阅读说明：本技术 一种快速测定il-2蛋白药物和抗cd25抗体药物的生物学活性方法 (Method for rapidly determining biological activity of IL-2 protein drug and anti-CD 25 antibody drug ) 是由 王兰 段茂芹 于传飞 张峰 刘春雨 付志浩 王文波 郭莎 郭潇 黄璟 徐苗 王 于 2020-12-30 设计创作，主要内容包括：本发明公开了一种快速测定IL-2蛋白药物和抗CD25抗体药物的生物学活性方法,所述方法通过构建慢病毒(pLV-STAT5-Luc-PGK-Zencin)转染的C8166效应细胞,经筛选后得到稳定表达STAT5报告基因的单克隆细胞株,用IL-2蛋白药物刺激激活报告基因的表达,并用抗CD25抗体药物阻断IL-2刺激激活的信号通路,根据测定的信号值拟合四参数曲线测定IL-2蛋白药物及抗CD25抗体药物的生物学活性。本发明提供的检测方法可评价药物对细胞信号下游的相关指示、无需任何人原代组织来源的细胞或其他组分、显色稳定、实验周期短、操作简便、成本低。(The invention discloses a method for rapidly determining biological activities of an IL-2 protein drug and an anti-CD 25 antibody drug, which comprises the steps of constructing C8166 effect cells transfected by lentivirus (pLV-STAT5-Luc-PGK-Zencin), obtaining a monoclonal cell strain stably expressing STAT5 reporter gene after screening, stimulating and activating the expression of the reporter gene by the IL-2 protein drug, blocking a signal path stimulated and activated by IL-2 by the anti-CD 25 antibody drug, and determining the biological activities of the IL-2 protein drug and the anti-CD 25 antibody drug by fitting a four-parameter curve according to the determined signal values. The detection method provided by the invention can evaluate the related indication of the drug to the downstream of the cell signal, does not need any human primary tissue-derived cell or other components, and has the advantages of stable color development, short experimental period, simple and convenient operation and low cost.)

1. A method for rapidly determining the biological activity of an IL-2 protein drug and/or an anti-CD 25 antibody drug, the method comprising: constructing an effector cell for stably expressing STAT5 reporter gene, stimulating and activating the expression of the reporter gene by using IL-2, blocking a signal path stimulated and activated by the IL-2 by using an anti-CD 25 antibody drug, and fitting a four-parameter curve according to the measured signal value of the reporter gene to determine the biological activities of the IL-2 protein drug and the anti-CD 25 antibody drug.

2. A method for rapidly determining the biological activity of an IL-2 protein drug and/or an anti-CD 25 antibody drug, comprising the steps of:

(1) constructing and obtaining effector cells for stably expressing STAT5 reporter genes;

(2) diluting an IL-2 protein medicament, and adding the diluted IL-2 protein medicament into the effector cells in the step (1) for incubation;

(3) diluting an anti-CD 25 antibody drug sample, and transferring the diluted antibody drug and an IL-2 protein drug with a certain concentration to the effector cells in the step (1) for incubation;

(4) adding an enzyme reaction substrate, and fitting a four-parameter curve according to the measured signal value to determine the biological activity of the IL-2 protein drug and the anti-CD 25 antibody drug;

preferably, the dilution described in step (2) is a 5-fold dilution;

preferably, the dilution described in step (3) is a 5-fold dilution.

3. The method according to claim 1 or 2, wherein the effector cells stably expressing the STAT5 reporter gene are obtained by transfecting C8166 cells, HDLM-2 cells, Mo7e cells, DS-1 cells, 293 cells, NSO cells, HeLa cells, SP2 cells, COS cells, 549A cells or human hepatoma cells with lentiviruses and adding corresponding antibiotics for screening;

preferably, the effector cells stably expressing the STAT5 reporter gene are obtained by transfecting C8166 cells with a vector containing the STAT5 reporter gene and adding corresponding antibiotics for screening;

more preferably, the antibiotic is Zencin.

4. The method according to claim 3, wherein the vector is pLV-STAT 5-PGK-Zencin.

5. The method of claim 1 or 2, wherein the anti-CD 25 antibody drug comprises a basilix antibody drug, a dalberg antibody drug;

preferably, the anti-CD 25 antibody drug is a basilix antibody drug.

6. The method of claim 1 or 2, wherein the IL-2 protein drug is a recombinant IL-2 protein drug.

7. The method according to claim 1 or 2, wherein the chemiluminescence values are detected using a luciferase kit, and an inverted S-shaped four-parameter curve is fitted to the obtained relative chemiluminescence unit values;

preferably, the relative chemiluminescent unit values are read using chemiluminescence on a microplate reader.

8. A C8166 effector cell stably expressing a STAT5 reporter gene, wherein the effector cell is constructed by the method of claim 3.

9. Use of the effector cell of claim 8 for the detection of an IL-2 protein drug and/or an anti-CD 25 antibody drug.

10. Use of the method of any one of claims 1-7 for quality control of an IL-2 protein drug and/or an anti-CD 25 antibody drug.

Technical Field

The invention belongs to the field of biological activity detection of biological medicines, and particularly relates to a method for rapidly determining biological activities of an IL-2 protein medicine and an anti-CD 25 antibody medicine.

Background

The therapeutic monoclonal antibody (monoclonal antibody for short) as an important biotechnological product has the advantages of high specificity, strong targeting property, definite curative effect and the like, is an antibody which is generated by a single B cell clone, is highly uniform and only aims at a certain specific epitope, and has obvious curative effect in treating tumors, autoimmune diseases, infectious diseases and transplant rejection at present. In 2018, the sales of monoclonal antibody drugs accounts for 55.3% of the biological drug market in the world. In addition, among the first twenty medicines sold worldwide in 2019, the single-resistant medicines in 13 biological medicines occupy 9 seats, and the single-resistant medicines are one of the most important subdivision fields of the global pharmacy at present and also become an important part of the global medicine market.

In the face of the rapid development of antibody drugs, a corresponding antibody drug quality evaluation technical system is urgently needed to be established, and the determination of the biological activity on the cell level is one of the most effective modes for determining the effective components and content of the drugs and the drug potency and is also an important quality control index for ensuring the effectiveness of the antibody drugs, so that the method plays an important role in the discovery and development of the antibody drugs. At present, cell-based biological activity measurement methods are mostly adopted in biological activity detection methods, and mainly comprise a cell proliferation inhibition method, a cytotoxicity method, an antibody-dependent cell-mediated cytotoxicity method, a complement-dependent cytotoxicity method and a cell competition ELISA method.

Interleukin 2(IL-2) is a 15.5kD glycoprotein produced by T cells and NK cells, predominantly by CD4⁺T cell production, other immune cells such as CD8⁺T cells, NK cells can also produce small amounts of IL-2. IL-2 was discovered in 1976 to be able to sustain long-term growth of T cells in vitro and was therefore designated as T cell growth factor, which was uniformly designated as IL-2 by the second International lymphokine conference in 1979. IL-2 exerts its physiological effects through the interleukin-2 receptor (IL-2R), which is a diverse group of complexes classified by their affinity for IL-2 into low, medium, and high affinity receptors, each of which is composed of one or more of the three subunits, IL-2R α (CD25), IL-2R β (CD122), and IL-2R γ (CD 132). At present, the traditional Chinese medicine composition is widely used for treating tumors, viral hepatitis and the like clinically, and has a remarkable curative effect.

At present, CTLL-2 is adopted in Chinese pharmacopoeia to detect the biological activity of IL-2 protein drugs, the cells are suspension cells, and the cells can be used for analysis about 3 weeks after recovery. In addition, 10% CTLL-2 cell supernatant needs to be added into a complete culture medium during cell recovery, and the supernatant needs to be reserved for standby in the ordinary culture process. The cell culture solution is RPMI1640+ 10% FBS + 1% streptomycin +100IU/mL IL-2, and the cycle of the method is about 3-4 daysThe cells were washed 3 more times before plating on the first day, and the samples were loaded at 37 ℃ with 5% CO after plating₂Culturing for 18-24 hours under the condition. Adding MTT solution for 4-6 h, adding lysis solution, and continuing to add 5% CO at 37 deg.C₂Culturing for 18-24 hours under the condition, and performing aseptic operation in the whole process. And (4) mixing the cell sap uniformly, reading by using an enzyme-labeling instrument, and recording a measurement result. The method has the defects of long detection period, complex operation and the like.

The anti-CD 25 monoclonal antibody is used as a representative of a novel immunosuppressant, specifically acts on CD25, can effectively reduce the incidence rate of acute rejection by blocking the activation and proliferation of T cells, remarkably improves the survival rate of transplants and transplant recipients, reduces the dosage of hormone and shortens the using time of the hormone. At present, methods adopted for detecting the biological activity of basiliximab, dallizumab and related biological similar drugs include a competitive ELISA method and a flow cytometry method, wherein when the competitive ELISA method is used for detecting the biological activity of an anti-CD 25 monoclonal antibody, a downstream signal path cannot be reflected, and the applicability of a stability indication for release test cannot be evaluated; when the flow cytometry method is used for detecting the biological activity of the anti-CD 25 monoclonal antibody, related equipment and a large amount of consumables are needed, and the economic burden is increased for enterprises.

Currently, there remains a need in the art for novel assays for determining the biological activity of IL-2 protein drugs and anti-CD 25 monoclonal antibody drugs. In view of the above, the invention provides a method for evaluating biological activity according to chemiluminescence signal values of downstream cell signaling pathways, and IL-2 protein drugs and anti-CD 25 monoclonal antibody drugs are detected, so that an activity result can be obtained in the same day, the period is short, the operation is simple and convenient, the cost is low, no cells or other components from any human primary tissues are needed, and the color development is stable. On one hand, objective factors such as the possibility of cell contamination caused by long-time incubation are avoided, and on the other hand, the related indication of the drug to the cell signal downstream can be evaluated.

Disclosure of Invention

Aiming at the technical problems existing in the determination of the biological activities of IL-2 protein drugs and anti-CD 25 monoclonal antibody drugs at present, the invention aims to provide a method for rapidly determining the biological activities of IL-2 protein drugs and anti-CD 25 antibody drugs. The method comprises the steps of constructing C8166 effect cells transfected by lentivirus (pLV-STAT5-PGK-Zencin), obtaining a monoclonal cell strain stably expressing STAT5 reporter gene after two rounds of pressure screening, stimulating and activating the expression of the reporter gene by IL-2, blocking a signal path stimulated and activated by IL-2 by anti-CD 25 antibody drugs, and determining the biological activity of the IL-2 protein drugs and the anti-CD 25 antibody drugs by fitting a four-parameter curve according to the determined signal value.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect of the present invention, there is provided a method for rapidly determining the biological activity of an IL-2 protein drug and/or an anti-CD 25 antibody drug, the method comprising: constructing an effector cell for stably expressing STAT5 reporter gene, stimulating and activating the expression of the reporter gene by using IL-2, blocking a signal path stimulated and activated by the IL-2 by using an anti-CD 25 antibody drug, and fitting a four-parameter curve according to the measured signal value of the reporter gene to determine the biological activities of the IL-2 protein drug and the anti-CD 25 antibody drug.

In a second aspect, the present invention provides a method for rapidly determining the biological activity of an IL-2 protein drug and/or an anti-CD 25 antibody drug.

Further, the method comprises the steps of:

(1) constructing and obtaining effector cells for stably expressing STAT5 reporter genes;

(2) diluting an IL-2 protein medicament, and adding the diluted IL-2 protein medicament into the effector cells in the step (1) for incubation;

(3) diluting an anti-CD 25 antibody drug sample, and transferring the diluted antibody drug and an IL-2 protein drug with a certain concentration to the effector cells in the step (1) for incubation;

(4) adding an enzyme reaction substrate, and fitting a four-parameter curve according to the measured signal value to determine the biological activity of the IL-2 protein drug and the anti-CD 25 antibody drug;

preferably, the dilution described in step (2) is a 5-fold dilution;

preferably, the dilution described in step (3) is a 5-fold dilution.

Further, the effector cells for stably expressing the STAT5 reporter gene are obtained by transfecting C8166 cells, HDLM-2 cells, Mo7e cells, DS-1 cells, 293 cells, NSO cells, HeLa cells, SP2 cells, COS cells, 549A cells or human hepatoma cells through lentiviruses and adding corresponding antibiotics for screening;

preferably, the effector cells stably expressing the STAT5 reporter gene are obtained by transfecting C8166 cells with a vector containing the STAT5 reporter gene and adding corresponding antibiotics for screening;

more preferably, the antibiotic is Zencin.

The C8166 cells belong to human T lymphocyte leukemia cells, and express IL-2R alpha (CD25), IL-2R beta (CD122) and IL-2R gamma (CD132) receptors on the surface, and after the receptors are combined with ligands, downstream signal channels are activated, and enter a nuclear recognition and combination sequence, so that a signal value is generated. And (2) carrying out lentivirus packaging on the synthesized and constructed pLV-STAT5-PGK-Zencin, then transferring the packaged product into a C8166 cell, activating a JAK-STAT5 signal channel after a ligand is combined with a receptor, and starting the expression of a luciferase reporter gene connected with a STAT5 sequence. The anti-CD 25 antibody competes with IL-2 for binding to CD25, blocking the signaling pathway, and therefore, the concentration of anti-CD 25 antibody is inversely proportional to the amount of luciferase expressed in the effector cells.

Further, the vector is pLV-STAT 5-PGK-Zencin.

In the embodiment of the invention, the preparation method of the effector cell for stably expressing the STAT5 reporter gene comprises the steps of introducing a vector containing a STAT5-Luciferase sequence into a C8166 cell, and adding a corresponding antibiotic for screening;

preferably, the vector containing the STAT5-Luciferase sequence is constructed pLV-STAT 5-PGK-Zencin.

Further, the anti-CD 25 antibody drugs include a balixb antibody drug, a dalized bead antibody drug;

preferably, the anti-CD 25 antibody drug is a basilix antibody drug.

Furthermore, the IL-2 protein drug is a recombinant IL-2 protein drug.

Further, the first or second aspect of the present invention is a method of detecting chemiluminescence values using a luciferase kit, fitting an inverted S-shaped four-parameter curve based on the relative chemiluminescence unit values obtained;

preferably, the relative chemiluminescent unit values are read using chemiluminescence on a microplate reader.

In embodiments of the invention, chemiluminescence values are detected using a luciferase kit including (but not limited to): the detection of the reporter gene is carried out according to the instruction manual by a Bright-Glo Luciferase kit of Promega, a Luciferase reporter gene detection kit of Biyunnan, a Luciferase reporter gene detection kit of Biovision and other kits based on Luciferase luminescence detection;

preferably, the luciferase kit is a Bright-Glo luciferase kit from Promega.

Luciferase (Luciferase) is a generic term for a class of enzymes that are derived from organisms capable of emitting light in nature and that catalyze the oxidative emission of Luciferin (Luciferase) or fatty aldehydes in the organism. Luciferases can be classified into Firefly Luciferase (FL) and Bacterial Luciferase (BL) depending on the species of the organism from which they are derived. Currently, the luciferase gene derived from firefly in North America is most widely used, and the gene can code for a luciferase protein producing 550 amino acids. FL gene is from firefly, in Mg²⁺、ATP、O₂In the presence of (2), the D-luciferin (D-luciferin) is catalyzed to be subjected to oxidative decarboxylation to generate the activated oxyluciferin, and photons are released to generate fluorescence of 550-580 nm.

Luciferase reporter gene detection is an important tool for analyzing the interaction relationship between potential cis-elements (such as promoters, enhancers, silencers and the like) and trans-acting factors in the flanking region of a structural gene in the field of modern molecular biology research. The luciferase reporter gene system is a reporter system for detecting the activity of firefly luciferase (firefly luciferase) by using Luciferin (luciferase) as a substrate. Luciferase catalyses the oxidation of Luciferin to Oxyluciferin, which in turn fluoresces biologically (Bioluminescence) and is then measured by a chemiluminometer or a scintillation counter.

In the examples of the present invention, Relative chemiluminescence unit values (RLU) were read on a microplate reader using chemiluminescence, and an inverted S-shaped four-parameter curve was fitted by data processing. The inverted S-shaped four-parameter curve can reflect indexes such as upper and lower asymptotes, an IC50 value, a slope and the like;

preferably, the data processing is performed using graphpad7.0 to fit an inverse S-shaped four-parameter curve.

In the examples of the present invention, the relative potency of the samples was obtained by comparing the Half inhibition concentration values (IC 50) of the four parameter curves of the samples with the reference. The calculation formula is as follows: relative potency is reference IC 50/sample IC50 × 100%.

In a third aspect of the invention there is provided a C8166 effector cell stably expressing a STAT5 reporter.

Further, the effector cell is constructed by the method described in the second aspect of the present invention.

In the embodiment of the invention, the preparation method of the C8166 effector cells for stably expressing STAT5 reporter genes comprises the steps of introducing a vector containing a STAT5-Luciferase sequence into the C8166 cells, and adding corresponding antibiotics for screening;

preferably, the vector containing the STAT5-Luciferase sequence is constructed pLV-STAT 5-PGK-Zencin.

In a fourth aspect, the invention provides the use of an effector cell of the third aspect of the invention for the detection of an IL-2 protein drug and/or an anti-CD 25 antibody drug.

Further, the anti-CD 25 antibody drugs include a balixb antibody drug, a dalized bead antibody drug;

preferably, the anti-CD 25 antibody drug is a basilix antibody drug.

A fifth aspect of the invention provides the use of a method according to the first or second aspect of the invention for quality control of an IL-2 protein medicament and/or an anti-CD 25 antibody medicament.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Further, some terms are explained as follows:

the term "effector cell" as used in the context of the present application refers to a cell that naturally or artificially (e.g., by transfecting a vector containing a nucleic acid sequence encoding a protein of interest into a host cell) expresses a STAT5-luciferase reporter, and the effector cell in the present application functions as an effector cell in the JAK-STAT5 signaling pathway, or mimics an effector cell in the JAK-STAT5 signaling pathway.

The term "IL-2 protein drug" as used in the context of the present application, is a recombinant protein drug that activates the expression of the downstream gene of STAT5 in the effector cells described in the present application, which in turn initiates the expression of the luciferase reporter gene linked to the STAT5 sequence, whose main physiological role is to stimulate and maintain the differentiation and proliferation of T cells, and the biological activities of the tumor involved include: stimulating the proliferation and activation of Natural Killer (NK) cells and enhancing their activity; inducing cytotoxic lymphocytes to enhance cytolytic activity thereof; inducing lymphokine to activate and kill (LAK) cells, stimulating proliferation and enhancing activity of Tumor Infiltrating Lymphocytes (TIL), and the like.

The term "anti-CD 25 antibody drug" as used in the context of the present application refers to an antibody drug that targets CD25(IL-2 ra), and the anti-CD 25 antibody drugs described herein include balixb antibody drugs, dalized bead antibody drugs, and preferably balixb antibody drugs in the embodiments of the present invention. In the effector cells described herein, anti-CD 25 antibody drugs compete with IL-2 protein drugs for binding to CD25, thereby blocking the downstream signaling pathway of STAT 5.

The term "JAK-STAT 5 Signal pathway" used in the context of the present application refers to a Janus kinase (JAK) -Signal transducer and transcription activator 5(Signal transducer and activator of transcription5, STAT5) Signal pathway, which is a downstream pathway of cytokine Signal transduction, regulates development, differentiation, proliferation, apoptosis, etc. of cells, not only participates in regulating normal physiological processes, but also plays an important role in the occurrence and development of tumors, and especially has significance in hematological tumors. It is composed of three components, namely the tyrosine kinase-associated receptor, JAK and STAT 5.

The term "biological activity" as used in the context of the present application refers to the ability of a biological product to achieve a defined biological effect based on its specific ability or potential to evaluate the corresponding activity of the biological product using the biological effect of a specific cell line (target cell).

The term "four-parameter curve" as used in the context of the present application refers to a curve fitted according to the four-parameter equation Y ═ Bottom + (Top-Bottom)/(1+10^ ((LogEC50-X) × HillSlope)), which gives four parameters Top, Bottom, EC50, HillSlope, etc.

In order to determine the biological activity of IL-2 protein drugs and anti-CD 25 antibody drugs, an effector cell capable of stably expressing STAT5-luciferase reporter genes needs to be established, and the effector cell in the detection method can be obtained only by ensuring that the cell can meet the conditions and accessories required by JAK-STAT5 signal pathway transduction and the initiation of the expression of the luciferase reporter genes connected with the STAT5 sequence. Based on this, the inventors of the present application selected various host cells (C8166, HDLM-2, Mo7e) conventionally used in the art for expressing foreign genes for testing and screening of effector cells as the method of the present invention. As a result, it was found that even if a vector containing a STAT5-luciferase reporter sequence was successfully transfected into each of the above-mentioned host cells, it could not be guaranteed that the successfully transfected host cells could be used as effector cells for determining the biological activities of IL-2 protein drugs and anti-CD 25 antibody drugs, and among these cells, only C8166 cells were a parent cell line capable of satisfying the conditions and accessories required for JAK-STAT5 signaling pathway transduction and initiation of expression of a luciferase reporter linked to a STAT5 sequence.

Through a large amount of cell screening work, the inventor selects human T lymphocyte leukemia cell C8166 cell for constructing effector cells in the detection method. The detection effect of the effector cells constructed based on the C8166 cells is obviously better than that of effector cells constructed by other types of cells. Aiming at a C8166 cell which grows in a suspended manner, a slow virus transfection mode under a specific condition is selected, an effect cell which stably and highly expresses STAT5-luciferase reporter gene is successfully constructed, a cell strain which stably and highly expresses STAT5-luciferase reporter gene, highly expresses luciferase and has high signal to noise ratio is obtained by comparing the expression reactivity condition of different monoclonal effect cell strains to luciferase and the performance of measuring the signal to noise ratio and the like of the biological activity of IL-2 protein drugs and anti-CD 25 antibody drugs, and a rapid and accurate quantitative detection method for the biological activity of IL-2 protein drugs and anti-CD 25 antibody drugs is established.

The invention has the following advantages and beneficial effects:

(1) the invention establishes a rapid and accurate quantitative detection method aiming at the detection of the biological activity of IL-2 protein drugs and anti-CD 25 antibody drugs.

(2) The invention screens a plurality of host cells (including C8166, HDLM-2 and Mo7e) which are conventionally used for expressing exogenous genes, and finally screens the C8166 host cells with better luciferase expression reactivity to the stimulation of IL-2.

(3) Compared with the prior art, the detection method provided by the invention can detect results at different times after the detection components are added, and no cells or other components of any human primary tissue source are needed, so that the color development is more stable, and the quality is more controllable.

(4) The detection method provided by the invention has the advantages of short experimental period, simple and convenient operation, stable and reliable detection result, low cost and the like, and can obtain an activity result in the same day, thereby avoiding objective factors such as cell pollution possibility caused by long-time incubation and multi-step operation on the one hand, and evaluating related indication of a drug to cell signal downstream on the other hand.

(5) The detection method provided by the invention can be used for evaluating and screening IL-2 protein drugs and anti-CD 25 antibody drugs, and can be used as an effective measure in the early development and screening processes of IL-2 protein drugs and anti-CD 25 antibody drugs.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 is a four-parameter plot of a C8166 monoclonal cell strain against drug-stimulated effector cell luciferase with IL-2 protein after lentivirus transfection;

FIG. 2 is a graph of four parameters of drug-stimulated effector cell luciferase with IL-2 protein at various concentrations after optimization;

FIG. 3 is a graph of four parameters of drug stimulation of effector cell luciferase by anti-CD 25 antibody protein at various concentrations after optimization;

FIG. 4 is a four parameter plot of HDLM-2 cell line versus drug stimulation of the IL-2 protein by the effector cell luciferase following lentivirus transfection;

FIG. 5 is a graph of four parameters of Mo7e cell line on drug stimulation of effector luciferase with IL-2 protein after lentiviral transfection;

FIG. 6 is a four parameter plot of drug stimulation of effector cell luciferase by IL-2 protein after transfection of three different effector cells;

FIG. 7 is a graph of four parameters obtained by a conventional method for determining the biological activity of an IL-2 protein drug;

FIG. 8 is a graph of four parameters obtained by a conventional method for determining the biological activity of an anti-CD 25 antibody drug;

FIG. 9 is a PLA analysis diagram obtained by a conventional method for determining the biological activity of an anti-CD 25 antibody drug.

Detailed Description

The present invention is further illustrated below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention. As will be understood by those of ordinary skill in the art: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents. The following examples are examples of experimental methods not indicating specific conditions, and the detection is usually carried out according to conventional conditions or according to the conditions recommended by the manufacturers.

Example 1 construction and screening of monoclonal cell lines stably expressing STAT5-luciferase 1, Experimental materials

C8166 cells were derived from ATCC; pLV-STAT5-PGK-Zencin, available from Ghman bioengineering, Inc.; IL-2 was purchased from biosystemmsACRO; basilixib antibody (monoclonal antibody drug against CD25) is a monoclonal antibody chamber retention (noval); the Bright-Glo luciferase kit was purchased from Promega.

2. Lentiviral transduction

Adopting lentivirus packaging method to transduce C8166 cells, adding 50 μ L of constructed lentivirus plasmid into 96-well plate, and adding 50 μ L of the plasmid with density of 1 × 10 into the same well⁶one/mL of C8166 cells, 50. mu.L of 10% FBS +1640 medium and 50. mu.L of 1X 10 density medium were added to another well⁶The C8166 cells/mL were used as a blank, centrifuged at 1000rpm for 30 minutes, and then removed and incubated in an incubator for 30 minutes. mu.L of the mixture of lentiviral plasmid and cells was placed in a 24-well plate, 2mL of 1640 fresh medium containing 10% FBS was added, and after incubation in an incubator for 48 hours, 2mL of the cell mixture was transferred to a 12-well plate, 2mL of 1640 fresh medium containing 10% FBS was added, and Zencin was added to the cell culture medium to a final concentration of 200. mu.g/mL for pressure screening.

3. Screening of monoclonal cell lines

After the cell density and viability were restored, monoclonal clones were screened in 96-well plates at a density of 0.5/well by limiting dilution. During the cell growth period, observing and marking which wells are monoclonals, and when the confluency of C8166 cells in the monoclonals reaches more than 50%, transferring the cells in the wells to a 24-well plate for gradually expanding culture. Diluting to 2.5X 10⁵cell/mL cells, 100 u L per hole to 96 hole plate, IL-2 diluted to three concentrations of 0.5 u g/mL, 0.05 u g/mL, 0 u g/mL, each 10L. 37 ℃ and 5% CO₂After 6 hours of incubation, 50. mu.L of Bright-Glo was added to each well and the chemiluminescence was measured (see FIG. 1).

And selecting the cells with the highest signal-to-noise ratio for secondary screening of the monoclonal cells. Single colonies were screened in 96-well plates at a density of 0.5/well by limiting dilution. During the cell growth, observing and marking which wells are monoclonal, when the confluency of C8166 cells in the monoclonal wells reaches more than 50%, transferring the cells in the wells to a 24-well plate, and gradually enlarging the culture until the subculture is stable. Selecting cell sap with the survival rate of more than 80 percent, and diluting the cell sap to 2.5 multiplied by 10⁵cell/mL, 100. mu.L per well plated in 96-well plates, IL-2 diluted to 0.16. mu.g/mL, 3.2X 10^-2μg/mL，6.4×10^-3μg/mL，1.28×10^-3μg/mL，2.56×10^-4μg/mL，5.12×10^-5μg/mL，1.024×10^-5μg/mL，2.048×10^-6μg/mL，4.096×10^-7mu.g/mL, 10. mu.L per well. 37 ℃ and 5% CO₂After 24 hours of incubation, 100. mu.L of Bright-Glo was added to each well and the chemiluminescence was measured (see FIG. 2).

Selecting cell sap with the survival rate of more than 80 percent, and diluting the cell sap to 2.5 multiplied by 10⁵cell/mL, 100. mu.L per well plated in 96-well plates, 10. mu.L of IL-2 at 4ng/mL concentration per well, and anti-CD 25 antibody diluted to 20. mu.g/mL, 4. mu.g/mL, 0.8. mu.g/mL, 0.16. mu.g/mL, 3.2X 10^-2μg/mL，6.4×10^-3μg/mL，1.28×10^-3μg/mL，3.2×10^-5μg/mL，6.4×10^-6μg/mL，1.28×10^-6μg/mL，2.56×10^-7μ g/mL, 10 μ L per well. 37 ℃ and 5% CO₂After 24 hours of incubation, 100. mu.L of Bright-Glo was added to each well and the chemiluminescence was measured (see FIG. 3).

4. Results of the experiment

The result shows that the screened monoclonal cell strain C8166 stably expressing STAT5-luciferase has better luciferase expression reactivity to IL-2 stimulation (see the figure 1-3), so the monoclonal cell strain C8166 stably expressing STAT5-luciferase constructed by the method can be used as an experimental cell.

Example 2 comparison of different effector cells

1. Experimental Material

C8166 cells were derived from ATCC; pLV-STAT5-PGK-Zencin was purchased from Ghman bioengineering, Inc.; IL-2 was purchased from biosystemmsACRO; basilixib antibody (monoclonal antibody drug against CD25) is a monoclonal antibody chamber retention (noval); the Bright-Glo luciferase kit was purchased from Promega.

2. Lentiviral transduction

Adopting a lentivirus packaging method to transduce HDLM-2 and Mo7e cells, respectively taking 50 mu L of constructed lentivirus plasmids, adding the lentivirus plasmids into a 96-well plate, and adding 50 mu L of lentivirus plasmids into the same well, wherein the density of the lentivirus plasmids is 1 multiplied by 10⁶one/mL of HDLM-2 and Mo7e cells were added to two additional wells, respectively, with 50. mu.L of 10% FBS +1640 medium and 50. mu.L of 1X 10 density medium⁶The individual/mL HDLM-2 and Mo7e cells were used as blank control, centrifuged at 1000rpm for 30 minutes, and then taken out and incubated in an incubator for 30 minutes. mu.L of the mixture of lentiviral plasmid and cells was added to a 24-well plate, 2mL of 1640 fresh medium containing 10% FBS was added, and the incubation was carried out in an incubator for 48 hours. 2mL of the cell mixture was transferred to a 12-well plate, 2mL of 1640 fresh medium containing 10% FBS was added, and Zencin was added to the cell culture medium to a final concentration of 200. mu.g/mL for pressure screening.

3. Screening of monoclonal cell lines

After the cell density and viability were restored, monoclonal clones were screened in 96-well plates at a density of 0.5/well by limiting dilution. During cell growth, observing and marking which wells are monoclonal, when the confluency of HDLM-2 and Mo7e cells in the monoclonal wells reaches more than 50%, transferring the cells in the wells to a 24-well plate, and gradually expanding and culturing. Diluting to 2.5X 10⁵cell/mL, 100. mu.L per well plated in 96-well plates, IL-2 diluted to three concentrations of 0.5. mu.g/mL, 0.05. mu.g/mL, 0. mu.g/mL, 10. mu.L per well, 37 ℃, 5% CO₂After 6 hours of incubation, 50. mu.L of Bright-Glo was added to each well and the chemiluminescence was measured (see FIGS. 4 and 5).

4. Comparison of three different effector cells after transfection

Three different cells were added to a 96-well plate and diluted to 2.5X 10⁵cell/mL, 100. mu.L per well plated into 96-well plates, IL-2 diluted to three concentrations of 0.5. mu.g/mL, 0.05. mu.g/mL, 0. mu.g/mL, 10. mu.L per well, 37 ℃, 5% CO₂After 6 hours of incubation, 50. mu.L of Bright-Glo was added to each well and the chemiluminescence was measured (see FIG. 6).

5. Results of the experiment

The results show that when the vectors containing STAT5-luciferase reporter gene sequences are successfully transfected into C8166, HDLM-2 and Mo7e host cells respectively, only the C8166 host cells have better luciferase expression reactivity to IL-2 stimulation (see figures 4-6), and the C8166 host cells are the parent cell lines which can meet the conditions and accessories required by JAK-STAT5 signal pathway transduction and starting of expression of the luciferase reporter gene connected with the STAT5 sequences.

Example 3 verification of the detection method

1. Compared with the methodology of the traditional IL-2 protein drug activity detection method

CTLL-2 is adopted in Chinese pharmacopoeia to detect the biological activity of IL-2 protein drugs, the cells are suspension cells, and the cells can be used for analysis about 3 weeks after recovery. In addition, 10% CTLL-2 cell supernatant needs to be added into a complete culture medium during cell recovery, and the supernatant needs to be reserved for standby in the ordinary culture process. The cell culture solution is RPMI1640+ 10% FBS + 1% streptomycin +100IU/mL IL-2, the cycle of the method is about 3-4 days, the cells are washed for more than 3 times before being plated on the first day, the samples are added after being plated, the temperature is 37 ℃, and the CO content is 5%₂Culturing for 18-24 hours under the condition. Adding MTT solution for 4-6 h, adding lysis solution, and continuing to add 5% CO at 37 deg.C₂Culturing for 18-24 hours under the condition, and performing aseptic operation in the whole process. And (4) mixing the cell sap uniformly, reading by using an enzyme-labeling instrument, and recording a measurement result.

The traditional method has the defects of long detection period, complex operation, low signal-to-noise ratio and the like (see fig. 7).

2. Compared with the methodology of the traditional anti-CD 25 antibody drug activity detection method

The biological activity of the anti-CD 25 antibody is detected by cell competition ELISA method.

The biological activity assay method of the preparation is actually a cell biological activity assay method which competes for binding activity, cannot reflect downstream signaling pathways, lacks applicability for release and stability indication, and results require calculation of relative activity using PLA analysis (see fig. 8 and 9).

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.