CN102863530A - Fat cell differentiation metabolite IGF-1 antibody, chip including same and application of fat cell differentiation metabolite IGF-1 antibody - Google Patents
Fat cell differentiation metabolite IGF-1 antibody, chip including same and application of fat cell differentiation metabolite IGF-1 antibody Download PDFInfo
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Abstract
The invention relates to the technical field of bioengineering and discloses a fat cell differentiation metabolite IGF-1 monoclonal antibody and a chip including the same. Heavy-chain variable region amino acid sequences of the monoclonal antibody are shown as SEQ ID NO.1, and light-chain variable region amino acid sequences of the monoclonal antibody are shown as SEQ ID NO.2. Homogeneity and biological activity oneness of the monoclonal antibody enable antigen-antibody reaction results to be convenient for quality control, and standardization and normalization are benefited. The chip including the antibody can be directly applied to medical and scientific research institutions for research on detection and prevention of cardiovascular and cerebrovascular diseases, arteriosclerosis and the like, is of extremely high application values in the clinical diagnosis field and solves the technical problems of time and labor consumption, poor result repeatability and the like in the traditional technology. Using the chip for physical examination and clinical diagnosis for masses of people is timesaving, laborsaving, simple, convenient and rapid, and the chip has wide industrial application prospect.
Description
Technical field
The present invention relates to technical field of bioengineering, particularly a kind of adipocytes differentiation metabolic product IGF-1 antibody and comprise chip and the application of this antibody.
Background technology
Normal metabolism of fat is the important physiological process that body growth is grown, its mechanism is that complicated multidirectional animal economy changes, relate to rise or the downward modulation of numerous albumen, cytokine, and unusual metabolism of fat will cause the generation of the diseases such as hepar damnification, hypertension, endocrine disturbance, obesity, the pathogeny of these diseases is very long evolutions, and is inseparable with customs such as the life of individuality and diet.Medical level and the diagnostic techniques of China also only are confined to traditional detection method to the diagnosis of single disease at present, and the development of Adipocyte Differentiation mark chip can laterally be compared relative disease, control the generation of its complication, more effectively instruct clinical application, improved treatment level, shortened and international counterparts between gap.Along with the quickening of Chinese aging process, the sickness rate of above disease can increasing year by year, and the development of this chip will made positive and great contribution aspect the prevention of normal health check-up, disease and the control.
The combination that the present invention includes the protein of IGF-1 in interior 45 kinds of monoclonal antibodies and adipocytes differentiation metabolic product has high degree of specificity, contains Adipocyte Factor, the disease-related factor and cytodifferentiation marker gene etc.Made antibody chip has been realized disease high throughput testing technology platforms such as diabetes, atherosclerosis, hypertension, obesities, independent research adipocytes differentiation metabolic product antibody chip detection kit (antibody chip) is by Chinese's independent development fully, use this chip, can be in several ways, multiple channel, stage construction detect tiring and content of adipocytes differentiation metabolic product, detection sensitivity is brought up to the ng level by ug, thereby reach more accurate, effect more fully, its detected result can be qualitative also can be quantitative.Adipocytes differentiation metabolic product antibody chip detection kit (antibody chip) has great influence to the development that promotes China's biological high-technology Disciplinary Frontiers, the international competitiveness that promotes China's biochip technology, product and market is significant, and this method meets the molecular immunology principle fully.At present external nearly all main drugmaker has all adopted biochip technology to seek drug targets to some extent, the toxicity of inspection medicine or side effect and carry out medicament production quantitatively and qualitative.Carry out large-scale drug screening with chip technology and can omit a large amount of animal experiments, shorten the used time of drug screening, thereby drive the research and development of original new drug.
Summary of the invention
The object of the present invention is to provide the monoclonal antibody of adipocytes differentiation metabolic product IGF-1 and comprise chip and the application of this antibody.
Overall technology design of the present invention is: utilize cytogamy, subclone and enzyme-linked immunosorbent assay carry out the specificity screening technology, obtain high-titer, the anti-human adipocytes differentiation metabolic product monoclonal antibody of high specific, the height because this monoclonal antibody is tired, specificity is good, can directly apply to the point sample of chip, complement C3 antibody and examined product in conjunction with the CY3 mark have been set up the method that complete detection system detects 45 kinds of fatty metabolic differentiation protein contents. and this antibody chip has highly sensitive, specificity is good, simple to operate, the advantages such as high-throughput can be used for clinical and monitoring and Quality Control health check-up serum.
IGF-1 monoclonal antibody provided by 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 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 characteristic with adipocytes differentiation metabolic product IGF-1 specific binding.
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.
The antibody that also comprises the following adipocytes differentiation metabolic product of specific binding on the chip of the present invention: TNF-α, ASP, leptin, PAI-1, IL-6, resistin, adiponectin, visfatin, RBP-4, GLUT-4, LPL, PGAR, UCPs, PPAR-γ, C/EBP, ADD1, SREBP1, NPY, MC-4R, POMC, α-MSH, AgRP, orexin, GH, IGF-1, Acrp30, VEGF, HBEGF, HGF, the Ang II, ACE, AGT, FA transporter, eNOS, iNOS, ACC, FAS, ME, ATP-citratelyase, AP2, apoE, perilipin, ACBP, PEPCK, the A2-adrenergic receptor.
The present invention also provides the test kit that comprises described antibody chip, it is characterized in that, also comprises the C3 complement antibody of beacon molecule marker, and described beacon molecule is preferably CY3.
In embodiment, the invention discloses the preparation method of adipocytes differentiation metabolic product antibody chip, it comprises following processing step:
(1) animal immune: (HPLC detects purity greater than 90% to the IGF-1 polypeptide that autonomous design is synthetic, sequence PTGYG SSSRRAPQTG) 1ml is fully emulsified, the BALB/c healthy mice of immune and used myeloma cell's homology, high purity Adipocyte Differentiation metabolism protein 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 made cell suspension;
(3) cytogamy: will have 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, 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 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, select high-titer, the cell strain of high specific is enlarged culturing and frozen again;
(5) 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 the 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 the 1.44(absorbance unit when the A280nm) concentration is 1mg/ml.
(6) the C3 complement antibody is carried out the CY3 mark
(7) adipocytes differentiation metabolic product chip microarray point sample: (every chip comprises 4-8 array, 1 standard, 3-7 detection) standard array sensing range 0.01～100ng/ml, 45 kinds of antibody of detection arrays (2 points of every kind of antibody).
Processing parameter in concrete technology step of the present invention and each step is:
Step (1) IGF-1 polypeptide (HPLC detects purity greater than the 90%) 1ml that autonomous design is synthetic adds the complete freund adjuvant of equivalent and through fully emulsified, BALB/c healthy mice with used myeloma cell's homology, mouse age is in 8～12 weeks, every abdominal injection 0.2ml, and interval 2 week injection is once; Adopt the enzyme-linked immunosorbent assay method to measure antiserum(antisera).
Get non-immune BALB/c healthy mice peritoneal macrophage in the step (2), 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 highly active Sp2/0 myeloma cell in the step (3) and mix in the ratio of 1:10 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 (4) 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.
The BALB/c mouse of selecting in the step (5) or its parental generation mouse, first with the capable mouse peritoneal injection of pristane or whiteruss, after the week with 5 * 10
5Hybridoma is inoculated in the mouse peritoneal and goes, after one week of inoculation, 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.
What select in the step (6) is that CY3 is to the mark of complement C3 antibody
Select adipocytes differentiation metabolic product chip microarray point sample in the step (7): (every chip comprises 4-8 array, 1 standard, 3-7 detection) standard array sensing range 0.01～100ng/ml, 45 kinds of antibody of detection arrays (2 points of every kind of antibody).
The obtained progress of the present invention is: the anti-human IGF-1 monoclonal antibody that obtains is through AKTA protein purification instrument FPLC purifying, its height of tiring, specificity are very good, put on chip through limiting dilution with other 44 kinds of monoclonal antibodies relevant with adipocytes differentiation metabolic product, 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 quantitative and qualitative analysis and detect.
The present invention's IGF-1 polypeptide that autonomous design is synthetic is cultivated and thereby a large amount of histocyte original position specificity screenings obtains the anti-human IGF-1 monoclonal antibody of high-titer, high specific through immune animal, cytogamy, cloning, and this antibody can directly apply to clinical detection and fundamental research with the antibody chip that 44 kinds of monoclonal antibodies relevant with adipocytes differentiation metabolic product are made.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 adipocytes differentiation metabolic product, anti-human adipocytes differentiation metabolic product 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 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 adipocytes differentiation metabolic product IGF-1 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.Method 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 H3CDR) 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 adipocytes differentiation metabolic product antibody of the present invention
The preparation method of adipocytes differentiation metabolic product antibody comprises following processing step:
(1) animal immune: synthetic IGF-1 polypeptide (PTGYG SSSRRAPQTG) 1ml of autonomous design is fully emulsified, the BALB/c healthy mice of difference immunity and used myeloma cell's homology, high purity people's adipocytes differentiation metabolic product 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 made cell suspension;
(3) cytogamy: will have greater activity Sp2/0 myeloma cell and mix in the 1:10 ratio with splenocyte suspension, 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 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;
(5) monoclonal antibody specificity screening: choosing detects antibody titer greater than the supernatant in the positive hole of 1:10000 through enzyme-linked immunosorbent assay, carries out specificity screening with multiple other genetically engineered drug.
(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 the 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 the 1.44(absorbance unit when the A280nm) concentration is 1mg/ml.
Step (1) adds the complete freund adjuvant of equivalent with the synthetic IGF-1 polypeptide 1ml of autonomous design and through fully emulsified, and with the BALB/c healthy mice of used myeloma cell's homology, mouse age is in 9 weeks, every abdominal injection 0.2ml, and interval 2 week injection is once; Measure antiserum(antisera).
Step (2) 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 synthetic IGF-1 polypeptide of the autonomous design 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:
Every hole adds for the third time clear 50 μ l(1:5000 dilution of immune rear 3 days mouse peripheral blood), 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 clear 50 μ l of for the third time immune rear 3 days mouse peripheral blood 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, then use the stop buffer termination reaction, read optical density value through microplate reader 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.
Has the ratio of highly active Sp2/0 myeloma cell and splenocyte by 1:10's in the step (4)
Ratio is mixed, and 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 (5) be with cell cultures when covering at the bottom of 10% 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, screen positive expression with multiple batches of adipocytes differentiation metabolic product through ELISA.Then determine hypersecretion, high specific cell strain enlarged culturing or frozen.
The BALB/c mouse of selecting in the step (6) or its parental generation mouse, first with the capable mouse peritoneal injection of pristane or whiteruss, after the week with 5 * 10
5Hybridoma is inoculated in the mouse peritoneal and goes, after one week of inoculation, 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: the preparation method of antibody chip
Increase following processing step on embodiment 1 basis:
The CY3 mark complement C3 antibody of selecting in the step (7).
The adipocytes differentiation metabolic product chip microarray point sample of selecting in the step (8): (every chip comprises 4-8 array, 1 standard, 3-7 detection) standard array sensing range 0.01～100ng/ml, 45 kinds of antibody of detection arrays (2 points of every kind of antibody).
Embodiment 3: monoclonal antibody indices of the present invention detects
The monoclonal antibody that the IGF-1 hybridoma cell strain of embodiment 1 screening is secreted carries out sequential analysis, and its variable region of heavy chain is as follows:
EVQLLESGGG LVQPGGSLRL SCAASGFTFS NYIMWWVRQA PGKGLEWVSV ISSSGGMTRY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDN GDYVGEKGFD IWGQGTMVTV SSAA(is shown in SEQ ID No.1)
Its variable region of light chain is as follows:
QDIQMTQSPS SLSASVGDRV TITCRASQSI SNYLNWYQQK PGKAPKLLIY TASTLQSGVP SRFSGSASGT DFTLTINSLQ PEDFATYSCQ QSYNSPWTFG QGTKVEIKRTS(is shown in SEQ ID No.2)
Embodiment 4: index detects
The indices of the IGF-1 monoclonal antibody of the embodiment of the invention 1 preparation is as follows:
1, positive colony hole ascites enzyme-linked immunosorbent assay is tired greater than 1:56000
2,45 kinds of adipocytes differentiation metabolic product associated antibodies comprising of this chip are as follows:
Adipocyte Factor (adipokines): TNF-α, ASP, leptin, PAI-1, IL-6, resistin, adiponectin, visfatin, RBP-4, GLUT-4, LPL, PGAR, UCPs, PPAR-γ, C/EBP, ADD1, SREBP1, NPY
The factor with disease-related: MC-4R, POMC, α-MSH, AgRP, orexin, GH, IGF-1, Acrp30, VEGF, HBEGF, HGF, Ang II, ACE, AGT, FA transporter, eNOS, iNOS
Each differential period marker gene: ACC, FAS, ME, ATP-citratelyase, AP2, apoE, perilipin, ACBP, PEPCK, A2-adrenoreceptor.
This adipocytes differentiation metabolic product antibody chip is expressed all positive through the specificity screening of 30 batches adipocytes differentiation metabolic product product, carrying out the specificity screening positive expression with other adipocytes differentiation metabolic product of 11 kinds 18 batches is 0.Show that this chip specificity is very high, be fit to offer medical treatment and scientific research institution carries out evaluation and the Quality Control evaluation of adipocytes differentiation metabolic product, this antibody also can be made into immunohistochemical methods or the enzyme-linked immunosorbent assay test kit is carried out medical basic research.
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.IGF-1 monoclonal antibody, it is characterized in that, its weight chain variable region amino acid sequence is shown in SEQ ID NO.1, 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 IGF-1.
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. the purposes of each described monoclonal antibody of claim 1 to 2 in preparation diabetes, atherosclerosis, hypertension, fat detection reagent.
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, it is characterized in that, also comprise the antibody of the following adipocytes differentiation metabolic product of specific binding: TNF-α, ASP, leptin, PAI-1, IL-6, resistin, adiponectin, visfatin, RBP-4, GLUT-4, LPL, PGAR, UCPs, PPAR-γ, C/EBP, ADD1, SREBP1, NPY, MC-4R, POMC, α-MSH, AgRP, orexin, GH, IGF-1, Acrp30, VEGF, HBEGF, HGF, the Ang II, ACE, AGT, FA transporter, eNOS, iNOS, ACC, FAS, ME, ATP-citratelyase, AP2, apoE, perilipin, ACBP, PEPCK, the A2-adrenergic receptor.
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 Number | Priority Date | Filing Date | Title |
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| CN2012103633859A CN102863530A (en) | 2012-09-26 | 2012-09-26 | Fat cell differentiation metabolite IGF-1 antibody, chip including same and application of fat cell differentiation metabolite IGF-1 antibody |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012103633859A CN102863530A (en) | 2012-09-26 | 2012-09-26 | Fat cell differentiation metabolite IGF-1 antibody, chip including same and application of fat cell differentiation metabolite IGF-1 antibody |
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| CN102863530A true CN102863530A (en) | 2013-01-09 |
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| CN105622730A (en) * | 2016-02-18 | 2016-06-01 | 深圳华尔康生物科技有限公司 | IGF-1 specifically bound polypeptide and application thereof |
| US11143659B2 (en) | 2015-01-27 | 2021-10-12 | Arterez, Inc. | Biomarkers of vascular disease |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104098684A (en) * | 2013-04-03 | 2014-10-15 | 苏州偲聚生物材料有限公司 | Polypeptide, and detection member and detection kit both containing same |
| CN104098684B (en) * | 2013-04-03 | 2016-03-23 | 苏州偲聚生物材料有限公司 | Polypeptide, the detection means comprising this polypeptide and detection kit |
| US11143659B2 (en) | 2015-01-27 | 2021-10-12 | Arterez, Inc. | Biomarkers of vascular disease |
| US11821905B2 (en) | 2015-01-27 | 2023-11-21 | Arterez, Inc. | Biomarkers of vascular disease |
| CN105622730A (en) * | 2016-02-18 | 2016-06-01 | 深圳华尔康生物科技有限公司 | IGF-1 specifically bound polypeptide and application thereof |
| CN105622730B (en) * | 2016-02-18 | 2020-06-26 | 深圳华尔康生物科技有限公司 | Polypeptide specifically binding IGF-1 and application thereof |
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