CN107760760A - A kind of method of the receptor antibody pharmaceutical biology activity of quick measure IL 6/IL 6 - Google Patents
A kind of method of the receptor antibody pharmaceutical biology activity of quick measure IL 6/IL 6 Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
Abstract
The present invention relates to a kind of quick measure IL 6 and the method for the receptor antibody pharmaceutical biologies of IL 6 activity.Methods described includes structure and obtains the effector cell of stable expression SIE reporter genes, activation reporter gene expression is stimulated with IL 6, and the signal paths of IL 6 are blocked with IL 6/IL 6R antibody drugs, being fitted four parameter curves according to the reporter gene signal value of measure determines antibody biological activity.The present invention establishes more fast and accurately quantitative detecting method for IL 6/IL 6R antibody drug Determination of biological activity, and experimental period is short, easy to operate, and avoids for a long time caused by incubation the problem of the objective factor such as cell contamination possibility.
Description
Technical field
The present invention relates to bio-pharmaceutical Activity determination field, for IL-6/IL-6R monoclonal antibody drug biological activities
Measure establishes more fast and accurately quantitative detecting method.
Background technology
Monoclonal antibody has become the important component of Global Medicine market, and contribute to main market dilatation power
Measure, the monoclonal antibody drug before global best-selling drugs ranking in 2016 in ten heavy pound medicine headed by adalimumab occupies half
Rivers and mountains.In face of the fast development of antibody class medicine, there is an urgent need to establish corresponding antibody drug quality evaluation technical system, and live
Property measure be to the active ingredient and content of medicine and the measure of drug titers, be to ensure that the important of antibody class drug effectiveness
Quality control index.Activity test method uses the biological activity determination method based on cell more at present, including cell inhibitory effect method,
Cytotoxicity method, the cell toxicant method of Complement Dependent, the cytotoxicity method of antibody dependent cellular mediation and CELISA.
Torr pearl monoclonal antibody (Tocilizumab, the refined Metro of trade name) was a kind of IL-6 receptor blocking pharmacons, on January 8th, 2010
FDA approval listings are obtained, moderate to the severe activity rheumatoid for treating to antirheumatic (DMARDs) response deficiency is closed
Save scorching adult patients.Torr pearl monoclonal antibody is combined with membranous type or soluble IL-6 acceptors, is blocked the combination of itself and IL-6, is made IL-6
Signal path can not be activated, so as to suppress the related mechanism of panimmunity reaction, including the activation of B cell and T cell differentiation
Into inflammatory cell etc., the symptom of rheumatoid arthritis can be effectively improved.
The Determination of biological activity method of Torr pearl monoclonal antibody is mainly the method for cell inhibitory effect at present, and this method is according to right
IL-6 has the human leukemia cell line KT-3/DS-1 of growth dependence, adds Torr pearl monoclonal antibody and IL-6, both competitive bindings are thin
Cellular surface IL-6 acceptors, after being incubated 72 hours, made by determining the quantity of cell to quantify the cell inhibitory effect of Torr pearl monoclonal antibody
With.Length this method experimental period (72 hours Torr pearl monoclonal antibody action time, adding incubation time 3 hours after substrate), variation is larger.
The present invention uses transgenic cell measuring method for activity, and experimental day can obtain result, and experimental period is short, operation letter
Just, and avoid for a long time caused by incubation the problem of the objective factor such as cell contamination possibility.
The content of the invention
The invention provides a kind of method of quick measure IL-6/IL-6R antibody drug biological activities, methods described bag
Include structure and obtain the effector cell of stable expression SIE reporter genes, stimulate activation reporter gene expression with IL-6, and use IL-6/
IL-6R antibody drugs block IL-6 signal paths, and being fitted four parameter curves according to the reporter gene signal value of measure determines antibody
Biological activity.
It is an object of the invention to provide a kind of method of quick measure IL-6/IL-6R antibody drug biological activities, institute
The method of stating includes：
(1) structure obtains the effector cell of stable expression SIE reporter genes；
(2) IL-6 is added into (1) described cell moderate stimulation and activates reporter gene expression；
(3) IL-6/IL-6R antibody drugs sample and reference product equal proportion are diluted, the antibody after dilution is shifted respectively
In cell and IL-6 mixtures into step (2), it is incubated in 37 DEG C of incubators；
(4) luciferase substrate is added, being fitted four parameter curves according to the reporter gene signal value of measure determines that antibody is given birth to
Thing activity.
DS-1 cells belong to IL-6 dependent Mice bone-marrow-derived lymphocytes, and its surface expression has IL-6 acceptors (by ligand binding chain α
Chain and signal transduction chain gp130 compositions), IL-6 is combined with IL-6R, and the JAK albumen of activation and gp130 couplings simultaneously activates STAT3,
STAT3 albumen forms dimer, is identified into nucleus and combines GAS or SIE sequences, so as to maintain the survival of DS1 cells
And propagation.SIE-luciferase sequences importing DS-1 is intracellular, and activation JAK-STAT signals lead to after IL-6 is combined with IL-6R
Road, and start the expression for the luciferase reporter gene being connected with SIE sequences.Anti-IL-6R antibody and IL-6 competitive bindings IL-
6 acceptors, IL-6 signal paths are blocked, therefore anti-IL-6R antibody concentration is inversely proportional with the luciferase expression amount in effector cell.
In embodiments of the invention, the effector cell of stable expression SIE reporter genes be selected from DS-1 cells,
MH60.BSF2 cells, the cell of B9 cells 293, Chinese hamster ovary (CHO) cell, NSO, SP2 cell, HeLa cells, young hamster
Any of kidney (BHK) cell, MK cells (COS), human liver cancer cell, 549A cells, 3T3 cells, preferably stable table
Up to the DS-1 cells of SIE reporter genes.
In embodiments of the invention, reporter gene is luciferase luciferase reporter genes.
In embodiments of the invention, the step (1) includes：Transfected with the Reporter gene vector comprising SIE sequences
DS-1 cells, MH60.BSF2 cells, B9 cells, 293 cells, Chinese hamster ovary (CHO) cell, NSO, SP2 cell, HeLa
Cell, baby hamster kidney (BHK) cell, MK cells (COS), human liver cancer cell, 549A cells or 3T3 cells, and add corresponding
Antibiotic-screening obtains the monoclonal cell strain of stable expression reporter gene.
In embodiments of the invention, the preparation side of stable expression SIE-luciferase DS-1/SIE-luc cells
Method is, the vector introduction DS-1 containing SIE-luciferase sequences is intracellular.It is furthermore preferred that there are SIE-luciferase sequences
The carrier of row is commercially available pGL4.47 [luc2P/SIE/Hygro] plasmid.
In embodiments of the invention, effector cell's suspension is prepared, according to 2.5 × 104-2×105Individual/hole, preferably 5
×104The cell density in individual/hole.
In embodiments of the invention, IL-6 activation reporter gene expressions are added, IL-6 concentration is 10pg/mL-2 μ g/
ML, preferably IL-6 concentration are 100ng/mL.
In embodiments of the invention, IL-6/IL-6R antibody drugs for it is commercially available and grind for IL-6 or IL-6 by
The monoclonal antibody of body, it is furthermore preferred that Torr pearl monoclonal antibody (Tocilizumab), Sarilumab, Siltuximab, outstanding auspicious monoclonal antibody,
WBP216 (MEDI5117), Mai Botaike CMAB806, the Dan Ke that Haizheng Pharmaceutical Co official written reply number is 2016L09574 of the bright Kant of medicine
Grand antibody.
In embodiments of the invention, Torr pearl monoclonal antibody sample and reference product dilution initial concentration are 1pg/mL-1mg/mL,
Preferably 125 μ g/mL.
In embodiments of the invention, Torr pearl monoclonal antibody sample and the ratio of reference product equal proportion dilution are 1:2-1:5, it is excellent
Elect 1 as:3.
In embodiments of the invention, incubation time is 4-22 hours, preferably 6 hours.
In embodiments of the invention, values of chemiluminescence is detected using luciferase kit, is preferably, used
Bio-Glo luciferase kits (product article No. G7940), the Luciferase of Biovision companies of promega companies are glimmering
Light element enzyme reporter gene detection kit (product article No. K801-200), the luciferase reporter gene detection examination of green skies company
Agent box etc. detects values of chemiluminescence.
Luciferase (luciferase) comes from the biology that nature can light, be can be catalyzed in vivo it is glimmering
The general name of light element (luciferin) or the class of enzymes of fatty aldehyde oxyluminescence., can be by fluorescence according to the difference of source organism species
Plain enzyme is divided into firefly luciferase (FL) and bacterial luciferase (BL).At present, with the luciferase in North America firefly source
The most extensive, the luciferase protein of the gene codified 550 amino acid of generation of gene application.FL genes come from the light of firefly
Worm, in Mg2+、ATP、O2Participation under, be catalyzed D- fluoresceins (D-luciferin) oxidative deamination, the oxidation for producing activated state is glimmering
Light element, and photon is released, produce 550-580nm fluorescence.
Luciferase reporter gene detection is that analytical structure gene side areas is dived in modern molecular biology research field
Cis element (such as promoter, enhancer and silencer) and trans-acting factor interaction relationship a kind of important work
Tool.Luciferase reporter gene system is for substrate, to detect firefly luciferase with fluorescein (luciferin)
(fireflyluciferase) active a kind of reporting system.Luciferase can be catalyzed luciferin and be oxidized to
Oxyluciferin, during luciferin is aoxidized, bioluminescence (bioluminescence) can be sent and then passed through
Chemiluminescence Apparatus or liquid flashing determining instrument measure.
In embodiments of the invention, relative chemical flat light emission value is read using chemiluminescence on ELIASA
(relative light unit, RLU) is fitted the parameter curve of the type of falling S four by data processing.The parameter curve of the type of falling S four, can be anti-
Mirror the index such as asymptote, IC50 values and slope up and down.Preferably, data processing fitting is carried out using SoftMax Pro 5.4
The parameter curve of the type of falling S four.
In embodiments of the invention, comparative sample and the 503nhibiting concentration (half of the parameter curve of reference product four are passed through
Maximal inhibitory concentration, IC50) value, draw the relative potency of sample.Calculation formula is：Relative effect
Valency=reference product IC50/ sample IC50 × 100%.
Brief description of the drawings
Fig. 1 is effector cell's uciferase activity measurement result of different clones
Fig. 2 is four parametric plots under DS-1/SIE-luc cell difference incubation times
Fig. 3 is four parametric plots under DS-1/SIE-luc cell difference IL-6 concentration
Fig. 4 is four parametric plots under DS-1/SIE-luc cell different antibodies concentration
Fig. 5 is four parametric plots under DS-1/SIE-luc cell difference cell densities
Fig. 6 is that detection method determines four kinds of monoclonal antibody medicine activity figures
Fig. 7 is that detection method and Proliferation Ability method compare figure
Embodiment
With reference to specific embodiment, the present invention is expanded on further, is only used for explaining the present invention, and it is not intended that to this
The limitation of invention.It will be understood by those skilled in the art that：Can in the case where not departing from the principle and objective of the present invention
So that these embodiments are carried out with a variety of change, modification, replacement and modification, the scope of the present invention is limited by claim and its equivalent
It is fixed.The experimental method of unreceipted actual conditions in the following example, generally according to normal condition or according to the bar proposed by manufacturer
Part examinations.
The stable expression SIE-luciferase of embodiment 1 DS-1 cell lines screening
1.1 experiment material
DS-1 cell deriveds are in ATCC；PGL4.47 [luc2P/SIE/Hygro] plasmid is bought in Promega companies；Culture
Bought with recombination human interleukin -6 (IL-6) in Prospec companies；Activity experiment with recombination human interleukin -6 (IL-6) buy in
Nanjing Nuo Ai neoformations Technology Co., Ltd.；Bio-Glo luciferase kits are bought in Promega companies.
1.2 plasmid transfection
Using NEONTMElectroporation transfection system (invitrogen) carries out pGL4.47 [luc2P/SIE/ to DS-1 cells
Hygro] plasmid transfection.After transfection 24 hours, add 100ug/mL Hygromycin B and carry out pressurization screening.Treat cell density
After vitality restoration, by limiting dilution assay, according to the density in 0.5/hole, paving 96 orifice plates screening monoclonal.In cell growth
Period, it is monoclonal to observe and mark which hole, when DS-1 cell confluencies degree reaches more than 30% in monoclonal hole, by this hole
Inner cell is transferred to 24 orifice plates, progressively expands culture.
1.3DS-1/SIE-luc cell lines screen
Primary dcreening operation：Spread per 10000, hole cell to 384 orifice plates, IL-6 be diluted to 50ng/mL, 5ng/mL, 0.5ng/mL,
0ng/mL, 37 DEG C, 5%CO2Overnight incubation, 40 μ L Steady-Glo are added per hole within second day, detect values of chemiluminescence.
Table 1DS-1/SIE clones primary dcreening operation values of chemiluminescence
| Con(ng/ml) | DS-1/SIE-6 | DS-1/SIE-7 | DS-1/SIE-8 | DS-1/SIE-13 | DS-1/SIE-27 |
| 50 | 8280 | 1160 | 3553 | 8821 | 2591 |
| 5 | 3412 | 593 | 2075 | 3914 | 1702 |
| 0.5 | 739 | 159 | 687 | 958 | 490 |
| 0 | 181 | 39 | 185 | 284 | 275 |
| Signal to noise ratio | 46 | 30 | 19 | 31 | 9 |
Choose the higher clones of signal to noise ratio such as DS-1/SIE-6,7,8,13,27 and enter secondary screening.
Secondary screening：Being spread per 10000, hole cell to 384 orifice plates, IL-6 is diluted to initial concentration 50ng/ml, and 1:2 dilutions 11
Concentration gradient, 37 DEG C, 5%CO2Overnight incubation, 40 μ L steady-Glo are added per hole within second day, detect values of chemiluminescence.
By secondary screening, stimulation of the DS-1/SIE-13 cells to IL-6 has preferable luciferase expression reactivity (see Fig. 1),
It is experiment cell to choose DS-1/SIE-13 cells, and is named as DS-1/SIE-luc cells.
The detection method of embodiment 2 optimizes
2.1 incubation times optimize
Because DS-1/SIE-luc cells are IL-6 dependent cells, lacking IL-6 cells can not grow, therefore explore one
Individual more suitable incubation time avoids DS-1/SIE-luc cells dead due to lacking IL-6.
Method：DS-1/SIE-luc cells are pressed 5 × 104Individual/hole is added in 96 hole blanks；The μ g/ of IL-6 initial concentrations 2
ML, 1:3 10 concentration gradients of dilution；37 DEG C, 5%CO2 cultures.Respectively Bio- is added in 4h, 6h, 8h, 10h, 12h and 22h
Glo, detect values of chemiluminescence, according to the values of chemiluminescence of measure be fitted four parameter curves and signal to noise ratio, determine one compared with
For suitable incubation time.
As a result：With the growth of incubation time, four parameter curve lower platforms gradually reduce (see Fig. 2), thus it is speculated that are because IL-
6 lack, cell death, therefore we select 6h both to can guarantee that signal to noise ratio as incubation time, be also avoided that cell death causes
Signal to noise ratio virtual height.
2.2 concentration optimization
According to the IL-6 activation experiment results of embodiment 2.1, IL-6 set 250ng/mL, 100ng/mL, 25ng/mL,
Tetra- different concentration of 10ng/mL, the Inhibition test of IL-6R monoclonal antibodies is carried out respectively, be fitted according to the values of chemiluminescence of measure
Four parameter curves determine IL-6 concentration.
Method：DS-1/SIE-luc cells are pressed 5 × 104Individual/hole is added in 96 hole blanks；Torr pearl monoclonal antibody is (by China
Food and medicine calibrating research institute monoclonal antibody product chambers are retained) initial concentration is 125 μ g/mL, 1:3 concentration of gradient dilution 10
Point, add in above-mentioned cell plates；IL-6 is diluted to 250ng/mL, 100ng/mL, 25ng/mL, 10ng/mL respectively, shifts respectively
Into above-mentioned cell plates；37 DEG C, 5%CO2Culture.Bio-Glo is added after 6h, detects values of chemiluminescence.
As a result show, IL-6 concentration selection 100ng/ml, can now reach good signal to noise ratio (see Fig. 3), can also save
Save IL-6 dosages.
2.3 antibody dose-effect scopes optimize
Torr pearl monoclonal antibody is diluted to higher concentration 1mg/mL, 1：3 dilutions, 20 concentration points, sent out according to the chemistry of measure
Four parameter curves of light value fitting determine the sphere of action of Torr pearl monoclonal antibody.
Method：DS-1/SIE-luc cells are pressed 5 × 104Individual/hole is added in 96 hole blanks；IL-6 is diluted to 100ng/
ML, it is transferred in above-mentioned cell plates；Torr pearl monoclonal antibody is diluted to 1mg/mL, and 1:3 concentration points of gradient dilution 20, will be anti-after dilution
Body is transferred in cell plates；37 DEG C, 5%CO2Culture.Bio-Glo is added after 6h, detects values of chemiluminescence.
As a result Fig. 4 is seen：According to IC50Value and upper lower platform calculate, and determine the final concentration of 125 μ g/mL of Torr pearl monoclonal antibody starting point,
1:4 dilutions, if ten concentration points, can ensure that upper lower platform respectively has two concentration points, 1 points of linear segment.
2.4 cell density optimization
Set 2.5 × 104Individual/hole, 5 × 104Individual/hole, 1 × 105Individual/hole, 1 × 105Individual/different cell in four, hole is close
Degree, the Inhibition test of IL-6R monoclonal antibodies is carried out respectively, four parameter curves being fitted according to the values of chemiluminescence of measure determine optimal thin
Born of the same parents' density.
Method：DS-1/SIE-luc cells are pressed 2.5 × 10 respectively4Individual/hole, 5 × 104Individual/hole, 1 × 105Individual/hole, 2 ×
105Individual/hole is added in 96 hole blanks；Add 100ng/mL IL-6；Torr pearl monoclonal antibody initial concentration is 125 μ g/mL, 1:4 is dilute
10 concentration gradients are released, are transferred in above-mentioned cell plates；37 DEG C, 5%CO2Culture.Bio-Glo is added after 6h, detects chemistry hair
Light value.
As a result：The signal to noise ratio being calculated according to upper lower platform, DS-1/SIE-luc cell densities 5 × 104Individual/hole is to 2
×105When in the range of individual/hole, signal to noise ratio is more or less the same, and cell density is little to SNR influence (see Fig. 5).Select cell close
Degree 5 × 104Individual/hole carries out subsequent experimental.
The checking of the detection method of embodiment 3
3.1 specificities are verified
This method is the biological activity method for anti-IL-6/IL-6R antibody, and therefore, the checking to its specificity is adopted
With the monoclonal antibody medicine of different target spots：Torr pearl monoclonal antibody (Tocilizumab, target spot IL-6R) Rituximab (Rituximab,
Target spot is CD20), (Bevacizumab, target spot are for Cetuximab (Cetuximab, target spot EGFR) and Avastin
VEGF).Under identical experiment condition, using our DS-1/SIE-luc reporter gene live body systems, these four lists are determined
The activity of antiradiation drug.
Method：DS-1/SIE-luc cells are pressed 5 × 104Individual/hole is added in 96 hole blanks；Add 100ng/mL's
IL-6；Four kinds of monoclonal antibodies are diluted to initial concentration 125 μ g/mL respectively, 1:4 10 concentration gradients of dilution, are transferred to above-mentioned respectively
In cell plates；37 DEG C, 5%CO2Culture.Bio-Glo is added after 6h, detects values of chemiluminescence.
As a result Fig. 6 is seen：This method does not have dose-effect curve, explanation to the antibody drug of CD20, EGFR, VEGF target spot
This method is unsuitable for the other drugs in addition to IL-6/IL-6R target spots, it was demonstrated that this method specificity is preferable.
3.2 with the methods comparison of conventional method
The Determination of biological activity method of Torr pearl monoclonal antibody is mainly the method for cell inhibitory effect at present, and this method is according to right
IL-6 has the human leukemia cell line KT-3/DS-1 of growth dependence, adds Torr pearl monoclonal antibody and IL-6, both competitive bindings are thin
Cellular surface IL-6 acceptors, after being incubated 72 hours, made by determining the quantity of cell to quantify the cell inhibitory effect of Torr pearl monoclonal antibody
With.We determine the relative potency of same batch of sample, each experiment weight using conventional method and DS1-SIE reporter gene methods respectively
It is multiple 6 times.
Transgenic cell method：DS-1/SIE-luc cells are pressed 5 × 104Individual/hole is added in 96 hole blanks；Add
100ng/mL IL-6；Torr pearl monoclonal antibody initial concentration is 125 μ g/mL, 1:4 10 concentration gradients of dilution, are transferred in cell plates；
37 DEG C, 5%CO2Culture.Bio-Glo is added after 6h, detects values of chemiluminescence.
Proliferation Ability method：DS-1 cells are pressed 2 × 104Individual/hole is inoculated in 96 orifice plates；Add 10ng/mL IL-6；Support
Pearl monoclonal antibody initial concentration is 400 μ g/mL, 1:4 10 concentration gradients of dilution, are transferred in cell plates；37 DEG C, 5%CO2Culture 72
Hour.CCK8 nitrite ions are added into cell plates with 20 μ L/ holes, continue culture 3 hours, detection 450nm/650nm wavelength surveys its suction
Luminosity.
As a result display is (see figure:7)：Transgenic cell method measurement result has with conventional method Proliferation Ability method measurement result
Good uniformity, and the relative variability coefficient of transgenic cell method will be less than conventional method.Transgenic cell method greatly reduces
Experiment duration, can greatly improve operating efficiency.
Claims (15)
1. a kind of method of quick measure IL-6/IL-6R antibody drug biological activities, methods described obtain stabilization including structure
The effector cell of SIE reporter genes is expressed, stimulates activation reporter gene expression with IL-6, and hindered with IL-6/IL-6R antibody drugs
Disconnected IL-6 signal paths, four parameter curves are fitted according to the reporter gene signal value of measure and determine antibody biological activity.
2. a kind of method of quick measure IL-6/IL-6R antibody drug biological activities, methods described include：
(1) structure obtains the effector cell of stable expression SIE reporter genes；
(2) IL-6 is added into (1) described cell moderate stimulation and activates reporter gene expression；
(3) IL-6/IL-6R antibody drugs sample and reference product equal proportion are diluted, the antibody after dilution is transferred to step respectively
Suddenly in the cell and IL-6 mixtures in (2), it is incubated in 37 DEG C of incubators；
(4) luciferase substrate is added, being fitted four parameter curves according to the reporter gene signal value of measure determines antibody biology
Activity.
3. method according to claim 1 or 2, wherein the effector cell of stable expression SIE reporter genes is thin selected from DS-1
Born of the same parents, MH60.BSF2 cells, the cell of B9 cells 293, Chinese hamster ovary celI, NSO cells, SP2 cells, HeLa cells, bhk cell, COS are thin
Any of born of the same parents, human liver cancer cell, 549A cells, 3T3 cells, the DS-1 cells of preferably stable expression SIE reporter genes.
4. method according to claim 1 or 2, wherein reporter gene are luciferase luciferase reporter genes.
5. method according to claim 1 or 2, wherein IL-6/IL-6R antibody drugs be Torr pearl monoclonal antibody, Sarilumab,
Siltuximab, outstanding auspicious monoclonal antibody, WBP216, Mai Botaike CMAB806, the Haizheng Pharmaceutical Co official written reply number of the bright Kant of medicine are
2016L09574 monoclonal antibody etc. lists and in the antibody drug for IL-6 or IL-6 acceptors ground.
6. method according to claim 1 or 2, wherein the step (1) includes：With the reporter gene for including SIE sequences
Carrier transfection DS-1 cells, MH60.BSF2 cells, the cell of B9 cells 293, Chinese hamster ovary celI, NSO cells, SP2 cells, HeLa are thin
Born of the same parents, bhk cell, COS cells, human liver cancer cell, 549A cells or 3T3 cells, and add corresponding antibiotic and screen to obtain stabilization
Express the monoclonal cell strain of reporter gene.
7. method according to claim 1 or 2, it is characterised in that：Effector cell's suspension is prepared, according to 2.5 × 104-2×
105Individual/hole, preferably 5 × 104The cell density in individual/hole.
8. method according to claim 1 or 2, it is characterised in that：Add IL-6 activation reporter gene expressions, IL-6 concentration
For 10pg/mL-2 μ g/mL, preferably IL-6 concentration is 100ng/mL.
9. method according to claim 1 or 2, it is characterised in that：Torr pearl monoclonal antibody dilution initial concentration is 1pg/mL-1mg/
ML, preferably 125 μ g/mL.
10. method according to claim 1 or 2, it is characterised in that：The ratio of Torr pearl monoclonal antibody equal proportion dilution is 1:2-1:
5, preferably 1:3.
11. method according to claim 1 or 2, it is characterised in that：Incubation time is 4-22 hours, preferably 6 hours.
12. method according to claim 1 or 2, it is characterised in that：Chemiluminescence is detected using luciferase kit
Value, it is preferably, the Luciferase using the Bio-Glo luciferase kits of promega companies, Biovision companies is glimmering
Light element enzyme reporter gene detection kit, the detection chemiluminescence of the luciferase reporter gene detection kit of green skies company
Value.
13. method according to claim 1 or 2, it is characterised in that：On ELIASA relativization is read using chemiluminescence
Flat light emission value is learned, the parameter curve of the type of falling S four is fitted by data processing.
14. method according to claim 1 or 2, it is characterised in that：Pass through comparative sample and the parameter curve of reference product four
503nhibiting concentration value, draw the relative potency of sample.
15. application of the claim 1-14 methods describeds in the quality control of IL-6/IL-6R antibody drugs.
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| CN109912710A (en) * | 2019-03-07 | 2019-06-21 | 中国人民解放军军事科学院军事医学研究院 | PDGF-BB Determination of biological activity method based on reporter gene |
| CN110133241A (en) * | 2019-05-21 | 2019-08-16 | 中国食品药品检定研究院 | A kind of new method measuring recombinant human soluble gp130-Fc fusion protein biological activity |
| CN110358738A (en) * | 2019-08-22 | 2019-10-22 | 中国食品药品检定研究院 | A kind of stable measurement active method of anti-IgE antibodies pharmaceutical biology |
| CN110551756A (en) * | 2019-09-12 | 2019-12-10 | 宝船生物医药科技(上海)有限公司 | reporter gene cell strain and construction method and application thereof |
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| CN110358738B (en) * | 2019-08-22 | 2021-08-13 | 中国食品药品检定研究院 | Method for stably determining biological activity of anti-IgE antibody medicine |
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| CN110551756A (en) * | 2019-09-12 | 2019-12-10 | 宝船生物医药科技(上海)有限公司 | reporter gene cell strain and construction method and application thereof |
| CN111996172A (en) * | 2020-09-08 | 2020-11-27 | 中国食品药品检定研究院 | Method for determining biological activity of IL-4 targeted therapeutic antibody |
| CN112430623A (en) * | 2020-11-24 | 2021-03-02 | 中国食品药品检定研究院 | Method for rapidly determining pharmaceutical biological activity of CGRP/CGRP receptor antibody |
| CN112725409A (en) * | 2020-12-30 | 2021-04-30 | 中国食品药品检定研究院 | Method for rapidly determining biological activity of IL-2 protein drug and anti-CD 25 antibody drug |
| CN112725409B (en) * | 2020-12-30 | 2021-10-01 | 中国食品药品检定研究院 | Method for rapidly determining biological activity of IL-2 protein drug and anti-CD 25 antibody drug |
| CN114480406A (en) * | 2021-09-16 | 2022-05-13 | 广东翠点生物科技有限公司 | IL-1 signal path response element and application thereof |
| CN114480406B (en) * | 2021-09-16 | 2024-01-30 | 广东翠点生物科技有限公司 | IL-1 signal path response element and application thereof |
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