.
" 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 are all take sequence alignment, define as the basis by existing structure knowledge.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 " refers to be positioned at the amino-acid residue of same position (namely they correspond to each other) when two or more aminoacid sequence comparison.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 such as Kabat 1991 Sequences of Proteins of Immunological Interest, DHHS, Washington, DC), sometimes can in one or two amino acid of antibody, make one, two or three breach and/or insert 1,2,3 or 4 residue or about 15 residues (particularly in L3 and H3 CDR) at the most, thereby finish once comparison.
" but the position of substitution " refers to a specific position of antibody, and 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, the present invention is described in further detail below in conjunction with specific embodiment.
Embodiment 1: the preparation of monoclonal antibody of the present invention
The preparation method of antibody comprises following processing step:
(1) cytogamy: will have greater activity Sp2/0 myeloma cell and mix in the 1:10-100 ratio with the splenocyte suspension of synthetic VEGF polypeptide (sequence the is IFQEYPDEIE YIFKPS) sensitization of autonomous design respectively, adding polyoxyethylene glycol (U.S. Sigma company product) merges cell each other, in the mixed cell suspension of two kinds of cells, drip nutrient solution, carry out cell cultures with HAT selective medium (U.S. sea cloning companies produces);
(2) screening hybridoma: in the time of cell cultures to the 5-10 to be merged 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, filter out the antibody-secreting hole of high-titer according to the secretion situation of antibody, with cell enlarged culturing in the hole, then carry out antigen specific immune histological chemistry in-site detecting, select high-titer, the cell strain of high specific is enlarged culturing and frozen again;
(3) monoclonal antibody specificity screening: choosing detects antibody titer greater than the supernatant in the positive hole of 1:10000 through enzyme-linked immunosorbent assay, respectively 8 kinds of union of fracture mark antigens is carried out specificity and the screening of tiring.
(4) monoclonal antibody purification storage: specificity is good and tire the nutrient solution sucking-off of clone hole high, use AKTA-FPLC protein purification instrument (manufacturing of AM General company) that IgG monoclonal antibody culture supernatant solution is collected the 1.44(absorbance unit when the A280nm) concentration is 1-10mg/ml.
(5) CY3 traget antibody: with the fluorescein-labelled complement C3 of CY3 antibody.
The monoclonal antibody microarray point sample of (6) 8 kinds of union of fracture Research of predicting markers: 96 points of a chip point, two points of each monoclonal antibody, the content 0.01-1ng/ml of each point.
Step (1) is carried out complete rear employing enzyme-linked immunosorbent assay method and is measured antiserum(antisera), and the method is comprised of following operation steps:
A, coated:
Carbonate buffer solution with 50mmol/L, pH=9 is coated with 96 hole polyethylene boards with the VEGF synthetic peptide in the step (1), 4 micrograms/hole, and vacuum is drained, and seals 4 ℃ and saves backup.
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:
7 days cell culture supernatant 50-100 μ l(1:5000-10000 dilution after every hole adding cytogamy), every plate is established a normal control, positive control and blank (phosphate buffered saline buffer), washing;
D, adding ELIAS secondary antibody every hole 100-200 μ l, washing;
E, colour developing:
Every hole adds substrate 50-100 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.
Step (2) is in the time of to be merged cell cultures to the 5-10 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, filter out the antibody-secreting hole of high-titer according to the secretion situation of antibody, with cell enlarged culturing in the hole, then carry out antigen specific immune histological chemistry in-site detecting, select high-titer, the cell strain of high specific is enlarged culturing and frozen again;
Step (3) choosing detects antibody titer greater than the supernatant in the positive hole of 1:10000 through enzyme-linked immunosorbent assay, carries out specificity and the screening of tiring with other 7 kinds of union of fracture mark antigens respectively.
Step (4) is good with specificity and tire the nutrient solution sucking-off of clone hole high, use AKTA-FPLC protein purification instrument (manufacturing of AM General company) that IgG monoclonal antibody culture supernatant solution is collected the 1.44(absorbance unit when the A280nm) concentration is 1-10mg/ml.
Embodiment 2: the preparation method of antibody chip
Increase following processing step on embodiment 1 basis:
Step (5) the fluorescein-labelled complement C3 of CY3 antibody.
The design of step (6) standard substance point sample matrix: every kind of antibody repeats 2-5 point, chooses 6-18 different antibody concentration gradient, and 6-18 matrix adopts SmartArrayerTM micro-array chip point sample system to carry out point sample.
Step (7): the design of sample point sample matrix: 8 kinds of antibody, every kind of antibody repeats two points, amounts to 16 points, adopts and uses sampling liquid as negative control, adopts the antibody of anticomplement C3 as positive control, and each matrix amounts to 20 points.Amount to 24 dot matrix on every chip.Adopt SmartArrayerTM micro-array chip point sample system to carry out point sample.
Complete rear the vacuumizing at 30-40 ℃ of step (8) point sample spent the night, and carries out drying; Vacuumize plastic packaging after drying is complete, 4-8 ℃ saves backup.
The point sample parameter
Embodiment 3: monoclonal antibody indices of the present invention detects
The monoclonal antibody that the VEGF hybridoma cell strain of embodiment 1 screening is secreted carries out sequential analysis, and its variable region of heavy chain is as follows:
QVQLV QSGAE VKKPG ASVKV SCKAS GGTFS SYAIS WVRQA PGQGL EWMGG FDPED GETIY AQKFQ GRVTM TEDTS TDTAY MELSS LRSED TAVYY CATGR SMVRG VIIPF NGMDV WGQGT TVTVS SY(is shown in SEQ ID No.1)
Its variable region of light chain is as follows:
DIR MTQSP SSLSA SVGDR VTITC RASQSISSYL NWYQQ KPGKA PKLLI YAASS LQSGV PSRFS GSGSG TDFTL TISSL QPEDFATYYC QQSYS TPLTF GGGTK VEIKP(is shown in SEQ ID No.2)
Embodiment 4: index detects
Its indices of monoclonal antibody of the VEGF of the embodiment of the invention 1 preparation is as follows:
1, highly active Sp2/0 myeloma cell mixes in the ratio of 1:10-100 with the ratio of splenocyte
2, positive colony hole enzyme-linked immunosorbent assay is tired greater than 1:16000
4,8 kinds of union of fracture mark monoclonal antibodies are as follows: IGF-1, IGF-2, BMP-2, EGF, PDGF, bFGF, VEGF, BDNF.
Choose case patients serum 240 examples and altogether be divided into 4 groups: Normal group, cerebral trauma group, fracture group, fracture merge every group of 60 examples of cerebral trauma group.Antibody chip with embodiment 2 preparations of the present invention detects, and it is the highest that the antibody of seven indexs of IGF-I IGF-II bFGF BMP-2 VEGF EGF BDNF merges the expression of fracture group in cerebral trauma, secondly is the cerebral trauma group, fracture group, normal group.PDGF antibody is just in time opposite with the above results.Normal group is the highest, secondly is the fracture group, the cerebral trauma group, and cerebral trauma merges the fracture group.Can find out that from statistics the parts of fine intracellular cytokine has the potential of accelerating union of bone fracture, the fracture that is secondary to traumatic brain injury can healing acceleration.Illustrate that this chip specificity is high, be fit to offer medical treatment and effect assessment that scientific research institution carries out union of fracture.
The above only is 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 improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (8)
1. the monoclonal antibody of a VEGF 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 with SEQ ID NO.1 and have 95% consistence at least, 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 with SEQ ID NO.2 and have at least 95% consistence and described monoclonal antibody to have the characteristic of specific binding VEGF.
2. monoclonal antibody according to claim 1, it is characterized in that, 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.
3. each described monoclonal antibody of claim 1 to 2 is estimated purposes in the reagent of union of fracture 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. an antibody chip comprises each described monoclonal antibody of claim 1 to 2.
7. antibody chip according to claim 6 is characterized in that, also comprises the antibody of the following antigen of specific binding: IGF-1, IGF-2, BMP-2, EGF, PDGF, bFGF, BDNF.
8. comprise the test kit of claim 6 or 7 described antibody chips, it is characterized in that, also comprise the C3 complement antibody of beacon molecule marker, described beacon molecule is preferably CY3.
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Application publication date: 20130109 |