Detection method of biological activity of granulocyte colony-stimulating factor

Abstract

The present invention provides a method for detecting the biological activity of granulocyte colony-stimulating factor, comprising three steps: establishing a cell line, preparing a standard and test sample, and then determining the activity. The method is more convenient to use, requiring no special cell preparation and shortening the experimental operation time by 1 to 2 days. Furthermore, the method improves detection throughput, enabling sample dilution to 6 to 12 concentration points and allowing for vertical layout in a 96-well plate, increasing the number of samples tested per plate.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a method for detecting the biological activity of granulocyte colony stimulating factor. Background Art

[0002] Human granulocyte colony stimulating factor (hG-CSF) is a hematopoietic growth factor that regulates the maturation and growth of granulocytes, particularly neutrophils, in mammals. It stimulates the differentiation and maturation of granulocyte-macrophage colony-forming units (CFU-GM) into granulocyte-colony-forming units (CFU-G), promoting the release of mature neutrophils into the peripheral blood and thereby promoting neutrophil growth. Its clinical use in the treatment of neutropenia caused by various causes has significant economic and social significance.

[0003] In 1985, Welte.K successfully purified and refined human granulocyte colony-stimulating factor (hG-CSF) from the culture supernatant of human bladder cancer cell line 5637. Then Welte.K and Souza.LM of AMGEN Company in the United States further determined the N-terminal amino acid sequence of hG-CSF, cloned the hG-CSF gene from the 5637 cell line, and used genetic engineering technology to insert the gene into Escherichia coli to obtain hG-CSF, and developed recombinant human granulocyte colony-stimulating factor (rhG-CSF), with the trade name Filgratin.

[0004] Granulocyte colony-stimulating factor (G-CSF) is a World Health Organization international standard. The biological activity of recombinant human granulocyte colony-stimulating factor (rhG-CSF) is an important quality control indicator in scientific research and production. Currently, the biological activity of G-CSF, PEG-GCSF, and other products is tested using the NFS60 cell proliferation assay. This method has the following disadvantages: the procedure is complex, such as the need to add stimulatory factors in advance to co-culture cells; the cell color development process uses CCK8, a small solution volume of 10μL / well, which requires high operational requirements and is prone to errors, leading to experimental failure; and the entire assay takes a long time, requiring three days to complete.

[0005] Therefore, there is an urgent need to provide a method for detecting the biological activity of granulocyte colony stimulating factor that is simple to operate and takes a short time. Summary of the Invention

[0006] The purpose of the present invention is to address the deficiencies of the above prior art and to provide a method for detecting the biological activity of granulocyte colony stimulating factor that is simple to operate, short in time and has high detection throughput.

[0007] The technical solution of the present invention is:

[0008] The method for detecting the biological activity of granulocyte colony stimulating factor is characterized by comprising three steps: construction of a cell line, preparation of a standard substance and a test substance, and determination.

[0009] Preferably, the construction of the cell line comprises:

[0010] (1) Constructing a plasmid containing GCSFR-Geneticin, transfecting the plasmid containing GCSFR-Geneticin into HEK293 cells, and obtaining a polyclonal cell line GCSFR-HEK293 by pressure screening;

[0011] (2) pGL4.47-STAT3-Luc-HygromycinB was introduced into GCSFR-HEK293 cells, and the monoclonal cell line GCSFR-STAT3-Luc-HEK293 was obtained by pressure screening.

[0012] Preferably, the pressurization in step (1) is performed under the conditions of 650-750 μg / mL Geneticin for screening.

[0013] Preferably, the pressurization in step (2) is performed under the conditions of 650-750 μg / mL Geneticin and 250-350 μg / mL Hygromycin B.

[0014] Geneticin, also known as geneticin, is a resistance screening agent commonly used in transfection. Hygromycin B, also known as hygromycin B, is commonly used to screen and maintain cells transfected with the hygromycin resistance gene.

[0015] Furthermore, the preparation of the standard and test samples includes:

[0016] (1) GCSFR-STAT3-Luc-HEK293 cells were cultured in complete medium until the cells reached 85-95% confluence;

[0017] (2) Digest the cells with 0.2-0.3% trypsin, collect them by centrifugation, and count them. Adjust the cell density to (6±1)×10 with working culture medium. 5 cells / mL to obtain a cell suspension, and then take 50±5μL of the cell suspension into a 96-well plate and adjust the volume to (3±0.5)×10 4 cells / well;

[0018] (3) Prepare the dilution standard rhG-CSF at 2000±500 IU / mL and the test sample at 20±10 ng / mL. Then, perform 3-fold serial dilutions with working culture medium to obtain 8 to 12 concentration gradients. Then, take 50±5 μL of each of the 8 to 12 concentration gradient dilution samples and apply them to the cell plate.

[0019] Preferably, the cells in step (1) have a confluence of 89-92%.

[0020] More preferably, the cells in step (1) have a confluence of 90%.

[0021] Furthermore, the complete culture medium in step (1) is a DMEM culture medium containing 10±1% FBS, 250-350 μg / mL Hygromycin B, and 650-750 μg / mL Geneticin.

[0022] Furthermore, the working culture medium in steps (2) and (3) is DMEM culture medium containing 1±0.1% FBS.

[0023] Furthermore, the determination includes:

[0024] (1) Place the cell plate in an incubator at 37 ± 2°C and 5 ± 0.5% CO2 for 5.5–7.5 h. Then, add 50 ± 5 μL of the colorimetric developer, Bright Glo, to the cell plate and read the luminescence value using a microplate reader.

[0025] (2) The measured luminescence value and concentration are subjected to a four-parameter fitting curve to calculate the activity of the test sample.

[0026] The formula for four-parameter fitting is The formula involves four parameters: a, b, c, and d; it is a commonly used data processing method in immunoassays. Four-parameter curve fitting can be performed using software such as GraphPad Prism.

[0027] Use software to analyze the data and obtain the EC values of the standard and test samples. 50 The biological activity of the test article was calculated according to the following formula:

[0028] Biological activity of the test sample (IU / mL) = (Pr×Ds×Es) / (Dr×Er)

[0029] Where Pr is the biological activity of the standard;

[0030] Ds is the pre-dilution multiple of the test sample;

[0031] Dr is the pre-dilution multiple of the standard;

[0032] Es is the dilution multiple of the test sample equivalent to the half-effective dose of the standard sample;

[0033] Er is the half-effective dilution multiple of the standard.

[0034] Preferably, the incubation in step (1) is 5.8 to 6.5 hours.

[0035] More preferably, the incubation in step (1) is 6 hours.

[0036] The present invention utilizes a reporter gene assay based on the principle that the reporter gene cell line stably expresses the G-CSF receptor (GCSFR) and luciferase controlled by the STAT3 action sequence. The test sample binds to the cellular GCSFR and activates the STAT3 pathway. STAT3, upon binding to the action sequence, activates the reporter gene to express luciferase. Upon addition of a reaction substrate, the luciferase catalyzes the substrate, generating a chemiluminescent signal. The luminescence value is then measured to reflect the biological activity of the test sample.

[0037] The granulocyte colony stimulating factors of the present invention include, but are not limited to, G-CSF, rhG-CSF, PEG-GCSF, PEG-rhG-CSF stock solution or finished product.

[0038] Beneficial effects:

[0039] The detection method of the present invention is more convenient to operate, cells do not need special treatment, the volume of the color developer added is 50 μL, and no accidental addition will occur; the operation time is shortened, and the experimental operation time is shortened by 1 to 2 days.

[0040] At the same time, the detection throughput of the method of the present invention is improved, the sample is diluted to 6 to 12 concentration points (preferably 8), and can be arranged vertically in a 96-well plate, thereby increasing the number of samples detected on each cell plate. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is the fitting curve of test sample 1 determined by the reporter gene method; RS represents the standard, and Sample represents the test sample.

[0042] Figure 2 The graph shows the assay results of sample 1 using the NFS60 cell proliferation assay; RS represents the standard, and Sample represents the sample. DETAILED DESCRIPTION

[0043] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally carried out under conventional conditions or under conditions recommended by the manufacturer.

[0044] Example 1

[0045] Experimental reagents:

[0046] Complete culture medium: 10% FBS (mass fraction) + DMEM + 300 μg / mL Hygromycin B + 700 μg / mL Geneticin

[0047] Working culture medium: 1% FBS (mass fraction) + DMEM

[0048] Plasmid pGL4.47-STAT3-Luc-HygromycinB: purchased from Promega, catalog number E4047

[0049] Color developer Bright glo: purchased from Promega

[0050] 0.25% pancreatin: purchased from Gibco. Unless otherwise specified, pancreatin solution refers to the weight-to-volume ratio in this invention, specifically 0.25g pancreatin in 100mL solution

[0051] Experimental samples:

[0052] Standard: rhG-CSF from China Food and Drug Administration, batch number 98 / 01

[0053] Test products: G-CSF or PEG-GCSF from Jiangsu Aosaikang Pharmaceutical Co., Ltd. Experimental instruments:

[0054] CO2 incubator: Thermo, HERA cell 150i

[0055] Cell counter: CounterStar, IC1000

[0056] Microplate reader: Biotck, synergy H1, Gen5 software system

[0057] Clean bench: Suzhou Jiabao, JB-CJ-2FC

[0058] Ultracentrifuge: Eppendorf, 5810R

[0059] The specific experimental steps are as follows:

[0060] 1. Construction of cell lines:

[0061] (1) Construct a plasmid containing GCSFR-Geneticin, transfect the plasmid containing GCSFR-Geneticin into HEK293 cells, and select under pressure at 700 μg / mL Geneticin to obtain a polyclonal cell line GCSFR-HEK293;

[0062] (2) pGL4.47-STAT3-Luc-HygromycinB was introduced into GCSFR-HEK293 cells and screened under pressure under the conditions of 700 μg / mL Geneticin and 300 μg / mL HygromycinB to obtain the monoclonal cell line GCSFR-STAT3-Luc-HEK293.

[0063] The constructed plasmid nucleic acid sequence containing GCSFR-Geneticin is shown in SEQ ID NO: 1.

[0064] 2. Preparation of Standards and Test Samples:

[0065] (1) GCSFR-STAT3-Luc-HEK293 cells were cultured in complete medium until the cells reached 90% confluence;

[0066] (2) Digest the cells with 0.25% trypsin, collect them by centrifugation, and count them. Adjust the cell density to 6×10 5 cells / mL to obtain a cell suspension, and then take 50 μL of the cell suspension into a 96-well plate and adjust the volume to 3×10 4 cells / well;

[0067] (3) Prepare the dilution standard rhG-CSF at 2000 IU / mL and the test sample at 20 ng / mL, then perform 3-fold serial dilutions with working culture medium to obtain 8 concentration gradients. Then, take 50 μL of each of the 8 concentration gradient dilution samples and place them on the cell plate.

[0068] 3. Determination:

[0069] (1) Place the cell plate in an incubator at 37°C and 5% CO2 for 6 h, then add 50 μL of the colorimetric reagent Bright Glo to the cell plate and read the luminescence value using a microplate reader.

[0070] (2) Then use the software to perform four-parameter fitting curve.

[0071] Obtain EC values for standards and test products 50 The biological activity of the test article was calculated according to the following formula:

[0072] Biological activity of the test sample (IU / mL) = (Pr×Ds×Es) / (Dr×Er)

[0073] Where Pr is the biological activity of the standard;

[0074] Ds is the pre-dilution multiple of the test sample;

[0075] Dr is the pre-dilution multiple of the standard;

[0076] Es is the dilution multiple of the test sample equivalent to the half-effective dose of the standard sample;

[0077] Er is the half-effective dilution multiple of the standard.

[0078] Example 2

[0079] The three groups of test products (G-CSF) were tested using the method of Example 1 and the NFS60 cell proliferation method to verify the accuracy of the method of the present invention.

[0080] Experimental reagents:

[0081] Basal culture medium: 90% RPMI-1640 medium (mass fraction) + 10% FBS (mass fraction) + 55mM 2-Mercaptoethanol

[0082] Complete medium: basal medium + final concentration 20ng / mL rhG-CSF

[0083] RPMI-1640 culture medium and 2-Mercaptoethanol were purchased from Gibco

[0084] M-NFS-60 cells: from ATCC

[0085] Experimental samples:

[0086] Standard: rhG-CSF from China Food and Drug Administration, batch number 98 / 01

[0087] The specific steps of the NFS60 cell proliferation method are as follows:

[0088] 1. Cell Culture and Assay Plate Preparation

[0089] M-NFS-60 cell culture:

[0090] 1) Pre-warm culture medium

[0091] 2) Take one tube of frozen M-NFS-60 cells, dissolve them quickly at 37°C, and add them to a 75 cm 2 The cells were cultured in a cell culture flask at 37°C and 5% CO2 until they reached 80% confluence, during which time the cells were centrifuged and the medium was changed once.

[0092] Cell assay plate preparation:

[0093] 1) Take cells that have grown to 80% confluence, collect the cells and centrifuge at 1200 rpm for 7 minutes, discard the culture medium and wash them three times with culture medium.

[0094] 2) Suspend the cells in basal medium, take 100 μL of cell suspension and mix with 100 μL of trypan blue, count the cells using a cell counter, and adjust the cell density to 2×10 5 Cells were seeded in a 50 μL / well cell assay plate, and 100 μL / well PBS was added to the edge wells of the assay plate.

[0095] 2. Preparation of Standards and Test Articles (96-well Cell Culture Plate: Plate A)

[0096] Dissolve the standard according to the instructions and add 1 mL of basal culture medium to the ampoule. The activity unit is 5 × 10 6 IU / mL, dissolve and store in aliquots at -80℃.

[0097] 1) Pre-diluted standard with a starting concentration of 2000 IU / mL

[0098] 2) Pre-dilute the test sample with a starting concentration of 20,000 pg / mL

[0099] 3. Preparation of working solution (performed in 96-well plate: Plate B)

[0100] 1) Dilute the standard diluted to 2000 IU / mL and the test sample diluted to 20,000 pg / mL into working solution, setting 9 gradients and 3-fold dilution;

[0101] Dilution steps: Transfer the pre-diluted standard and test sample into the first group of plate B, add 200 μL of basal culture medium to the rest, then take 100 μL of solution from the first group and transfer it to the second group, and repeat this process for a total of 9 dilution gradients. Take another group and add only basal culture medium as a negative control.

[0102] 2) Pipette the above-prepared standard and test sample working solutions and negative control into the cell assay culture plate at a volume of 50 μL / well (3 replicate wells in parallel, with the standard and test sample contained on one assay plate).

[0103] 4. Response and reading

[0104] Place the test plate in a 37°C, 5% CO2 incubator for 36 to 48 hours, remove the culture plate, and observe the cell growth under a microscope. After confirming that the cells are growing well, add 10 μL of CCK-8 to each well, mix thoroughly, and continue to culture at 37°C, 5% CO2 for 3 to 4 hours. Take it out and equilibrate it at room temperature for 10 to 15 minutes. Set the absorption wavelength of the microplate reader to 450 nm and read the absorbance value.

[0105] 5. Data storage and calculation

[0106] Use software to analyze the data and obtain the EC values of the standard and test samples. 50The biological activity of the test article was calculated according to the following formula:

[0107] Biological activity of the test sample (IU / mL) = (Pr×Ds×Es) / (Dr×Er)

[0108] Where Pr is the biological activity of the standard;

[0109] Ds is the pre-dilution multiple of the test sample;

[0110] Dr is the pre-dilution multiple of the standard;

[0111] Es is the dilution multiple of the test sample equivalent to the half-effective dose of the standard sample;

[0112] Er is the half-effective dilution multiple of the standard.

[0113] Accuracy verification results:

[0114] The three groups of test products (G-CSF) were numbered and tested using the method of the present invention and the NFS60 cell proliferation method, respectively. The test results are shown in Table 1.

[0115] Table 1

[0116]

[0117] System suitability: Correlation coefficient R 2 Value ≥ 0.95; CV between wells ≤ 20%.

[0118] The verification results in Table 1 show that the detection method of the present invention has good accuracy.

[0119] The detection method of the present invention is more convenient to operate, does not require special treatment of cells, does not cause accidental addition of color developing agents, shortens the operation time, and shortens the experimental operation time by 1 to 2 days. At the same time, the detection throughput of the method of the present invention is improved, the sample is diluted to 6 to 12 concentration points, and can be arranged vertically in a 96-well plate, increasing the number of samples detected per cell plate. SEQUENCE LISTING <110> Jiangsu Aosaikang Pharmaceutical Co., Ltd. <120> Detection method of biological activity of granulocyte colony-stimulating factor <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 7449 <212> DNA <213> Artificial Sequence <400> 1 60. aatgtagtct tatgcaatac tcttgtagtc ttgcaacatg gtaacgatga gttagcaaca tgccttacaa ggagagaaaa agcaccgtgc atgccgattg gtggaagtaa ggtggtacga tcgtgcctta ttaggaaggc aacagacggg tctgacatgg attggacgaa ccactgaatt gccgcattgc agagatattg tatttaagtg cctagctcga tacataaacg ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg 360 420. taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag gactcggctt gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg 540 actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa gcggggggaga attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa atataatta 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aaaatctcta 4200 gcagtagtag ttcatgtcat cttattattc agtatttata acttgcaaag aaatgaatat 4260 cagagagtga gaggaacttg tttattgcag cttataatgg ttacaaataa agcaatagca 4320 tcaacaattt caaataaa gcatttttt cactgcattc tagttgtggt ttgtccaaac 4380 tcatcaatgt atcttatcat gtctggctct agctatcccg cccctaactc cgcccatccc 4440 gcccctaact ccgcccagtt ccgcccattc tccgcccat ggctgactaa ttttttttat 4500 ttatgcagag gccgaggccg cctcggcctc tgagctattc cagagtgt gaggaggctt 4560 tttttggaggc ctagggacgt acccaattcg ccctatagtg agtcgtatta cgcgcgctca 4620 ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc 4680 cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc 4740 ccttcccaac agttgcgcag cctgaatggc gaatgggacg cgccctgtag cggcgcatta 4800 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg 4860 cccgctcctt tcgctttctt cccttcctt ctcgccacgt tcgccggctt tccccgtcaa 4920 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc 4980 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt 5040 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca 5100 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc 5160 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta 5220 acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat 5280 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc 5340 aataatattg aaaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttatccct 5400 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag 5460 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta 5520 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc 5580 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca 5640 tacactattc tcagaatgac ttggttgagt actcaccagt cacaagaaag catcttacgg 5700 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg 5760 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca 5820 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa 5880 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa 5940 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata 6000 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat 6060 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc 6120 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata 6180 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt 6240 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga 6300 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag 6360 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa 6420 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag 6480 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg 6540 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat 6600 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta 6660 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg 6720 6780 gtgagctatg agaaagcgcc acgcttcccg aagagagaaa ggcggacagg tatccggtaa 6840 gcggcaggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc 6900 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt 6960 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct 7020 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc 7080 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg 7140 agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt 7200 ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc 7260 gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc 7320 ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct 7380 atgaccatga ttacgccaag cgcgcaatta accctcacta aagggaacaa aagctggagc 7440 tgcaagctt 7449

Claims

1. A method for detecting the biological activity of granulocyte colony stimulating factor, characterized in that: It includes three steps: construction of cell lines, preparation of standards and test samples, and determination; The construction of the cell line includes: (1) Constructing a plasmid containing GCSFR-Geneticin, transfecting the plasmid containing GCSFR-Geneticin into HEK293 cells, and obtaining a polyclonal cell line GCSFR-HEK293 by pressure screening; (2) pGL4.47-STAT3-Luc-HygromycinB was introduced into GCSFR-HEK293 cells, and the monoclonal cell line GCSFR-STAT3-Luc-HEK293 was obtained by pressure screening.

2. The detection method according to claim 1, wherein The pressurization described in step (1) is performed for screening under the conditions of 650-750 μg / mL Geneticin.

3. The detection method according to claim 1, wherein The pressurization described in step (2) is performed under the conditions of 650-750 μg / mL Geneticin and 250-350 μg / mL Hygromycin B for screening.

4. The detection method according to claim 1, wherein The preparation of the standard and test samples includes: (1) GCSFR-STAT3-Luc-HEK293 cells were cultured in complete medium until the cells reached 85-95% confluence; (2) Digest the cells with 0.2-0.3% trypsin, collect them by centrifugation, and count them. Adjust the cell density to (6±1)×10 with working culture medium. 5 cells / mL to obtain a cell suspension, and then take 50±5μL of the cell suspension into a 96-well plate and adjust the volume to (3±0.5)×10 4 cells / well; (3) Prepare the dilution standard rhG-CSF at 2000±500 IU / mL and the test sample at 20±10 ng / mL. Then, perform 3-fold serial dilutions with working culture medium to obtain 8 to 12 concentration gradients. Then, take 50±5 μL of each of the 8 to 12 concentration gradient dilution samples and apply them to the cell plate.

5. The detection method according to claim 4, wherein The complete culture medium described in step (1) is DMEM culture medium containing 10±1% FBS, 250-350 μg / mL Hygromycin B, and 650-750 μg / mL Geneticin.

6. The detection method according to claim 4, wherein The cells described in step (1) reached a confluence of 89-92%.

7. The detection method according to claim 4, wherein The working culture medium described in steps (2) and (3) is DMEM culture medium containing 1±0.1% FBS.

8. The detection method according to claim 1, wherein The determination comprises: (1) Place the cell plate in an incubator at 37 ± 2°C and 5 ± 0.5% CO2 for 5.5–7.5 h. Then, add 50 ± 5 μL of the colorimetric developer, Bright Glo, to the cell plate and read the luminescence value using a microplate reader. (2) The measured luminescence value and concentration are subjected to a four-parameter fitting curve to calculate the activity of the test sample.

9. The detection method according to claim 8, wherein Incubate as described in step (1) for 5.8 to 6.5 hours.

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