CN102109527A - Method for measuring biological activity of recombinant human p43 protein - Google Patents
Method for measuring biological activity of recombinant human p43 protein Download PDFInfo
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Abstract
The invention provides a method for measuring the biological activity of a recombinant human p43 protein, comprising the following steps: (1) leading the recombinant human p43 protein to induce mouse mononuclear phagocyte Raw264.7 to generate TNF alpha; (2) measuring the content of the TNF alpha generated in the step (1) through an enzyme-linked immunization; and (3) obtaining the biological activity of the recombinant human p43 protein according to the content of the TNF alpha obtained by the step (2). The method utilizes that the p43 has a functional reaction of an activating immunization system under the active state, and has the advantages that the specificity is strong, the reaction is fast, the repeatability is good, and the biological activity of the p43 can be reflected quantitatively.
Description
Technical field
The present invention relates to the active detection technique of biological products field, be specifically related to a kind of detection method of recombinant human p 43 protein biologic activity.
Background technology
Malignant tumour is the formidable enemy who threatens human health, and it is main method that medical circle is mainly taked kill cancer cell to the treatment of malignant tumour, that is: operative treatment, chemotherapy, radiotherapy etc.These methods also injuring normal cell in kill cancer cell, and make tumour cell produce drug resistance easily.Thereby, people exploring always a kind of can selectivity ground kill tumor cell and injuring normal cell not, be difficult for making simultaneously tumour to produce the chemical sproof cancer method of controlling again.The new drug that is used for the treatment of tumour at present continues to bring out.In recent years, along with the fast development of Protocols in Molecular Biology, people pay close attention to the method for utilization genetically engineered drug treatment malignant tumour gradually.Traditional chemotherapy of tumors uses cytotoxic drug mostly, also normal cell is caused damage in the kill tumor cell, weakens people's physique.So last many causing death because bearing treatment of a lot of Advanced Carcinoma Patient died.Malignant tumour has a common characteristic, and that is exactly that local cell insanely, is not subjected to containment ground propagation, just means a large amount of nutrition supplies of needs and keep this propagation.When tumour forms, around reaching, the inside of knurl body can form the blood vessel of a large amount of supply nutrients.If can cut off these keepings " pipeline " so, the source of nutrition of blocking-up tumour, tumour also will die of exhaustion.
The new vessels of current rise suppresses therapy and has brought dawn for thoroughly treating tumour.Discover the particularly a large amount of nutrition supply of growth needs of solid tumor of tumour, around tumour, can form a large amount of new vesselses.Anti-angiogenic therapy mainly is the growth by the vascular endothelial cell that suppresses rapid abnormal propagation, specificity suppresses tumor vascular endothelial cell propagation, thereby prevention tumor-blood-vessel growth, make tumour capillary generation atrophy, cut off the nutrition supply of tumour, make apoptosis of tumor cells or degenerate to initial dormant state, thereby reach the purpose for the treatment of cancer.
Through the continuous effort in nearly ten years, found the multiple neovascularization inhibitor that promises to be anti-tumor medicine now.Wherein the most representative is human Endostatin (human endostatin).Relevant human Endostatin (Endostatin) in the world at first report be the research group of U.S. Harvard Medical School Folkman (Folkman) that publishes on the Cell magazine in January, 1997 paper about the mouse endostatin gene, this discovers that Angiostatin and Endostatin can eliminate malignant tumour in the mouse body, and not recurrence again.These two kinds of medicines are to be thereby that tumour provides the vessel growth of nutrition to treat tumour with the prevention.Caused international sensation at that time, Endostatin becomes the research focus in oncotherapy field rapidly.U.S. FDA gives special approval to that under the situation of not finishing preclinical study 30 examples mix genetic engineering injection with human Endostatin (Angiostatin) and mouse Endostatin (Endostatin) to the unresponsive patients with advanced cancer of other medicines and carry out clinical testing at the beginning of 99.At present, it is clinical that Endostatin has passed through the first phase of FDA, is in the second stage of clinical stage at present.But because the dissolubility of Endostatin is relatively poor, make its preparation cost raise, also can only adopt the liquid drugs injection formulation in the use simultaneously, this makes its application be subjected to very big restriction.
P43 albumen is the accessory factor of mammal aminoacyl tRNA synthetase, directly regulate the physiology course that endothelial cell forms capillary on the one hand, p43 also is subjected to the upstream effect of cytokines and secretes from various kinds of cell on the other hand, further activated macrophage and dendritic cells, thus tumor growth suppressed.The anti-angiogenesis activity of p43 albumen and the effect of inhibition tumor growth obtain to confirm in external and zoopery.People's gene reorganization p43 albumen demonstrates the potentiality that it is developed to the novel therapeutic cancer drug, resists multiple primary and the metastatic entity tumor is all effective, and can with the chemotherapy and radiation Synergistic treatment.
P43 albumen is as the family member of human body aminoacyl tRNA synthetase system and be that endothelial mononuclear cell activates propeptide and found by people such as Sophie Q in 1997.P43 albumen is single chain protein, 312 amino acid of total length.Korea S Imagene company (WO01/95927) has carried out deep research to structure, the biologically active of human p 43 protein, the result shows that human p 43 protein can suppress the growth of the vascular endothelial cell of rapid abnormal propagation, and suppressing the generation of chick chorioallantoic membrane new vessels, this explanation p43 albumen has potential antitumor action.
Qualitative and to measure the biologic activity of recombinant human p 43 protein quantitatively significant for the research and development of this medicine.The Determination of biological activity of the class of angiogenesis inhibition at present medicine does not also have good solution, the principle of existing assay method mainly is based upon on the basis of such medicament adjusting angiogenesis relevant physiological function, and concrete experimental technique comprises that the test of chick chorioallantoic membrane angiogenesis, mouse tumor inhibition test, Matrigel test and the cornea rebirth blood vessel in external endothelial cell migration test, endothelial cell proliferation test and the body generates test.After deliberation, existing these measuring method for activity all exist repeatability relatively poor, and it is bigger artificially to reach the experiment material influence factor, problems such as the difficult control of operation.
Therefore, be badly in need of a kind of method and the product that can accurately measure the recombinant human p 43 protein biologic activity at present.
Summary of the invention
The present invention has the function of inducing mouse mononuclear macrophage Raw264.7 TNF secretion α according to the p43 albumen under the activated state, and then, provide a kind of easy, method of the biologic activity of qualitative and quantitatively determining recombined human p43 albumen accurately and fast by determining recombinant human p 43 protein and inducing the dose-effect relationship that produces between TNF α.
One aspect of the present invention provides a kind of method of measuring the biologic activity of recombinant human p 43 protein,
The invention provides a kind of method of measuring the biologic activity of recombinant human p 43 protein, said method comprising the steps of:
(1) makes recombinant human p 43 protein inducing mouse mononuclear macrophage Raw264.7, produce TNF α;
(2) the TNF alpha content that generates by euzymelinked immunosorbent assay (ELISA) determination step (1);
(3) the TNF alpha content that obtains according to step (2) obtains the biologic activity of recombinant human p 43 protein.
In a preferred embodiment of the present invention, the abductive approach in the described step (1) comprises with damping fluid gradient dilution testing sample, and makes and be no less than five dilutabilitys, dilution mixed with the mouse monokaryon macrophage respectively hatch cultivation again.
In a preferred embodiment of the present invention, described damping fluid is a serum-free DMEM nutrient culture media.
In a preferred embodiment of the present invention, described euzymelinked immunosorbent assay (ELISA) is the double antibodies sandwich method.
In a preferred embodiment of the present invention, described step (3) is carried out according to three ones of Pharmacopoeia of the People's Republic of China versions in 2005 and " biotech drug research and development and quality control " second edition.
In a preferred embodiment of the present invention, described step (3) is following carries out: with the logarithm value lgC of p43 concentration
P43Being the X value, is the Y value with corresponding TNF α concentration, adopts Nonlinear regression curve fitting, obtains the dose-response curve of recombinant human p 43 protein, thereby obtains four parameter analysis results.
In a preferred embodiment of the present invention, the inverse of the high dilution of the recombinant human p 43 protein that can induce 50% maximum reaction that is obtained by described step (3) is as activity value.
The present invention also provides a kind of method of the p43 of preparation protein product, and described method comprises the method for the biologic activity of measurement recombinant human p 43 protein of the present invention.
The present invention utilizes functional response that the p43 under the activated state has activated immune system to carry out the p43 Determination of biological activity, have special strong, reaction fast, good reproducibility, and advantage that can quantitative reaction p43 biologic activity.
The accompanying drawing summary
Fig. 1 is the docs-effect S type curve that the recombinant human p 43 protein sample promotes mouse monokaryon macrophage Raw264.7 TNF secretion α among the embodiment 1.
Embodiment
In the present invention, if not special explanation, percentage (%) or part all refer to percent by weight or the weight portion with respect to composition.
In the present invention, if not special explanation, each related component or its preferred ingredient can be combined to form new technical scheme mutually.
In the present invention, if not special explanation, all embodiments mentioned in this article and preferred implementation can be combined to form new technical scheme mutually.
In the present invention, if not special explanation, all technical characterictics mentioned in this article and preferred feature can be combined to form new technical scheme mutually.
In the present invention, if there is not opposite explanation, each components contents sum is 100% in the composition.
In the present invention, if there is not opposite explanation, the umber sum of each component can be 100 weight portions in the composition.
In the present invention, unless other explanations are arranged, numerical range " a-b " expression a represents that to the breviary of any real number combination between the b wherein a and b are real numbers.For example the whole real numbers between " 0-5 " have all been listed in numerical range " 0-5 " expression herein, and " 0-5 " just the breviary of these combinations of values represents.
In the present invention, unless other explanations are arranged, integer numerical range " a-b " expression a represents that to the breviary of the combination of the arbitrary integer between the b wherein a and b are integers.For example integer numerical range " 1-N " expression 1,2 ... N, wherein N is an integer.
In the present invention, unless other explanations are arranged, the multicomponent mixture of described each element of " its combination " expression, for example two kinds, three kinds, four kinds and up to the multicomponent mixture of maximum possible.
If do not particularly point out, the used term " a kind of " of this instructions refers to " at least a ".
If do not particularly point out, the benchmark of percentage of the present invention (comprising percent by weight) all is the general assembly (TW) of described composition.
" scope " disclosed herein is with the form of the lower limit and the upper limit.Can be respectively one or more lower limits and one or more upper limit.Given range limits by a selected lower limit and a upper limit.The border that the selected lower limit and the upper limit define special scope.All can carry out restricted portion by this way and comprise with capable of being combined, and promptly any lower limit can be combined to form a scope with any upper limit.For example, listed the scope of 60-120 and 80-110, be interpreted as that the scope of 60-110 and 80-120 also reckons with at special parameter.In addition, if if the minimum zone value of listing 1 and 2 and listed maximum magnitude value 3,4 and 5, then Xia Mian scope could all reckon with: 1-3,1-4,1-5,2-3,2-4 and 2-5.
In this article, except as otherwise noted, the ratio of each component or weight all refer to dry weight.
In this article, except as otherwise noted, described " biologic activity " is described according to " biotech drug research and development and quality control " second edition, is 1 unit (U) with contained sample size in the dilution of sample degree that can induce 50% maximum reaction.
One aspect of the present invention provides a kind of method of measuring the biologic activity of recombinant human p 43 protein, said method comprising the steps of:
(1) makes recombinant human p 43 protein inducing mouse mononuclear macrophage Raw264.7, produce TNF α;
(2) the TNF alpha content that generates by euzymelinked immunosorbent assay (ELISA) determination step (1);
(3) the TNF alpha content that obtains according to step (2) obtains the biologic activity of recombinant human p 43 protein.
Though without wishing to be bound by theory, but the mechanism of abductive approach is to join in the cell culture medium with certain density p43 albumen or other materials usually, promptly cultivate the Raw263.7 cell with the nutrient culture media that contains finite concentration p43, if the p43 that adds has biologic activity, then in the regular hour, can induce Raw264.7 emiocytosis TNF α.In the present invention, the abductive approach in the above-mentioned steps (1) is conventional, and those of ordinary skill in the art can directly obtain this abductive approach in conjunction with its professional technique again according to description of the invention.Concrete abductive approach can be referring to Park H, Park SG, Kim J, Ko YG, Kim S.Signalingpathways for TNF production induced by human aminoacyl-tRNAsynthetase-associating factor, p43.Cytokine.2002 Nov 24; 20 (4): 148-53.
In a preferred embodiment of the present invention, described abductive approach comprises:
Being applicable to the damping fluid gradient dilution testing sample of cellular incubation, and making and be no less than five dilutabilitys, again dilution is mixed and hatches cultivation with the specific cells (for example mouse monokaryon macrophage) of some respectively.Behind certain optimization incubation time, this cell can produce can be for the effect (for example TNF α) that detects.
In the present invention, the euzymelinked immunosorbent assay (ELISA) in the above-mentioned steps (2) is conventional, and those of ordinary skill in the art can directly obtain this euzymelinked immunosorbent assay (ELISA) in conjunction with its professional technique again according to description of the invention.Though without wishing to be bound by theory, but the ultimate principle of euzymelinked immunosorbent assay (ELISA) foundation is the specific reaction between antigen and antibody usually, its basic skills is that known antigen or antibody are adsorbed on surface of solid phase carriers, and the antigen-antibody reaction of enzyme labeling is carried out at solid phase surface.Euzymelinked immunosorbent assay (ELISA) of the present invention can include but not limited to the double antibodies sandwich method.Described double antibodies sandwich method for example can comprise specific antibody (antibody of anti-TNF alpha) and solid phase carrier (ELISA Plate) are connect, form insolubilized antibody; Unconjugated antibody and impurity are removed in washing; Add sample to be checked (cell culture supernatant that contains TNF α), insulation reaction; Antigen in the sample (TNF α) combines with insolubilized antibody, forms the solid phase antigen antibody complex; Other not bound substances are removed in washing; Add enzyme labelled antibody (anti-TNF alpha antibodies of enzyme labeling), insulation reaction; Antigen on the solid-phase immunity compound (TNF α) combines (anti-TNF alpha antibodies of enzyme labeling) with enzyme labelled antibody; The amount of being examined antigen in the thorough unconjugated enzyme labelled antibody of washing, the enzyme amount that have on the solid phase carrier this moment and sample is relevant; Add the substrate colour developing.Substrate for enzymatic activity on the solid phase becomes coloured product.The depth of color and the target protein in the sample are proportionate.Can predict the amount of antigen in the sample by colorimetric.
In the present invention, the method that obtains the biologic activity of recombinant human p 43 protein by the TNF alpha content is conventional, and those of ordinary skill in the art can directly obtain concrete method according to its professional knowledge.Concrete method of testing can be carried out referring to three ones of the versions in 2005 of the Pharmacopoeia of the People's Republic of China for example and " biotech drug research and development and quality control " second edition.For example, according to three requirements of Pharmacopoeia of the People's Republic of China version in 2005, adopt GraphPad Prism software to handle.According to testing result, with the logarithm value lgC of p43 concentration
P43Being the X value, is the Y value with corresponding TNF α concentration, adopts Nonlinear regression curve fitting, obtains the dose-response curve of recombinant human p 43 protein, thereby obtains four parameter analysis results.The biologic activity of sample is described according to " biotech drug research and development and quality control " second edition, with the high dilution of the testing sample that can induce 50% maximum reaction tire as a reference (or titre), can be the contained units of testing sample promptly also with the amount of cell factor contained in this dilutability as 1 active unit (U) with this dilution inverse.
The present invention also provides a kind of method of the p43 of preparation protein product, and described method comprises the method for the biologic activity of measurement recombinant human p 43 protein of the present invention.
Embodiment
1. the preparation of recombinant human p 43 protein finished product
1.1 material
20% sweet mellow wine, Shanghai Baxter Healthcare Ltd.; Ultrafilter and 0.22 μ m sterile filters are PALL company product; The Biohazard Safety Equipment brand is Heraeus, and model is KS12;-70 ℃ of low temperature refrigerator brands are Thermo, and model is HETO ULTRA FREEZE UF5410; The freeze dryer brand is Thermo, and model is Heto Lyolab3000.
1.2 method
Carry out ultrafiltration dialysis with the phosphate buffer PBS of the pH7.4 recombinant human p 43 protein stoste (preparation method of p43 albumen stoste is referring to CN101225371A) after to purifying, add 20% sweet mellow wine, the final concentration of sweet mellow wine is 5%, gained solution is through 0.22 μ m sterile filters filtration sterilization, being sub-packed in the 2ml volume does not have in the thermal source cillin bottle, the semi-manufacture of packing were put-70 ℃ of pre-freezes 12 hours, the semi-manufacture that pre-freeze is good are put into freeze dryer,-50 ℃ of freeze-drying, freeze-drying time is 24 hours, after the freeze-drying finished product under aseptic condition, jump a queue, with rear pressing cover, label.The preparation specification is the p43 sample that 4mg/ props up.
2. sample detection
2.1 detection step
2.1.1Raw264.7 cellular incubation
2.1.1.1 material
Mouse monokaryon macrophage RAW264.7 is available from Shanghai Inst. of Life Science, CAS; The high sugar of DMEM, hyclone (FBS), penicillin streptomycin is two to be resisted, PBS and TryPLE Express are Invitrogen, the GIBCO product; 25cm
2Tissue Culture Flask is a Corning company product; CO
2The cell culture incubator brand is Heraeus, and model is HERAcell 150; The Biohazard Safety Equipment brand is Heraeus, and model is KS12.
2.1.1.2 method
The Raw264.7 cell is cultivated with the nutrient culture media (complete medium) of 10%FBS DMEM, and condition of culture is 5%CO
2, 37 ℃ of incubators; Cell is adherent growth, changes complete medium every other day, treats that the culture flask inner cell grows into about 90% degree of converging, and can carry out activity and detect, and each generation growth conditions good cell all can be used for active the detection.
Be seeded to 96 orifice plates 2.1.2 survey living cells
2.1.2.1 material
96 porocyte culture plates are Corning company product; Blood counting chamber is available from Qiujing Bio-Chemical Reagent Instrument Co., Ltd., Shanghai; The inverted microscope brand is Olympus, and model is IX51.
2.1.2.2 method
Wash about 90% degree of converging and cell in good condition with PBS, add TryPLE Express digestive juice in the room temperature vitellophag, mirror is observed the digestion process down, treat that the cell retraction becomes bowlder and stops digestion with complete medium, with suction pipe piping and druming mixing cell, mirror is counting down, with complete medium diluting cells to 1 * 10
5/ ml.With 100 μ l/well inoculation, at 5%CO
2, leave standstill in 37 ℃ of incubators and cultivated 4 hours.
2.1.3 diluted sample dosing
2.1.3.1 material
Positive control: lipopolysaccharides LPS (E.Coli 0111:B4) is diluted to 80 μ g/ml available from Sigma with serum-free DMEM nutrient culture media, and packing is frozen in-70 ℃; Negative control: with serum-free DMEM nutrient culture media dilution p43 sample to 2000 μ g/ml, boiling water bath sex change 5min; Blank: serum-free DMEM nutrient culture media; 20 μ l, 200 μ l, 1000 μ l pipettor rifle heads, the 1.5ml centrifuge tube is the aseptic no thermal source product of Axygen company; Pipettor is a Gilson company product.
2.1.3.2 method
1) cell inoculation to 96 orifice plate dilutes p43 sample, feminine gender and positive control with serum-free DMEM nutrient culture media: parallel 4000ug/ml, 2000 μ g/ml, 500 μ g/ml, 125 μ g/ml, 31.25 μ g/ml, 15.625 μ g/ml, 7.8125 μ g/ml, eight concentration of 3.90625 μ g/ml of being diluted to of p43 sample after about 3 hours; Negative control contains 2000 μ g/ml boiling water bath sex change p43 samples; Positive control contains 0.8 μ g/mlLPS; Do blank with serum-free DMEM nutrient culture media.
2) sample of respectively organizing with complete medium and the preparation of last step mixes by 7: 1 dilution proportion, make that the final concentration of p43 sample is 500 μ g/ml, 250 μ g/ml, 62.5 μ g/ml, 15.625 μ g/ml, 3.90625 μ g/ml, 1.953125 μ g/ml, 0.9765625 μ g/ml, 0.48828125 μ g/ml, the final concentration of negative control is 250 μ g/ml, and the final concentration of positive control is 0.1 μ g/ml.
3) former nutrient culture media in 96 orifice plates is abandoned in suction, adds each sample of dilution back with every hole 150 μ l volumes, establishes three holes again for every group, at 5%CO
2, leave standstill in 37 ℃ of incubators and cultivated 18 hours.
2.1.4 the TNF alpha content detects in the culture supernatant
2.1.4.1 material
Mouse TNF α Elisa Kit is Shanghai Excell Biology Product Co., Ltd.'s product; The manual eight passage volley of rifle fires are HTL company product; Biochemical incubator brand is Heraeus, and model is BK-600; The microplate reader brand is Thermo, and model is Multiskan Spectrum.
2.1.4.2 method
1) medicine was handled cell after 18 hours, got to organize respectively in every group of each three hole that nutrient solution mixes and the centrifuging and taking supernatant is stand-by, and the back operation is all according to Mouse TNF α Elisa kit instructions.
2) take out the ELISA kit, balance is to room temperature, and each reagent of mixing.
3) dilution TNF α standard items: get 1ml reagent dilution and add in 1 freeze-drying TNF α standard items, leave standstill 15 minutes with abundant dissolving and mixing, get the 2000pg/ml initial pipe, double dilution standard product to final concentration is 1000pg/ml subsequently, 500pg/ml, 250pg/ml, 125pg/ml, 62.5pg/ml 31.25pg/ml gets the reagent dilution simultaneously and makees 0pg/mlTNF α standard items.
4) with injection water dilution 20 * concentrated cleaning solution.
5) add the standard items of the 3rd step dilution and the sample of the 1st step collection respectively with every hole 100 μ l volumes, every group of sample established three multiple holes, and the film shrouding was hatched 90 minutes for 37 ℃.
6) with 1: 1000 ratio diluted concentrated biological elementization antibody.
7) wash plate 5 times, each 340 μ l cleansing solutions.
8) every hole adds the biotinylated antibody working fluid of 100 μ l the 6th step preparations, and the film shrouding was hatched 60 minutes for 37 ℃.
9) concentrate enzyme conjugates with 1: 1000 dilution proportion liquid.
10) wash plate 5 times, each 340 μ l cleansing solutions.
11) every hole adds enzyme that 100 μ l the 9th step prepares in conjunction with working fluid, and the film shrouding was hatched 30 minutes for 37 ℃.
12) wash plate 5 times, each 340 μ l cleansing solutions.
13) every hole adds 100 μ l chromogenic substrates, and the film shrouding was hatched 15 minutes for 37 ℃.
14) every hole adds 100 μ l stop buffers, measures light absorption in the 450nm wavelength in 10 minutes, and carries software and select Quadratic Polynomial method drawing standard curve by microplate reader.Software calculates the TNF α concentration of each sample automatically.
2.2 result
Testing result is as shown in the table:
The testing result that table 1 recombinant human p 43 protein sample is induced TNF α
2.3 interpretation of result
Test figure adopts GraphPadPrism software to handle according to three requirements of Pharmacopoeia of the People's Republic of China version in 2005.According to table 1 testing result, with the logarithm value lgC of p43 concentration
P43Being the X value, is the Y value with corresponding TNF α concentration, adopts Nonlinear regression curve fitting, obtains the dose-response curve of recombinant human p 43 protein sample shown in Figure 1.Income analysis result is as shown in table 2:
Table 2 recombinant human p 43 protein sample induces TNF α to detect four parameter analysis results of data
Table is annotated: Bottom, Top: critical value up and down, HillSlope: slope, EC
50: median effective dose concentration, R
2: related coefficient
The reaction amount effect relationship of recombinant human p 43 protein inducing mouse mononuclear macrophage Raw264.7 TNF secretion α, and related coefficient is greater than 0.98.Adopt the sample after said method detects the heated and inactivated processing, can not detect its biologic activity.
The biologic activity of sample is described according to " biotech drug research and development and quality control " second edition, available energy is induced the high dilution of the testing sample of 50% maximum reaction tire as a reference (or titre), can be the contained units of testing sample promptly also with the amount of cell factor contained in this dilutability as 1 active unit (U) with this dilution inverse.
Calculate in view of the above, the concentration when this batch recombinant human p 43 protein sample is induced 50% maximum the reaction in the present embodiment is 5.505 * 10
-3Mg/ml, reaction volume are 0.1ml, promptly contain 5.505 * 10 among the 1U
-4Mg p43 albumen, its specific activity are 1816.5U/mg.
According to the method revision test of embodiment 16 times, the specific activity average of results sample is 1585.762U/mg to same batch of sample, and relative standard deviation is 24.9304%, sees Table 3.The reappearance that detects the recombinant human p 43 protein biologic activity with this method is better, meets in the cell in vitro survey experiment alive to be controlled near 30% requirement for measurement result.
Table 3 recombinant human p 43 protein biologic activity is criticized a reappearance and is detected
Table is annotated: n: detect batch R
2: related coefficient, EC
50: median effective dose concentration, SD: standard deviation, RSD: relative standard deviation
Claims (8)
1. method of measuring the biologic activity of recombinant human p 43 protein said method comprising the steps of:
(1) makes recombinant human p 43 protein inducing mouse mononuclear macrophage Raw264.7, produce TNF α;
(2) the TNF alpha content that generates by euzymelinked immunosorbent assay (ELISA) determination step (1);
(3) the TNF alpha content that obtains according to step (2) obtains the biologic activity of recombinant human p 43 protein.
2. the method for claim 1 is characterized in that, the abductive approach in the described step (1) comprises with damping fluid gradient dilution testing sample, and makes and be no less than five dilutabilitys, dilution is mixed with the mouse monokaryon macrophage respectively hatch cultivation again.
3. the method for claim 1 is characterized in that, described damping fluid is a serum-free DMEM nutrient culture media.
4. the method for claim 1 is characterized in that, described euzymelinked immunosorbent assay (ELISA) is the double antibodies sandwich method.
5. the method for claim 1 is characterized in that, described step (3) is carried out according to three ones of Pharmacopoeia of the People's Republic of China versions in 2005 and " biotech drug research and development and quality control " second edition.
6. the method for claim 1 is characterized in that, described step (3) is following carries out: with the logarithm value lgC of p43 concentration
P43Being the X value, is the Y value with corresponding TNF α concentration, adopts Nonlinear regression curve fitting, obtains the dose-response curve of recombinant human p 43 protein, thereby obtains four parameter analysis results.
7. the method for claim 1 is characterized in that, the inverse of the high dilution of the recombinant human p 43 protein that can induce 50% maximum reaction that is obtained by described step (3) is as activity value.
8. method for preparing the p43 protein product, described method comprises the method for the biologic activity of the described measurement recombinant human p 43 protein of claim 1.
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| CN116718781A (en) * | 2023-08-09 | 2023-09-08 | 苏州依科赛生物科技股份有限公司 | Method for detecting cell adhesion promoting activity of recombinant human vitronectin |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116718781A (en) * | 2023-08-09 | 2023-09-08 | 苏州依科赛生物科技股份有限公司 | Method for detecting cell adhesion promoting activity of recombinant human vitronectin |
| CN116718781B (en) * | 2023-08-09 | 2023-10-03 | 苏州依科赛生物科技股份有限公司 | Method for detecting cell adhesion promoting activity of recombinant human vitronectin |
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