Method for detecting human chorionic gonadotropin activity

Abstract

The invention relates to a method for detecting the activity of human chorionic gonadotropin (hCG). A CHAPS solution is used as a sample diluent of hCG, and a lower first-hole concentration can be used. The method has good drug specificity and cell specificity, good stability indication property, small intermediate precision coefficient of variation, high average recovery rate, a linear fitting correlation coefficient close to 1, and a moderate relative activity detection range (e.g., 50% to 200%). The method can realize accurate and sensitive testing of the biological activity of hCG, and provides a key technology for quality control of drug production.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a method for detecting the activity of human chorionic gonadotropin. Background Art

[0002] Human chorionic gonadotropin (hCG) is a glycoprotein secreted by the trophoblast cells of the placenta. Both human chorionic gonadotropin and luteinizing hormone (LH) secreted by the pituitary gland are glycoprotein hormones. hCG and LH transmit hormone information by binding to the same receptor on the target cell. LH / hCG receptors are located on the cell membrane and are widely distributed in the body. They are expressed not only in the gonadal target tissues, but also in some non-gonadal tissues such as the prostate, breast, adrenal glands, thyroid gland, and cerebral cortex. Relevant studies in recent years have found that LH / hCG receptors are also present on the surface of some malignant tumor cells, such as endometrial cancer, cervical cancer, ovarian cancer, breast cancer, gastric cancer, etc. The hCG receptor molecule is relatively large, and the extracellular amino terminal region is composed of about 340 amino acids, rich in leucine, and plays an important role in the recognition of hormones. hCG binds to the LH / hCG receptor on the ovarian cell membrane, activates the adenylate cyclase system and stimulates the synthesis of steroid hormones, and participates in the reproductive process of mammals, such as the implantation of fertilized eggs, the regulation of gonadal development and functional activities.

[0003] In clinical practice, recombinant human chorionic gonadotropin (recombinant hCG) has been used to treat infertility, luteal insufficiency, functional uterine bleeding, cryptorchidism, male sexual dysfunction, threatened abortion or habitual abortion and other diseases. In the production process of related drugs, quality needs to be strictly controlled, among which biological activity is one of the important indicators of quality control. According to the physicochemical properties and pharmacodynamic characteristics of recombinant hCG, the activity determination of recombinant hCG can be divided into in vitro cell assay, in vivo assay, enzymatic reaction assay and immunological activity method, but each has its shortcomings. The current commonly used detection method is the mouse uterine weight gain method included in the "Chinese Pharmacopoeia", which is cumbersome to operate, has a long experimental cycle, and is greatly affected by individual differences.

[0004] Therefore, it is necessary to develop new methods for detecting the biological activity of recombinant hCG. Summary of the invention

[0005] Based on this, one of the purposes of the present invention is to provide a method for detecting the activity of human chorionic gonadotropin (hCG), which is an in vitro cell assay for hCG activity, wherein a CHAPS diluent is used to prepare an hCG test sample, the method has good drug specificity and cell specificity, good stability indicating property, small intermediate precision coefficient of variation, high average recovery rate, a linear fitting correlation coefficient close to 1, and a moderate relative activity detection range, and can realize accurate and sensitive testing of the biological activity of hCG, thereby providing a key technology for quality control of drug production.

[0006] In a first aspect of the present invention, a method for detecting human chorionic gonadotropin activity is provided, comprising the following steps:

[0007] Obtaining hCG analysis standards, hCG test products and tool cells, wherein the tool cells are cells capable of expressing G protein-coupled receptors and cyclic nucleotide-gated channels;

[0008] preparing an analytical standard solution, wherein the analytical standard solution is prepared from a CHAPS diluent and the hCG analytical standard;

[0009] preparing a test solution, wherein the test solution is prepared from a CHAPS diluent and the hCG test solution;

[0010] Incubating the tool cells with Ro20-ACTOne dye in the dark to obtain a cell dye sample; wherein the Ro20-ACTOne dye is an ACTOne membrane potential dye containing the phosphodiesterase inhibitor Ro20-1724; and

[0011] The analytical standard solution and the test solution are respectively mixed with the cell dye sample, incubated, and the relative activity of the hCG test sample is determined based on the staining results of the cell sample.

[0012] In some embodiments, the CHAPS diluent is a mixture of CHAPS and DPBS solution, and the concentration of CHAPS relative to the DPBS solution is 0.04% to 0.06% (w / v).

[0013] In some embodiments, the detection method comprises the following steps:

[0014] Obtaining the hCG analysis standard, the hCG test product and the tool cell;

[0015] The tool cells are incubated with the Ro20-ACTOne dye in a dark environment and incubated in multiple culture wells under the same conditions to obtain multiple cell dye samples, and initial fluorescence signal values ​​F0 of the multiple cell dye samples are collected;

[0016] Adding the same volume of the analytical standard solution at different hCG concentrations to multiple cell dye samples to obtain multiple cell sample solutions with a first hCG concentration series; adding the same volume of the test solution at different hCG concentrations to other multiple cell dye samples to obtain multiple cell sample solutions with a second hCG concentration series; incubating each cell sample solution in the dark under the same conditions, and testing the endpoint fluorescence signal value Ft after the incubation; wherein the analytical standard solution of each concentration is prepared by the CHAPS diluent and the hCG analytical standard, and the test solution of each concentration is prepared by the CHAPS diluent and the hCG test solution;

[0017] According to the F0 and Ft of the multiple cell sample solutions of the first hCG concentration series, the half effective concentration EC of the hCG analysis standard is obtained by analysis. 50(s) According to the F0 and Ft of the multiple cell sample solutions of the second hCG concentration series, the half effective concentration EC of the hCG test sample is obtained by analysis. 50(t) According to EC 50(s) and EC 50(t) The relative activity of the hCG test sample is calculated by the ratio of 50(s) and the EC 50(t) All the results were obtained by using four-parameter logistic fitting analysis method.

[0018] In some embodiments, the detection method satisfies one or more of the following characteristics:

[0019] (ta1) the highest hCG concentration in the first hCG concentration series and the highest hCG concentration in the second hCG concentration series are each independently greater than or equal to 50 ng / mL;

[0020] (ta2) the lowest hCG concentration in the first hCG concentration series and the lowest hCG concentration in the second hCG concentration series are each independently less than or equal to 0.0008 ng / mL;

[0021] (ta3) the dilution factor between any two adjacent hCG concentrations in the first hCG concentration series and the dilution factor between any two adjacent hCG concentrations in the second hCG concentration series are each independently selected from 1.5 to 5;

[0022] (ta4) the number of hCG concentration distributions in the first hCG concentration series and the second hCG concentration series are each independently 8, 9, 10, 11, 12, 13, 14 or 15;

[0023] (ta5) the hCG concentration distribution range of the first hCG concentration series and the second hCG concentration series each independently includes 0.00076 to 50 ng / mL, and the dilution factor each independently is 2 or 4;

[0024] (ta6) the first hCG concentration series and the second hCG concentration series each independently include the following hCG concentration values: the dilution factors of the concentration values ​​ranked from high to low relative to the first well concentration are 1, 4, 4, 4, 2, 2, 2, 2, 4, 4 and 4, respectively, wherein the first well concentration is 50 ng / mL;

[0025] (ta7) the hCG concentration distribution of the first hCG concentration series and the second hCG concentration series is set to be the same; and (ta8) in the step of incubating each cell sample solution in the dark under the same conditions, the incubation temperature is 25 to 30° C. and the incubation time is 30 to 60 minutes.

[0026] In some embodiments, the hCG analysis standard and the hCG test sample are both recombinant human chorionic gonadotropin.

[0027] In some embodiments, the detection method satisfies one or more of the following characteristics:

[0028] (tb1) The tool cell is HEK293 / LHCGR recombinant cell;

[0029] (tb2) In the step of incubating the tool cells with the Ro20-ACTOne dye in the dark, the inoculation density of the tool cells in each culture chamber is 1.4×10 3 ~1.6×10 3 Pieces / mm 2 ;and

[0030] (tb3) In the step of incubating the tool cells and the Ro20-ACTOne dye in the dark, the incubation temperature is 25-30° C. and the incubation time is 120±15 min.

[0031] In some embodiments, the Ro20-ACTOne dye is prepared by a method comprising the following steps: adding 0.04 to 0.06 μmol of Ro20-1724 per milliliter of 1×ACTOne membrane potential dye.

[0032] In some embodiments, the Ro20-ACTOne dye is prepared by a method comprising the following steps: mixing 40-60 mM Ro20-1724 solution and 1×ACTOne membrane potential dye in a volume ratio of 1:(1000±200), wherein the Ro20-1724 solution is a DMSO solution of Ro20-1724.

[0033] In some embodiments, in the step of incubating the tool cells with the Ro20-ACTOne dye in the dark, the amount of the Ro20-ACTOne dye added is 1 volume;

[0034] In the step of adding the same volume of the analytical standard solution with different hCG concentrations to the plurality of cell dye samples, the amount of the analytical standard solution added to each cell dye sample is 0.25 times the volume;

[0035] In the step of adding the same volume of the test solution with different hCG concentrations to the plurality of cell dye samples, the amount of the test solution added to each cell dye sample is 0.25 times the volume.

[0036] In some embodiments, the detection method satisfies one or more of the following characteristics:

[0037] (tc1) said F0 and said F t The acquisition conditions included: excitation wavelength (535±5) nm, emission wavelength (595±5) nm;

[0038] (tc2) the four-parameter logistic fitting analysis is based on the curve of the change of Ft / F0 ratio with the logarithmic value of hCG concentration;

[0039] (tc3) performing the four-parameter logistic fitting analysis includes the step of eliminating abnormal data; and

[0040] (tc4) The detection method described in the invention also includes testing the half effective concentration EC of hCG quality control product. 50(z) , wherein the method of obtaining the hCG quality control product is the same as that of the hCG analysis standard product; the detection method also includes a step of judging the system adaptability, and when the following two conditions are met at the same time, the system adaptability is judged to be passed: condition A, analyzing the EC of the hCG analysis standard product, the hCG quality control product and the hCG test product in duplicate wells 50 The repeatability coefficient of variation CV% should not be greater than 30%; and condition B, the relative activity of the quality control product is between 70% and 130%.

[0041] In some embodiments, the relative activity test results of the hCG test product meet one or more of the following characteristics:

[0042] The intermediate precision coefficient of variation of the tests of 50%, 75%, 100%, 125% and optional 200% theoretical concentration values ​​are all less than 30%;

[0043] The average recovery of relative activity was 98% to 107%;

[0044] The recovery rate of each determination result of relative activity is selected from 89% to 116%;

[0045] The linear fitting correlation coefficient between the measured relative activity and the theoretical relative activity is R 2 ≥0.9995; and

[0046] The relative activity detection range includes 50% to 200%.

[0047] The method for detecting the activity of human chorionic gonadotropin (hCG) provided by the present invention is an in vitro cell assay method for hCG activity, using genetically modified LHCG recombinant cells, which can express G protein-coupled receptors (GPCRs) and cyclic nucleotide-gated channels (CNG); hCG drugs can bind to and activate GPCRs on the surface of LHCGR recombinant cells, and generate cyclic adenosine monophosphate (cAMP) through a series of reactions. In the presence of phosphodiesterase inhibitor Ro20, the higher the hCG drug concentration, the higher the intracellular cAMP concentration, the higher the degree of cell membrane depolarization, the stronger the fluorescence signal, and the fluorescence signal is significantly related to the dose of hCG. Using this principle, the cell biological activity of hCG can be indirectly determined. According to the ratio Ft / F0 of the fluorescence signal value F0 before adding the sample (adding hCG) and the fluorescence signal value Ft after adding the sample, the concentration of the sample (hCG concentration, denoted as C hCG ) logarithmic value change curve (Ft / F0-C hCG The half effective concentration EC can be calculated by four-parameter curve fitting method. 50 By comparing the data of the test sample with that of the analytical standard, the relative activity of the test sample can be obtained.

[0048] The inventors have found through extensive research that using a solution of 3-[3-(cholesterol aminopropyl) dimethylamino]-1-propanesulfonic acid (CHAPS) as a sample diluent for hCG can make the test system more stable and sensitive, and can be used with a lower first well concentration (the first well concentration of the preset hCG concentration gradient can be 50 ng / mL) for hCG biological activity testing. Furthermore, LHCG recombinant cells based on HEK293 cells (HEK293 / LHCG recombinant cells) can be specifically used for in vitro activity detection of hCG.

[0049] The hCG activity test method provided by the present invention has good drug specificity and cell specificity, good stability indication properties, small intermediate precision coefficient of variation (CV%), high average recovery rate, linear fitting correlation coefficient close to 1, and a moderate relative activity detection range (such as 50% to 200%). It can achieve accurate and sensitive testing of hCG biological activity and provide key technologies for quality control of drug production. In some embodiments, the relative activity of the hCG test product has the following characteristics: CV% is less than 30%, the average recovery rate is 98% to 107%, and the linear fitting correlation coefficient can reach R 2 ≥0.9995, relative activity detection range is 50%~200%. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0051] Figure 1 , a graph showing the change in Ft / F0 fluorescence signal corresponding to CHAPS dilutions of different concentrations as a function of hCG drug concentration in one embodiment of the present invention;

[0052] Figure 2 , a graph showing the change in Ft / F0 fluorescence signal of dilutions of different components with the concentration of hCG drug in one embodiment of the present invention;

[0053] Figure 3 , a dilution curve diagram of the 96-well plate in one embodiment of the present invention, in which the dilution factors of the 2nd to 11th examples are 2.5 times (columns 2-3), 2 times (columns 4-8), and 2.5 times (columns 9-11) respectively;

[0054] Figure 4 , a dilution curve diagram of the 96-well plate in one embodiment of the present invention, in which the dilution factors of the 2nd to 11th examples are 4 times (columns 2-4), 2 times (columns 5-8), and 4 times (columns 9-11) respectively;

[0055] Figure 5 , a graph showing the Ft / F0 fluorescence signals of different types of drugs as a function of hCG drug concentration in one embodiment of the present invention;

[0056] Figure 6 , a graph showing the changes in Ft / F0 fluorescence signals of different types of cells with hCG drug concentration in one embodiment of the present invention;

[0057] Figure 7 , a graph showing the Ft / F0 fluorescence signal of the stability indicating property in one embodiment of the present invention as a function of the hCG drug concentration, using different test time points;

[0058] Figure 8 , linear analysis graph of measured relative activity and theoretical relative activity. DETAILED DESCRIPTION

[0059] The present invention will be further described in detail below in conjunction with the accompanying drawings, embodiments and examples. It should be understood that these embodiments and examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. The purpose of providing these embodiments and examples is to make the understanding of the disclosure of the present invention more thorough and comprehensive. It should also be understood that the present invention can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various changes or modifications without violating the connotation of the present invention, and the equivalent form obtained also falls within the protection scope of the present invention. In addition, in the following description, a large number of specific details are given in order to provide a more comprehensive understanding of the present invention. It should be understood that the present invention can be implemented without one or more of these details.

[0060] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing implementation modes and embodiments and are not intended to limit the present invention.

[0061] the term

[0062] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0063] The terms "and / or", "or / and", and "and / or" used in this article have a selection range that includes any one of two or more related listed items, and also includes any and all combinations of related listed items, and the arbitrary and all combinations include any combination of two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A and B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0064] In the present invention, "plurality", "multiple", "multiple times", etc., unless otherwise specified, refer to a number greater than 2 or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0065] As used herein, "combination thereof", "any combination thereof", "any combination thereof" etc. include all suitable combinations of any two or more of the listed items.

[0066] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc., shall be based on the ability to implement the technical solution of the present invention, solve the technical problem of the present invention, and achieve the expected technical effect of the present invention.

[0067] Herein, "preferred", "better", "more preferred" and "suitable" are only used to describe implementation methods or examples with better effects, and it should be understood that they do not constitute a limitation on the scope of protection of the present invention. If multiple "preferred" items appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "preferred" item is independent.

[0068] In the present invention, “further”, “furthermore”, “particularly”, etc. are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present invention.

[0069] In the present invention, "optionally", "optional", "optional" means optional, that is, it means to be selected from either of the two parallel schemes of "yes" or "no", or it means to be selected from either of the two parallel schemes of "including" or "including". If there are multiple "optional" in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent. For example, "optionally including" means including or including.

[0070] In the present invention, the terms "first", "second", etc. in "the first aspect", "the second aspect", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0071] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0072] In the present application, the sub-steps of the method are not necessarily executed in the order described unless otherwise specified. Unless otherwise specified herein, the execution of these steps is not strictly limited in order and can be executed in other orders. Moreover, each step can independently include multiple sub-steps or multiple stages, which are not necessarily executed at the same time, but can be executed at different times, and the execution order is not necessarily sequential, but can be executed in turn or alternately or simultaneously with other steps or at least a part of the sub-steps or stages of other steps.

[0073] In the present invention, when it comes to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values ​​in the numerical interval is considered to be continuous, and includes the two numerical endpoints (i.e., the minimum value and the maximum value) of the numerical interval, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers in the numerical interval, including the two endpoint integers of the numerical range, and each integer between the two endpoints, is equivalent to directly listing each integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical ranges disclosed herein should be understood to include any and all sub-ranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. "Numerical interval" allows broadly including interval types such as percentage intervals, ratio intervals, and ratio intervals.

[0074] The temperature parameters in the present invention, if not specifically limited, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the precision range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0075] In the present invention, the term "room temperature" generally refers to 25°C to 30°C, preferably 20°C ± 5°C. In some embodiments of the present invention, room temperature refers to 25°C to 30°C.

[0076] In the present invention, when it comes to the unit of the data range, if there is a unit only after the right endpoint, it means that the units of the left endpoint and the right endpoint are the same. For example, 40-60mM means that the units of the left endpoint "40" and the right endpoint "60" are both mM.

[0077] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as a reference separately. Unless they conflict with the invention purpose and / or technical solution of the present application, the cited documents involved in the present invention are cited with all contents and all purposes. When the present invention involves cited documents, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the present invention involves cited documents, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into this application as references, but are limited to the ability to implement the present invention. It should be understood that when the content of the citation conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0078] In the present invention, unless otherwise limited, hCG represents human chorionic gonadotropin. Further, unless otherwise limited, it refers to human chorionic gonadotropin artificially synthesized or artificially separated, and its protein sequence may be the same as or different from the wild type.

[0079] In the present invention, unless otherwise specified, LHCGR recombinant cells refer to recombinant cells that are transformed by genetic recombination and are capable of expressing G protein-coupled receptors (GPCRs) and cyclic nucleotide-gated channels (CNG).

[0080] In the present invention, unless otherwise specified, HEK293 / LHCG recombinant cells refer to HEK293 recombinant cells that are obtained by cell gene recombination and are capable of expressing G protein-coupled receptors (GPCRs) and cyclic nucleotide-gated channels (CNG).

[0081] In the present invention, unless otherwise specified, CHAPS represents 3-[3-(cholesterylaminopropyl)dimethylamino]-1-propanesulfonic acid.

[0082] In the present invention, unless otherwise specified, Ro20 refers to Ro20-1724, which is a phosphodiesterase inhibitor that can selectively inhibit cyclic adenosine monophosphate-phosphodiesterase (cAMP-PDE) to prevent it from hydrolyzing cAMP.

[0083] In the present invention, if not otherwise specified, Ft / F0-C hCG The curve refers to the ratio of the fluorescence signal value F0 before adding the sample (the sample is hCG) to the fluorescence signal value Ft after adding the sample. hCG ) is the change curve of the logarithmic value.

[0084] In the present invention, if not otherwise specified, "first hole concentration" refers to the concentration used to make Ft / F0-C hCG The concentration corresponding to the culture well with the highest hCG concentration in a series of hCG concentration gradients of the curve.

[0085] In the present invention, unless otherwise specified, the fluorescence signal value is a relative light unit (RLU).

[0086] In the present invention, unless otherwise specified, the detection object refers to hCG, the analysis standard refers to the hCG analysis standard, the test sample refers to the hCG test sample, and the quality control sample refers to the hCG quality control sample.

[0087] In the present invention, the relative activity of hCG test products and hCG quality control products, unless otherwise specified, refers to the relative activity relative to the hCG analytical standard product, which can be measured by the EC of the hCG analytical standard product. 50 EC with hCG test article 50 ratio, or the EC of the hCG analytical standard 50 EC with hCG quality control 50 The ratio of .

[0088] A first aspect of the present invention

[0089] In the first aspect of the present invention, a method for detecting the activity of human chorionic gonadotropin (hCG) is provided. The method is an in vitro cell assay for hCG activity, using CHAPS solution as a sample diluent for hCG, which can be used with a lower first well concentration. The method has good drug specificity and cell specificity, good stability indication properties, and a small coefficient of variation of intermediate precision (including EC 50 The intermediate precision coefficient of variation), the average recovery rate is high, the linear fitting correlation coefficient is close to 1, and the relative activity detection range is moderate. It can achieve accurate and sensitive testing of the biological activity of hCG and provide a key technology for quality control of drug production.

[0090] Human chorionic gonadotropin (hCG) and luteinizing hormone (LH) secreted by the pituitary gland are both glycoprotein hormones, both composed of two polypeptide chains, α and β. The α subunits of follicle stimulating hormone (FSH), hCG, LH and thyroid stimulating hormone (TSH) are the same, while the β subunit is a highly specific part. hCG and LH transmit hormone information by binding to the same receptor on target cells. LH / hCG receptors are located on the cell membrane and are widely distributed in the body. They are expressed not only in gonadal target tissues, but also in some non-gonadal tissues such as prostate, breast, adrenal glands, thyroid glands, and cerebral cortex. Related studies have also found that LH / hCG receptors are also present on the surface of some malignant tumor cells, such as endometrial cancer, cervical cancer, ovarian cancer, breast cancer, gastric cancer, etc. In terms of molecular structure, the spatial structure of hCG receptors is the same as other G protein-coupled receptors (GPCRs), and they all belong to the G protein-coupled receptor rhodopsin / β2 adrenergic receptor family A. LH / hCG receptors have something in common with many other G protein receptors, all of which have 7 transmembrane peptide regions, 4 cytoplasmic regions (including 3 cytoplasmic loop regions and a carboxyl terminal region), and an extracellular region consisting of 3 loop regions and an amino terminal. The hCG receptor molecule is relatively large, and the extracellular amino terminal region is composed of about 340 amino acids, rich in leucine, and plays an important role in hormone recognition. hCG binds to the LH / hCG receptor on the ovarian cell membrane, activates the adenylate cyclase system and stimulates the synthesis of steroid hormones, and participates in the reproductive process of mammals, such as the implantation of fertilized eggs, the regulation of gonadal development and functional activities.

[0091] For information on the structure of hCG, please refer to the literature "Zhang Caitian, Dai Xiaohua, Cui Yugui. Research progress on luteinizing hormone / human chorionic gonadotropin receptor [J]. Foreign Medicine (Family Planning / Reproductive Health), 2007(03):169-171." and its cited literature.

[0092] hCG drugs can bind to GPCRs on the cell surface and activate them. The activated G protein then activates adenylate cyclase on the cell membrane, catalyzing ATP to remove a pyrophosphate to generate cyclic adenosine monophosphate (cAMP). The increased cAMP in the cell can then activate CNG channels, leading to ion outflow and cell membrane depolarization. The degree of this depolarization can be detected by appropriate cell membrane fluorescent dyes (such as ACTOne dye containing phosphodiesterase inhibitor Ro20). In addition, cAMP as a substrate can be hydrolyzed by intracellular cyclic adenosine monophosphate-phosphodiesterase (cAMP-PDE) into inactive 5'-AMP, reducing the intracellular cAMP content. Therefore, hCG drugs can bind to GPCRs on the surface of LHCGR recombinant cells and activate them, generating cAMP through a series of reactions. In the presence of phosphodiesterase inhibitor Ro20, Ro20 can selectively inhibit cAMP-PDE, preventing it from hydrolyzing cAMP. At this time, the higher the hCG drug concentration, the higher the increased cAMP concentration in the cell, the higher the degree of cell membrane depolarization, and the stronger the fluorescence signal. The detected fluorescence signal is significantly correlated with the dose of hCG. Using this principle, the cellular biological activity of hCG can be indirectly measured.

[0093] According to the ratio of the fluorescence signal value F0 before adding the sample (adding hCG) to the fluorescence signal value Ft after adding the sample, Ft / F0 changes with the added sample concentration (hCG concentration, denoted as C hCG ) logarithmic value change curve (Ft / F0-C hCG The half effective concentration EC can be calculated by four-parameter curve fitting method. 50 By comparing the data of hCG test sample with that of hCG analytical standard, the relative activity of hCG test sample can be obtained.

[0094] In the present application, the activity of hCG samples (such as test samples or quality control samples) can be characterized by relative activity relative to the analytical standard unless otherwise specified.

[0095] In this application, unless otherwise specified, "relative activity" refers to the relative activity of an hCG sample (such as a test sample or a quality control sample) relative to an analytical standard.

[0096] In this application, unless otherwise specified, "intermediate precision" refers to the precision under different experimental conditions including one or more variables such as different experimenters, different experimental dates, and different concentrations during method validation.

[0097] In this application, unless otherwise specified, the "intermediate precision coefficient of variation" refers to the relative standard deviation between the results measured by different experimenters at different times in the same laboratory environment.

[0098] In this application, unless otherwise specified, "recovery rate" may represent the accuracy of the test method, and unless otherwise specified, the numerical value is equal to the percentage ratio of the measured value to the theoretical value. Further, unless otherwise specified, "recovery rate" refers to the recovery rate of the relative activity of the hCG sample (such as the test sample or the quality control sample).

[0099] In this application, unless otherwise specified, "average recovery rate" refers to the average recovery rate of the relative activity of hCG samples (such as test samples or quality control samples).

[0100] In this application, unless otherwise specified, "linear fitting correlation coefficient" refers to the correlation coefficient of the linear fitting curve between the measured relative activity of the hCG sample and the theoretical relative activity. The closer the coefficient is to 1, the better the linearity is, indicating that the test result is more accurate.

[0101] In some embodiments, LHCGR recombinant cells are cultured on a 96-well fluorescent plate for a period of time (e.g., overnight, such as 12 to 18 hours), and after adding a membrane dye and incubating at room temperature in the dark for 2 hours, the background fluorescence value F0 is first read using a microplate reader at an excitation wavelength of 535 nm and an emission wavelength of 595 nm. Then, the samples to be tested (analytical standards or test samples, and quality control products, different samples to be tested are tested in different culture wells) are added to different culture wells for incubation, and then the sample fluorescence signal value Ft is read on the microplate reader at an excitation wavelength of 535 nm and an emission wavelength of 595 nm. According to the ratio of the sample fluorescence signal value Ft to the background fluorescence value F0 (Ft / F0), the half effective concentration EC of each sample to be tested is calculated using a four-parameter curve fitting method. 50 By comparing the test sample (or quality control sample) with the analytical standard, the relative activity of the test sample (or quality control sample) can be obtained.

[0102] In some embodiments, a method for detecting human chorionic gonadotropin activity is provided, comprising the following steps:

[0103] Obtaining hCG analysis standards, hCG test products and tool cells, wherein the tool cells are cells capable of expressing G protein-coupled receptors and cyclic nucleotide-gated channels;

[0104] preparing an analytical standard solution, wherein the analytical standard solution is prepared from a CHAPS diluent and the hCG analytical standard;

[0105] preparing a test solution, wherein the test solution is prepared from a CHAPS diluent and the hCG test solution;

[0106] Incubating the tool cells with Ro20-ACTOne dye in the dark to obtain a cell dye sample; wherein the Ro20-ACTOne dye is an ACTOne membrane potential dye containing the phosphodiesterase inhibitor Ro20; and

[0107] The analytical standard solution and the test solution are respectively mixed with the cell dye sample, incubated, and the relative activity of the hCG test sample is determined based on the staining results of the cell sample.

[0108] In some embodiments, a method for detecting human chorionic gonadotropin activity is provided, comprising the following steps:

[0109] S10: obtaining hCG analysis standards, hCG test products and tool cells, wherein the tool cells are cells capable of expressing G protein-coupled receptors and cyclic nucleotide-gated channels;

[0110] S20: incubating the tool cells with the Ro20-ACTOne dye in the dark, and performing the incubation in multiple culture wells under the same conditions to obtain multiple cell dye samples, and collecting the initial fluorescence signal values ​​F0 of the multiple cell dye samples; wherein the Ro20-ACTOne dye is an ACTOne membrane potential dye containing the phosphodiesterase inhibitor Ro20;

[0111] S30: adding the same volume of the analytical standard solution at different hCG concentrations to the plurality of cell dye samples to obtain a plurality of cell sample solutions having a first hCG concentration series; adding the same volume of the test solution at different hCG concentrations to the other plurality of cell dye samples to obtain a plurality of cell sample solutions having a second hCG concentration series; incubating each cell sample solution in the dark under the same conditions, and testing the endpoint fluorescence signal value Ft after the incubation; wherein the analytical standard solution of each concentration is prepared by CHAPS diluent and the analytical standard, and the test solution of each concentration is prepared by CHAPS diluent and the test solution;

[0112] S40: Analyze and obtain the half effective concentration EC of the hCG analysis standard according to the F0 and Ft of the multiple cell sample solutions of the first hCG concentration series. 50(s) According to the F0 and Ft of the multiple cell sample solutions of the second hCG concentration series, the half effective concentration EC of the hCG test sample is obtained by analysis. 50(t) According to EC 50(s) and EC 50(t) The relative activity of the hCG test sample is calculated by the ratio of 50(s) and the EC 50(t)All the results were obtained by using four-parameter logistic fitting analysis method.

[0113] In some embodiments, the fluorescence signal values ​​(F0 and Ft) are RLU (Relative Light Units).

[0114] In some embodiments, the conditions for obtaining the fluorescence signal value F0 are: excitation wavelength 535±5 nm (such as 535 nm), emission wavelength 595±5 nm (such as 595 nm).

[0115] In some embodiments, the conditions for obtaining the fluorescence signal value Ft are: excitation wavelength 535±5nm (such as 535nm), emission wavelength 595±5nm (such as 595nm).

[0116] In some embodiments, the F0 and the F t The acquisition conditions include: excitation wavelength 535±5nm (such as 535nm), emission wavelength 595±5nm (such as 595nm).

[0117] In some embodiments, the acquisition conditions of the fluorescence signal value F0 and the fluorescence signal value Ft are the same, which are: excitation wavelength 535±5nm (such as 535nm), emission wavelength 595±5nm (such as 595nm).

[0118] S10: Provide samples to be tested and tool cells.

[0119] In step S10, analytical standards and test products of human chorionic gonadotropin are provided, and a quality control product is optionally provided.

[0120] In the present invention, the analytical standard is obtained by the following method: using recombinant human chorionic gonadotropin (r-hCG) stock solution through dilution, packaging, detection and calibration.

[0121] In the present invention, the test sample can be any human chorionic gonadotropin, as long as it is artificially synthesized or artificially separated. The hCG of the test sample can have a wild-type sequence or a mutant, and the mutation mode can include but is not limited to insertion, deletion, displacement, substitution, etc.

[0122] In some embodiments, the human chorionic gonadotropin is recombinant human chorionic gonadotropin.

[0123] In some embodiments, the hCG test sample is obtained by: isolating and purifying the hCG test sample by culturing a cell line that recombinantly expresses human chorionic gonadotropin (r-hCG).

[0124] In the present invention, the method of obtaining hCG quality control products is the same as the method of obtaining hCG analytical standards. The analytical standards can be used to prepare a standard curve of hCG that varies with concentration. The quality control products can serve as system adaptability samples. Since the method of obtaining quality control products is the same as that of analytical standards, theoretically, the relative activity of the quality control products relative to the analytical standards should be 100%. It should be understood that a certain error range such as ±30% is allowed, corresponding to a relative activity of 70%-130%. By verifying the relative activity of the quality control products, it can be examined whether there is a system abnormality.

[0125] In some embodiments, the hCG analysis standard and the hCG test sample are both recombinant human chorionic gonadotropin.

[0126] In step S10, tool cells are also provided for detecting the activity of the hCG sample.

[0127] In some embodiments, the tool cell is a HEK293 / LHCGR recombinant cell with good cell specificity.

[0128] S20: providing a cell dye sample and obtaining an initial fluorescence signal value F0.

[0129] The cell suspension may be obtained by the method of step S200 described below. The inoculation conditions of step S300 may also be referred to, such as the culture vessel, inoculation density, etc.

[0130] In some embodiments, in the step of incubating the tool cells with the Ro20-ACTOne dye in the dark, the seeding density of the tool cells in each culture chamber is 1.4×10 3 ~1.6×10 3 Pieces / mm 2 .

[0131] In some embodiments, in the step of incubating the tool cells with the Ro20-ACTOne dye in the dark, the incubation temperature is 25-30° C., and the incubation time is 120±15 min.

[0132] In some embodiments, the Ro20-ACTOne dye is prepared by a method comprising the following steps: adding 0.04-0.06 μmol of Ro20 per milliliter of 1×ACTOne membrane potential dye.

[0133] In some embodiments, the Ro20-ACTOne dye solution is a 1×ACTOne membrane potential dye containing 0.04 to 0.06 mM phosphodiesterase inhibitor Ro20. In this case, the composition of the Ro20-ACTOne dye solution is: 0.04 to 0.06 μmol of Ro20 is contained in each mL of 1×ACTOne membrane potential dye. At this time, the concentration of Ro20 in the Ro20-ACTOne dye solution can be 40 to 60 μM, for example 40 μM, 45 μM, 50 μM, 55 μM, 60 μM, etc. In this application, "Ro20" refers to Ro20-1724 unless otherwise specified.

[0134] In some embodiments, the 1×ACTOne membrane potential dye is prepared by diluting 10×ACTOne membrane potential dye (commercially available) by 10 times. In some embodiments, the manufacturer of the 10×ACTOne membrane potential dye is CodexBio solutions.

[0135] In some embodiments, the Ro20-ACTOne dye is prepared by a method comprising the following steps: mixing 40-60 mM Ro20 solution with 1×ACTOne membrane potential dye in a volume ratio of 1:(1000±200) (for example, a volume ratio of 1:1000, 1:800, 1:900, 1:1200, 1:1100, etc.), further, the Ro20 solution is a DMSO solution of Ro20.

[0136] In some embodiments, the concentration of Ro20 in the DMSO solution of Ro20 can be 40 mM, 45 mM, 50 mM, 55 mM or 60 mM.

[0137] In some embodiments, in the step of incubating the tool cells with the Ro20-ACTOne dye in the dark, the amount of the Ro20-ACTOne dye added is 1 volume.

[0138] S30: Incubate the hCG sample with concentration gradient (prepared with CHAPS dilution solution) with the cell dye sample to obtain the final Point fluorescence signal value Ft.

[0139] After culturing the cells using the method of the following step S400, a cell dye sample may be added for incubation.

[0140] CHAPS diluent refers to a sample diluent containing CHAPS (3-[3-(cholesterol aminopropyl) dimethylamino]-1-propanesulfonic acid), which is used to dilute the hCG test sample to a specific concentration or volume for easy quantitative addition. Prior to the filing date of this application, the inventors had tried sample diluents with different compositions (such as 2.5% DMSO in DPBS solution), but found that the effects were not ideal and the stability of the detection method was not good. After a lot of exploration and hard research, it was unexpectedly discovered that the use of a solution containing CHAPS as a sample diluent has better detection effects and more stable experimental results. The reasons are speculated to be as follows: DMSO (structure as shown in the formula ), with high polarity, is a non-protonic polar solvent that is soluble in both water and organic solvents. It is known as a "universal solvent" small molecule substance, but is mostly used in the chemical industry and the dissolution of chemical substances. CHAPS (structure is ), CHAPS has both hydrophilic and hydrophobic groups, which can well protect the hydrophobic region of transmembrane proteins, thus providing a better and more stable solubilization effect for hCG, and can also play a role in protecting protein structure and increasing signal stability.

[0141] In some embodiments, the CHAPS diluent is a mixture of CHAPS and DPBS solution. Further, the concentration of CHAPS relative to the DPBS solution can be 0.04% to 0.06% (w / v), for example 0.04% (w / v), 0.05% (w / v), 0.06% (w / v), etc.

[0142] In some embodiments, the highest hCG concentration in the first hCG concentration series and the highest hCG concentration in the second hCG concentration series are each independently greater than or equal to 50 ng / mL. The detection method of the present invention allows for a lower first well concentration.

[0143] In some embodiments, the lowest hCG concentration in the first hCG concentration series and the lowest hCG concentration in the second hCG concentration series are each independently less than or equal to 0.0008 ng / mL, and further, may each independently be less than or equal to 0.00076 ng / mL.

[0144] In some embodiments, the dilution factor between any two adjacent hCG concentrations in the first hCG concentration series and the dilution factor between any two adjacent hCG concentrations in the second hCG concentration series are independently selected from 1.5 to 5, non-limiting examples of which are 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, etc. A person skilled in the art can select a suitable dilution factor according to the change of the activity of the hCG sample to be tested with the concentration, so as to obtain the dilution factor from Ft / F0-C hCG The corresponding EC can be analyzed from the curve50 .

[0145] In the present invention, unless otherwise specified, the "dilution factor" of a concentration series or concentration gradient is the multiple relationship between any two adjacent concentration values, and the dilution factor is the order in which the different concentrations are arranged in order from high to low. In any concentration series, multiple dilution factors may be the same or different.

[0146] In some embodiments, the number of hCG concentration distributions in the first hCG concentration series and the second hCG concentration series is independently ≥5, further independently ≥6, further independently ≥8, further independently ≥10, and can also be independently selected from 5-20, 5-15, 5-12, 6-20, 6-15, 6-12, 8-20, 8-15, 8-12, etc., and can further be independently 8, 9, 10, 11, 12, 13, 14 or 15. Those skilled in the art can select a suitable number of concentration distributions according to the change of the activity of the hCG sample to be tested with the concentration, so as to more accurately obtain the Ft / F0-C hCG The corresponding EC was obtained by analysis on the curve 50 .

[0147] In some embodiments, the hCG concentration distribution ranges of the first hCG concentration series and the second hCG concentration series each independently include 0.00076 to 50 ng / mL, and further, the dilution factors are each independently 2 or 4.

[0148] In some embodiments, the first hCG concentration series and the second hCG concentration series each independently include the following hCG concentration values: the dilution factors of each concentration value arranged from high to low relative to the concentration of the first well are 1, 4, 4, 4, 2, 2, 2, 2, 4, 4 and 4, respectively, wherein the concentration of the first well is 50 ng / mL; each concentration corresponds in sequence (some lower concentrations are allowed to be approximate values ​​after rounding): 50 ng / mL, 12.5 ng / mL, 3.125 ng / mL, 0.781 ng / mL, 0.39 ng / mL, 0.195 ng / mL, 0.098 ng / mL, 0.049 ng / mL, 0.0122 ng / mL, 0.003 ng / mL and 0.00076 ng / mL.

[0149] In some embodiments, the first hCG concentration series and the second hCG concentration series are each independently the following hCG concentration values: the dilution factors of each concentration value arranged from high to low relative to the concentration of the first well are 1, 4, 4, 4, 2, 2, 2, 2, 4, 4 and 4, respectively, wherein the concentration of the first well is 50 ng / mL; each concentration corresponds to 50 (some lower concentrations are allowed to be approximate values ​​after rounding): 50 ng / mL, 12.5 ng / mL, 3.125 ng / mL, 0.781 ng / mL, 0.39 ng / mL, 0.195 ng / mL, 0.098 ng / mL, 0.049 ng / mL, 0.0122 ng / mL, 0.003 ng / mL and 0.00076 ng / mL.

[0150] In some embodiments, the hCG concentration profiles of the first hCG concentration series and the second hCG concentration series may be the same or different, but are preferably the same.

[0151] In some embodiments, in the step of incubating each cell sample solution in the dark under the same conditions, the incubation temperature is 25 to 30° C., and the incubation time is 30 to 60 min.

[0152] In some embodiments, in the step of adding the same volume of the analytical standard solution with different hCG concentrations to the plurality of cell dye samples, the amount of the analytical standard solution added relative to each cell dye sample is 0.25 times the volume;

[0153] In the step of adding the same volume of the test solution with different hCG concentrations to the plurality of cell dye samples, the amount of the test solution added to each cell dye sample is 0.25 times the volume.

[0154] S40: Analyze and determine the relative activity of the test article.

[0155] In the present invention, unless otherwise specified, the relative activity of the hCG test sample can be determined by measuring the EC of the hCG assay standard. 50 (Can be recorded as EC 50(s) ) and the EC of hCG test substance 50 (Can be recorded as EC 50(t) ) is calculated.

[0156] In some embodiments, the four-parameter logistic fitting analysis is based on a curve of the Ft / F0 ratio versus the logarithmic value of hCG concentration.

[0157] In some embodiments, performing the four-parameter logistic fitting analysis includes the step of eliminating abnormal data.

[0158] In some embodiments, the detection method further comprises testing the half effective concentration EC of hCG quality control product. 50(z) The detection method further includes a step of judging the system adaptability. When the following two conditions are met at the same time, the system adaptability is judged to be passed: Condition A, analyzing the EC of the hCG analysis standard, the hCG quality control product and the hCG test product in duplicate wells 50 The repeatability intermediate precision coefficient of variation CV% should both be no greater than 30%; and condition B, the relative activity of the quality control product should be between 70% and 130%.

[0159] In some embodiments, the relative activity test results of the hCG test product meet one or more of the following characteristics:

[0160] The intermediate precision coefficient of variation of the tests of 50%, 75%, 100%, 125% and optional 200% theoretical concentration values ​​are all less than 30%;

[0161] The average recovery of relative activity was 98% to 107%;

[0162] The recovery rate of each determination result of relative activity is selected from 89% to 116%;

[0163] The linear fitting correlation coefficient between the measured relative activity and the theoretical relative activity is R 2 ≥0.9995; and

[0164] The relative activity detection range is 50% to 200%.

[0165] The above indicators can be obtained by the methods shown in the following examples.

[0166] In other embodiments of the present invention, a method for detecting human chorionic gonadotropin activity is provided, comprising the following steps:

[0167] S100: Provide analytical standards, test products and quality control products of human chorionic gonadotropin;

[0168] S200: resuspending the recombinant cells with growth activity in a culture medium to prepare a suspension of recombinant cells; wherein the recombinant cells are LHCGR recombinant cells, preferably, the LHCGR recombinant cells are HEK293 recombinant cells that are genetically modified to express G protein-coupled receptors and cyclic nucleotide-gated channels, that is, HEK293 / LHCGR recombinant cells;

[0169] S300: setting a first experimental group, a second experimental group, a third experimental group, a negative control group and a blank control group, and setting a plurality of independent culture wells in each group; inoculating the recombinant cells in the following manner: adding a suspension of the recombinant cells to each culture well of the first experimental group, the second experimental group, the third experimental group and the negative control group, respectively, and adding a culture solution to each culture well of the blank control group, and the added volume (denoted as V1) of each culture well of each group is the same;

[0170] S400: After the inoculation of the recombinant cells is completed, the recombinant cells are cultured in the culture wells of the first experimental group, the second experimental group, the third experimental group, the negative control group and the blank control group under the same conditions;

[0171] S500: After culturing to the desired cell confluence, add the same volume (denoted as V2) of Ro20-ACTOne dye solution to each culture well of the first experimental group, the second experimental group, the third experimental group, the negative control group and the blank control group; the Ro20-ACTOne dye solution added to each culture well is an ACTOne membrane potential dye containing the phosphodiesterase inhibitor Ro20; incubate in dark for the first time, and after the incubation is completed, read the fluorescence signal value F0 of each culture well;

[0172] S600: according to a preset hCG concentration gradient (such as selected from 0.00076 to 50 ng / mL), add an analytical standard whose human chorionic gonadotropin concentration meets the preset hCG concentration gradient to each culture well of the first experimental group, add a test sample whose human chorionic gonadotropin concentration meets the preset hCG concentration gradient to each culture well of the second experimental group, and add a quality control sample whose human chorionic gonadotropin concentration meets the preset hCG concentration gradient to each culture well of the third experimental group, and all three experimental groups are added in the form of solutions (preferably, the CHAPS diluent pre-dilutes the analytical standard, the test sample and the quality control sample into three groups of human chorionic gonadotropin solutions with the same concentration gradient); add a CHAPS diluent to each culture well of the negative control group and the blank control group; the added liquid volume (recorded as V3) of each culture well of each group is the same; incubate in dark for the second time, and after the incubation is completed, read the fluorescence signal value Ft of each culture well;

[0173] S700: According to the Ft value, F0 value and preset hCG concentration gradient of each culture well, the Ft / F0 concentration curve is prepared, and the four-parameter logical fitting analysis is performed to obtain the half effective concentration EC of the analytical standard, test sample and quality control product respectively. 50 , to analyze the EC of the standard 50 As a reference, the relative activities of the test sample and the quality control sample were obtained.

[0174] In some embodiments, the detection method further comprises a step S800 of judging the system suitability: when the following two conditions are met at the same time, the system suitability is judged to be passed: condition A, the EC of the duplicate wells of the analysis standard, quality control and test product 50 The coefficient of variation of repeatability, CV%, should be no more than 30%; and condition B, the relative activity of the quality control product should be between 70% and 130%. If both conditions A and B above cannot be met at the same time, the experiment is considered invalid and the system adaptability fails.

[0175] The various technical solutions of the present invention can also be carried out while omitting the quality control product testing step.

[0176] It should be understood that, although the above steps S100 to S800 are described in sequence, these steps are not necessarily executed in the order described. Unless otherwise specified herein, there is no strict order restriction on the execution of these steps, and they can be executed in other orders. Moreover, each step can independently include multiple sub-steps or multiple stages, and these sub-steps or stages are not necessarily executed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn or alternately or simultaneously with other steps or at least a part of the sub-steps or stages of other steps.

[0177] In some embodiments, the design of each group is as follows:

[0178] In the first experimental group, each well was added with a suspension of LHCGR recombinant cells in a volume of V1, a dye solution containing Ro20 in a volume of V2, and hCG analysis standards in preset concentration gradients were added to different wells in sequence (the volume was V3);

[0179] In the second experimental group, each well was added with a suspension of LHCGR recombinant cells in a volume of V1, a dye solution containing Ro20 in a volume of V2, and hCG test products in preset concentration gradients were added to different wells in sequence (the volume was V3);

[0180] In the third experimental group, each culture well was added with a suspension of LHCGR recombinant cells of volume V1, a dye solution containing Ro20 of volume V2, and hCG quality control products of preset concentration gradients were added to different culture wells in sequence (volume V3);

[0181] Each culture well of the negative control group: added with a volume V1 of the LHCGR recombinant cell suspension, a volume V2 of the Ro20-containing dye solution, and a volume V3 of the sample diluent (i.e., CHAPS diluent without hcG);

[0182] Each culture well of the blank control group: a blank culture medium (without cells) with a volume of V1 was added, a dye solution containing Ro20 with a volume of V2 was added, and a sample diluent with a volume of V3 (i.e., CHAPS diluent without hcG) was added;

[0183] Among them, each V1 is the same, each V2 is the same, and each V3 is the same.

[0184] In some embodiments, V1:V2:V3 is 2:2:1.

[0185] Step S100: Providing a sample to be tested

[0186] Please refer to the above step S10.

[0187] Step S200: preparing a suspension of recombinant cells.

[0188] In step S200, recombinant cells with growth activity are resuspended in a culture medium to prepare a suspension of recombinant cells; wherein the recombinant cells are LHCGR recombinant cells.

[0189] Further, LHCGR recombinant cells are genetically modified recombinant cells capable of expressing G protein-coupled receptors (GPCRs) and cyclic nucleotide-gated channels (NCGs). In some embodiments, LHCGR recombinant cells are obtained by genetically modifying HEK293 cells, i.e., HEK293 / LHCGR recombinant cells, which are genetically modified HEK293 recombinant cells capable of expressing G protein-coupled receptors and cyclic nucleotide-gated channels.

[0190] In some embodiments, the recombinant cells with growth activity are derived from LHCGR recombinant cells with a cell confluence of 60% to 90%, further HEK293 / LHCGR recombinant cells, such as 60%, 70%, 80%, 90%, etc.

[0191] In some embodiments, the suspension of recombinant cells is prepared by resuspending the cells collected after digestion in 10% FBS DMEM culture medium.

[0192] In some embodiments, trypsin (such as 0.25% trypsin) is used for digestion, and the cells are rounded and detached from the wall for 1 to 2 minutes, and an appropriate volume of new 10% FBS DMEM culture medium (preferably containing double antibodies) is added for neutralization. The cells are blown down into a centrifuge tube with a pipette, centrifuged, and the waste liquid is discarded and 10% FBS DMEM culture medium (preferably containing double antibodies) is added to resuspend the cells. In some embodiments, the centrifugation condition is 1000-1200 rpm for 5 minutes.

[0193] In some embodiments, the components of 10% FBS DMEM culture medium contain the following components (including but not limited to consisting of the following components): 90%±2% (v / v) DMEM medium, 10% (v / v) FBS, 1 μg / mL puromycin and 250 μg / mL G-418, all based on the concentration in the culture medium.

[0194] In some embodiments, the cell density of the recombinant cell suspension is (6-7)×10 5 / mL, for example 6.67×10 5 / mL, 6×10 5 / mL, 7×10 5 Pieces / mL, etc.

[0195] Step S300: inoculating recombinant cells.

[0196] In step S300, a first experimental group, a second experimental group, a third experimental group, a negative control group and a blank control group are set, and a plurality of independent culture wells are set in each group; the recombinant cells are inoculated as follows: a suspension of the recombinant cells is added to each culture well of the first experimental group, the second experimental group, the third experimental group and the negative control group, respectively, and a culture medium is added to each culture well of the blank control group, and the added liquid volume (denoted as V1) of each culture well of each group is the same.

[0197] In some embodiments, the culture vessel is a 96-well plate, and further can have a flat bottom.

[0198] In some embodiments, the cell density of the suspension of recombinant cells is 6.67×10 5 pcs / mL, V1 is 75 μL. Further, the culture vessel is a 96-well plate, and further can be a flat-bottomed plate.

[0199] In some embodiments, the seeding density of the recombinant cells in each culture well of the first experimental group, the second experimental group, the third experimental group, and the negative control group is 1.4×10 3 ~1.6×10 3 Pieces / mm 2 .

[0200] S400: Recombinant cell culture.

[0201] In step S400, after the inoculation of the recombinant cells is completed, the recombinant cells are cultured in the culture wells of the first experimental group, the second experimental group, the third experimental group, the negative control group and the blank control group under the same conditions.

[0202] In some embodiments, the recombinant cell culture conditions include: culturing in a cell culture incubator at 37° C. and 5.0% CO 2 until the cell confluence is 60% to 90%. Examples of cell confluence include 60%, 70%, 80%, 90%, etc.

[0203] In some embodiments, the culture time is overnight (e.g., selected from 12h-18h, further exemplified by 12h, 13h, 14h, 15h, 16h, 17h, 18h, etc.).

[0204] S500: Add staining solution and incubate.

[0205] In step S500, after culturing to the desired cell confluence, the same volume (denoted as V2) of Ro20-ACTOne dye solution is added to each culture well of the first experimental group, the second experimental group, the third experimental group, the negative control group, and the blank control group; wherein the Ro20-ACTOne dye solution added to each culture well is an ACTOne membrane potential dye containing Ro20; the first incubation is performed in dark, and after the incubation is completed, the fluorescence signal value F0 of each culture well is read. F0 is the background fluorescence signal value.

[0206] The Ro20-ACTOne dye solution can refer to the aforementioned step S20. For example, in some embodiments, the Ro20-ACTOne dye solution is prepared by the following method: 40-60 mM Ro20 solution and 1×ACTOne membrane potential dye are mixed at a volume ratio of 1:1000, and further, the Ro20 solution is a DMSO solution of Ro20.

[0207] In some embodiments, the conditions for the first light-proof incubation include: an incubation temperature of 25 to 30° C. and an incubation time of 120±15 min.

[0208] In some embodiments, the volume ratio of V1:V2 is 1:1.

[0209] S600: adding sample and incubating (adding hCG sample to be tested and then incubating).

[0210] In step S600, according to a preset hCG concentration gradient (such as selected from 0.00076 to 50 ng / mL), an analytical standard whose human chorionic gonadotropin concentration meets the preset hCG concentration gradient is added to each culture well of the first experimental group, a test sample whose human chorionic gonadotropin concentration meets the preset hCG concentration gradient is added to each culture well of the second experimental group, and a quality control sample whose human chorionic gonadotropin concentration meets the preset hCG concentration gradient is added to each culture well of the third experimental group (preferably, before adding the sample to be tested, the analytical standard, the test sample and the quality control sample are diluted in advance with CHAPS diluent to form three groups of human chorionic gonadotropin solutions with the same concentration gradient); CHAPS diluent is added to each culture well of the negative control group and the blank control group; the liquid addition volume (V3) of each culture well of each group is the same; a second light-proof incubation is performed, and after the incubation is completed, the fluorescence signal value Ft of each culture well is read.

[0211] In some embodiments, the first well concentration of the preset hCG concentration gradient is 50 ng / mL.

[0212] In some embodiments, the preset hCG concentration gradient is selected from the following concentration range: 0.00076-50 ng / mL. In some embodiments thereof, the preset hCG concentration gradient includes the following hCG concentrations: 50 ng / mL, 12.5 ng / mL, 3.125 ng / mL, 0.782 ng / mL, 0.39 ng / mL, 0.195 ng / mL, 0.098 ng / mL, 0.049 ng / mL, 0.0122 ng / mL, 0.003 ng / mL and 0.00076 ng / mL (the dilution factors relative to the concentration of the first well are 1, 4, 4, 4, 2, 2, 2, 2, 4, 4, 4, respectively).

[0213] In some embodiments, the concentration gradient of the hCG solution corresponding to the preset hCG concentration gradient is selected from 0.00076 to 50 ng / mL.

[0214] In some embodiments, the concentration gradient of the hCG solution corresponding to the preset hCG concentration gradient is selected from 0.0038 to 250 ng / mL. In some of the embodiments, the concentration gradient of the hCG solution corresponding to the preset hCG concentration gradient includes: 250 ng / mL, 62.5 ng / mL, 15.63 ng / mL, 3.91 ng / mL, 1.95 ng / mL, 0.98 ng / mL, 0.49 ng / mL, 0.24 ng / mL, 0.061 ng / mL, 0.015 ng / mL and 0.0038 ng / mL.

[0215] In some embodiments, the conditions for the second light-proof incubation include: an incubation temperature of 25 to 30° C. and an incubation time of 30 to 60 min.

[0216] In some embodiments, the volume V3 of liquid added to each well is 30 to 50 μL, for example 30 μL, 35 μL, 37.5 μL, 40 μL, 45 μL, 50 μL, etc.

[0217] In some embodiments, V1:V3 is 2:1.

[0218] In some embodiments, the volume ratio of the recombinant cell suspension added to each well (V1), the Ro20-ACTOne dye solution added to each well (V2), and the hCG solution added to each well (V3) is 1:1:0.5 (equivalent to 2:2:1).

[0219] In some embodiments, V1, V2, and V3 are 75 μL, 75 μL, and 37.5 μL, respectively.

[0220] S700: Calculate the relative activity of the test article.

[0221] In step S700, according to the Ft value, F0 value and the preset hCG concentration gradient of each culture well, the Ft / F0 concentration curve is prepared, and a four-parameter logistic fitting analysis is performed to obtain the half effective concentration EC of the analytical standard, the test sample and the quality control sample, respectively. 50 , to analyze the EC of the standard 50 As a reference, the relative activities of the test sample and the quality control sample were obtained.

[0222] In some embodiments, the Ft / F0 concentration curve has the Ft / F0 ratio as the ordinate and the logarithmic value of the human chorionic gonadotropin concentration in the culture well as the abscissa.

[0223] In some embodiments, a method for detecting hCG activity is provided, comprising the following steps:

[0224] Provide analytical standards, test products and quality control products of human chorionic gonadotropin;

[0225] Resuspending HEK293 / LHCGR recombinant cells grown to a cell confluence of 60% to 90% in 10% FBS DMEM culture medium to prepare a suspension of HEK293 / LHCGR recombinant cells; preferably, the 10% FBS DMEM culture medium contains: 90%±2% (v / v) DMEM medium, 10% (v / v) FBS, 1 μg / mL puromycin and 250 μg / mL G-418;

[0226] HEK293 / LHCGR recombinant cells were inoculated as follows: 1.4×10 3 ~1.6×10 3 Pieces / mm 2 The suspension of HEK293 / LHCGR recombinant cells was added to each well of the blank control group at the same inoculation density, and 10% FBS DMEM culture medium was added to each well of the blank control group. The volume V1 of each well of each group was the same;

[0227] After the inoculation of HEK293 / LHCGR recombinant cells was completed, the recombinant cells were cultured in each culture well of the first experimental group, the second experimental group, the third experimental group, the negative control group and the blank control group at 37°C and 5.0% CO2 until the cell confluence was 60% to 90%;

[0228] After culturing to the desired cell confluence, add the same volume V2 of Ro20-ACTOne dye solution to each culture well of the first experimental group, the second experimental group, the third experimental group, the negative control group and the blank control group; the Ro20-ACTOne dye solution added to each culture well is 1×ACTOne membrane potential dye containing 0.04-0.06 mM phosphodiesterase inhibitor Ro20; the first light-protected incubation is carried out at 25-30°C for 120±15 min. After the incubation is completed, read the fluorescence signal value F0 of each culture well;

[0229] According to the preset hCG concentration gradient (selectable from 0.00076 to 50 ng / mL), the analytical standard with a human chorionic gonadotropin concentration that meets the preset hCG concentration gradient is added to each culture well of the first experimental group, the test sample with a human chorionic gonadotropin concentration that meets the preset hCG concentration gradient is added to each culture well of the second experimental group, and the quality control sample with a human chorionic gonadotropin concentration that meets the preset hCG concentration gradient is added to each culture well of the third experimental group. The three experimental groups are all added with human chorionic gonadotropin in the form of solution (preferably, CHAPS diluent is used to pre-dilute the sample). The analytical standard, the test product and the quality control product are diluted into three groups of human chorionic gonadotropin solutions with the same concentration gradient respectively); CHAPS diluent is added to each culture well of the negative control group and the blank control group respectively, wherein the CHAPS diluent is a CHAPS DPBS solution, and the concentration of CHAPS relative to the DPBS solution is 0.04% to 0.06% (w / v); the added liquid volume V3 of each culture well of each group is the same; a second dark incubation is performed at 25 to 30° C. for 30 to 60 minutes, and the fluorescence signal value Ft of each culture well is read;

[0230] According to the Ft value, F0 value and preset hCG concentration gradient of each culture well, the Ft / F0 ratio was used as the ordinate and the logarithmic value of the human chorionic gonadotropin concentration in the culture well was used as the abscissa to prepare the Ft / F0 concentration curve, and the four-parameter logical fitting analysis was performed to obtain the half effective concentration EC of the analytical standard, test sample and quality control product, respectively. 50 , to analyze the EC of the standard 50 For reference, the relative activities of the test sample and the quality control sample were obtained respectively;

[0231] The acquisition conditions of the fluorescence signal value F0 and the fluorescence signal value Ft are: excitation wavelength 535±5nm (such as 535nm), emission wavelength 595±5nm (such as 595nm); the volume ratio of V1:V2:V3 is 1:1:0.5.

[0232] S800: Optional system suitability judgment

[0233] In some embodiments, the detection method may further include a step S800 of judging the system suitability: when the following two conditions are met at the same time, the system suitability is judged to be passed: Condition A, the EC of the duplicate wells of the analysis standard, quality control and test product 50 The coefficient of variation of repeatability, CV%, should be no more than 30%; and condition B, the relative activity of the quality control product should be between 70% and 130%. If both conditions A and B above cannot be met at the same time, the experiment is considered invalid and the system adaptability fails.

[0234] In some embodiments, performing the four-parameter logistic fitting analysis includes the step of eliminating abnormal data. In some embodiments, the criteria for proposing abnormal data include the following three indicators:

[0235] (1) Values ​​with clear technical problems can be eliminated, such as individual contaminated wells, non-monotonic dose-effect curve values, and other abnormal data;

[0236] (2)EC 50 You should try to avoid removing values ​​before and after the value, even though you should be careful when removing them; and

[0237] (3) The number of wells excluded from each plate (e.g., 96-well plate) should not exceed 3.

[0238] In some embodiments, the results are calculated as follows: using the analytical standard (STD) as a reference, the forced parallel mode Softmax software will automatically calculate the relative activities of the test and control products, and the results are recorded as integers before the percentage sign.

[0239] In some embodiments, the detection method further includes a step of judging the system suitability. When the following two conditions are met at the same time, the system suitability is judged to be passed: Condition A, the EC of the duplicate wells of the analysis standard, quality control and test product is 50 The repeatability coefficient of variation CV% should not be greater than 30%; and under condition B, the relative activity of the quality control product should be between 70% and 130%.

[0240] In some embodiments, the relative activity of the hCG test product meets one or more of the following characteristics: (1) the intermediate precision coefficient of variation CV% of the theoretical concentration value tests of 50%, 75%, 100% and 125% (optionally, also including 200%; alternatively, also including 150%) is less than 30%, (2) the average recovery rate of the relative activity is 98% to 107%, (3) the recovery rate range of each relative activity measurement result is selected from 89% to 116%, (4) the linear fitting correlation coefficient between the measured relative activity and the theoretical relative activity is R 2 ≥0.9995, and (5) the relative activity detection range includes 50% to 200%.

[0241] The detection method provided in the first aspect of the present invention has been experimentally verified to have specificity for detecting hCG, and has also verified the accuracy, precision, linearity, detection range and stability indication properties of the method. The results of the method verification show that the specificity for drug types and cell types is good, and the stability indication properties are good. In some embodiments, the daytime precision of the method and the CV% of the results of the determinations by different operators are both less than 30%, the average recovery rate is 98% to 107%, and the linear fitting correlation coefficient is R 2 =0.9995, the relative activity detection range is 50% to 200%, which is suitable for the determination of hCG biological activity.

[0242] Second aspect of the present invention

[0243] In a second aspect of the present invention, a human chorionic gonadotropin activity detection kit is provided, which can be used to conveniently implement the hCG activity detection method provided by the present invention, thereby avoiding the complicated reagent preparation process and accelerating the detection efficiency.

[0244] In some embodiments, a kit for detecting the activity of human chorionic gonadotropin is provided, comprising:

[0245] The analytical standard substance of human chorionic gonadotropin in the detection method described in the first aspect of the present invention; and

[0246] The non-test substance used to implement the detection method of the first aspect of the present invention may optionally include recombinant cells and / or culture fluid.

[0247] In some embodiments, the substance to be tested is selected from one or more of analytical standards, test products, and quality control products.

[0248] In some embodiments, the non-test substances do not include analytical standards, test products, and quality control products.

[0249] Among them, the definitions of analytical standard substance, recombinant cells, culture medium, analytical standard substance, test substance and quality control substance of human chorionic gonadotropin can be independently referred to the first aspect of the present invention.

[0250] Some specific embodiments are provided below.

[0251] The following will be described in detail with reference to the embodiments of the present invention in conjunction with the examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present invention, and can also be based on the experimental manual of this area (such as "Wang Junzhi. Biotechnology Drug Research and Development and Quality Control (Second Edition). Beijing: Science Press, 2007") or normal conditions, and can also be based on the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0252] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0253] Table 1. Reagent information

[0254]

[0255] Table 2. Name of consumables

[0256]

[0257] Note: In Table 2, "*" indicates that reagents and consumables with the manufacturer's catalog number recommended in the above table should be used first. If they are temporarily unavailable: biological reagents such as enzymes should be used from recommended manufacturers as much as possible, and other manufacturers that have been inspected and qualified can also be used; other chemical reagents and consumables can use analytically pure reagents and sterile consumables unless otherwise specified.

[0258] Table 3. Experimental equipment

[0259]

[0260] Solution preparation

[0261] G418 solution (50 mg / mL): Weigh 1 g of G418 powder (Amresco), dissolve in 20 mL of ultrapure water, sterilize by 0.22 μm filtration, store at -20°C after aliquoting, and the shelf life is 1 year. Alternatively, directly use 50 mg / mL G418 solution (Promega).

[0262] Puromycin solution (2 mg / mL): directly add 12.5 mL of ultrapure water to a 25 mg bottle of puromycin reagent to dissolve, filter and sterilize at 0.22 μm, and store at -20°C after aliquoting. The shelf life is 1 year.

[0263] Ro20 solution (50mM): directly add 7.2mL of DMSO to a 100mg Ro20 reagent bottle to dissolve it and prepare a 50mM Ro20 solution. After aliquoting, store it at -20℃. The shelf life is 2 years.

[0264] 10% CHAPS stock solution: weigh 5 g CHAPS powder, add 50 mL ultrapure water to dissolve, filter and sterilize at 0.22 μm, store at 5±3°C, valid for 2 years.

[0265] The composition of 10% FBS DMEM culture medium (containing double antibodies) is as follows:

[0266]

[0267] For every 400 mL of culture medium, take 358 mL of DMEM culture medium, 40 mL of fetal bovine serum, 2 mL of G418 solution (50 mg / mL), and 200 μL of puromycin solution (2 mg / mL), mix well, increase or decrease in proportion to the actual amount, and store at 5±3°C. The shelf life is 3 months.

[0268] Obtaining HEK293 / LHCGR Recombinant Cells

[0269] Cell plating

[0270] Take HEK293 / LHCGR recombinant cells with a growth confluence of 60% to 90%, discard the culture medium, add DPBS buffer or other appropriate solution to wash the cells and discard, then digest with 0.25% trypsin 1-2mL for 1-2min until the cells become round and detach from the wall, add an appropriate volume of new 10% FBS DMEM culture medium (containing double antibody) to neutralize. Use a pipette to blow down the cells into a centrifuge tube, centrifuge at 1000-1200rpm for 5min, discard the waste liquid and add 10% FBS DMEM culture medium (containing double antibody) to resuspend them. Count the cells and adjust the cell density to 6.67×10 5 / mL, use a multichannel pipette to add cells at 75μL / well in the 1st to 11th columns and rows BG of a black bottom transparent 96-well culture plate, add 75μL cells to each well of B12-D12 as a negative control, and add 75μL 10% FBS DMEM culture solution (containing double antibody) to each well of E12-G12 as a blank control. Add no less than 200μL PBS to each well of rows A and H to reduce edge effects. Place the cell plate in a 37°C, 5.0% CO2 incubator and culture overnight until the cell confluence reaches 60% to 90%.

[0271] Ro20-ACTOne dye solution preparation

[0272] Take 1mL 10×ACTOne Dye Dilution buffer, add 9mL sterile ultrapure water and mix well to prepare 1×ACTOne Dye Dilution buffer, then take 1mL 10×ACTOne Membrane Potential DyeSolution, add 9mL 1×ACTOne Dye Dilution buffer to dilute, then add 10μL 50mM Ro20 solution and mix well to prepare 1×ACTOne dye containing Ro20 (concentration of 50μM) for use. This step is prepared immediately before use, and can be increased or decreased in proportion to the actual amount used.

[0273] Add dye

[0274] The prepared 1×ACTOne dye containing 50 μM Ro20 was added to columns 1-11, rows BG, wells B12-D12 (negative control) and wells E12-G12 (blank control) of the cell plate at 75 μL / well, incubated at room temperature in the dark for 2 h ± 15 min, and the background fluorescence value F0 was read using a microplate reader at an excitation wavelength of 535 nm and an emission wavelength of 595 nm.

[0275] Preparation of sample diluent

[0276] Add 10 mL of DPBS to the sample loading tank, then add 50 μL of 10% CHAPS storage solution and mix well to prepare a sample diluent containing 0.05% CHAPS (which can be recorded as 0.05% CHAPS diluent). The amount can be increased or decreased in proportion to the actual amount used and should be prepared fresh before use.

[0277] Preparation of analytical standard (STD), test sample (SAM), and control sample (CTL) solutions

[0278] The analytical standard is made by the company itself, that is, it is obtained by diluting, packaging, testing and calibrating the recombinant human chorionic gonadotropin (r-hCG) stock solution; the hCG test sample is obtained by separating and purifying after cell culture using a recombinant human chorionic gonadotropin (r-hCG) expressing cell line; the quality control product is the same as the analytical standard.

[0279] Take analytical standard (LZM003), test sample, and quality control sample (replaced with analytical standard sample from the same batch), dilute to 250 ng / mL with sample diluent, and dilute in the dilution plate starting from this, 4 times (columns 2-4), 2 times (columns 5-8), and 4 times (columns 9-11), i.e., dilute to the following concentration gradient: 250, 62.5, 15.63, 3.91, 1.95, 0.98, 0.49, 0.24, 0.061, 0.015, and 0.0038 ng / mL, and the volume of each concentration gradient is not less than 50 μL. Among them, NA means no dilution, which corresponds to the hCG solution required for the first well with the maximum hCG concentration. Arrange the plate as shown in Table 5.

[0280] Table 4. Concentration gradient of hCG solution to be added

[0281]

[0282] Table 5.

[0283]

[0284] Note: STD-# = analytical standard dilution number, SAM-# = test sample dilution number, CTL-# = control sample dilution number.

[0285] Add sample

[0286] Take out the cell culture plate and use a multichannel pipette to add 37.5 μL / well of each dilution gradient of analytical standard, test sample, and quality control solution in the order of Table 5 (columns 11 to 1, sample concentration from low to high), and then add 37.5 μL of sample diluent to each well B12-G12. Incubate at room temperature in the dark for 30-60 minutes.

[0287] Detection

[0288] Take out the cell plate and put it into the fluorescence microplate reader. According to the standard operating procedures of the microplate reader, set and select the method, select the excitation wavelength of 535nm and the emission wavelength of 595nm to read the fluorescence signal value Ft. Calculate the ratio of the fluorescence signal value detected after adding the sample to the background fluorescence signal value before adding the sample (Ft / F0), with the Ft / F0 value as the ordinate and the logarithm of the LZM003 concentration as the abscissa, and use Softmax software for four-parameter logical fitting analysis. According to the four-parameter equation, the half effective concentration EC 50 , which is the value of parameter C in the curve equation.

[0289] Outlier removal criteria

[0290] Since the EC used in the relative activity calculation 50 The values ​​are derived from the fitting of multiple data. Eliminating some abnormal values ​​can improve the accuracy of the measurement results. The elimination criteria are as follows:

[0291] (1) Values ​​with clear technical problems can be eliminated, such as individual contaminated wells, non-monotonic dose-effect curve values, and other abnormal data.

[0292] (2)EC 50 You should try to avoid removing values ​​before and after the value, and be as cautious as possible when removing them.

[0293] (3) The number of holes to be removed from a board shall not exceed 3.

[0294] Result calculation

[0295] Using the analytical standard STD as a reference, the forced parallel mode Softmax software will automatically calculate the relative activity of the test and quality control products, expressed as a percentage, and the results will be recorded with integers before the percentage sign.

[0296] Relative activity of the test article = EC of the analytical standard 50 / EC of the test article 50 ;

[0297] Relative activity of the control = EC of the analytical standard 50 / EC of quality control products 50 .

[0298] System adaptability judgment

[0299] Analytical standards, quality control products and test products duplicate EC 50 The repeatability coefficient of variation CV% should not be greater than 30%, and the relative activity of the quality control product should be between 70% and 130%. If the above two conditions are met, the system adaptability is passed, otherwise the experiment will be invalid.

[0300] EC 50 The CV% can be calculated according to the coefficient of variation formula in statistics.

[0301] Example 1. LZM003 dilution buffer investigation

[0302] Purpose: To investigate the dilution buffer of LZM003 samples.

[0303] method:

[0304] 1. The inoculum size of HEK293 / LHCGR recombinant cells was 50,000 cells / well, and a 187.5 μL system was used. The starting concentration of LZM003 was 200 ng / mL, and a 2-fold gradient dilution was performed with 11 concentration gradients. The LZM003 samples were diluted with DPBS solution containing 1% CHAPS, DPBS solution containing 0.5% CHAPS, and DPBS solution containing 0.05% CHAPS, respectively, to investigate the effect on the results.

[0305] 2. The inoculum size of HEK293 / LHCGR recombinant cells was 50,000 cells / well, and a 187.5 μL system was used. The starting concentration of LZM003 was 200 ng / mL, and a 2-fold gradient dilution was performed with 11 concentration gradients. The LZM003 samples were diluted with a DPBS solution containing 2.5% DMSO and a DPBS solution containing 0.05% CHAPS, respectively, to investigate the effects of different types of diluents on the results.

[0306] Results: See Figure 1 , Figure 2 .

[0307] Conclusion: From Figure 1 It can be seen that the sample diluent containing 1% CHAPS significantly interfered with the reaction signal of LZM003, and the signal response value of LZM003 using the sample diluent containing 0.05% CHAPS was higher than that using the sample diluent containing 0.5% CHAPS. Figure 2 It can be seen that the sample diluent LZM003 EC containing 0.05% CHAPS 50 It is significantly smaller than that using sample dilution solution containing 2.5% DMSO, and the method has higher sensitivity.

[0308] Example 2. Optimization of LZM003 initial concentration and dilution gradient

[0309] Objective: To optimize the initial concentration and dilution gradient of LZM003.

[0310] method:

[0311] 1. The inoculation amount of HEK293 / LHCGR recombinant cells was 50,000 cells / well, and the 187.5 μL system was used. The initial concentration of LZM003 was 250 ng / mL (corresponding to the concentration of 50 ng / mL in the first well). Columns 2-3 were diluted 2.5 times, columns 4-8 were diluted 2 times, and columns 9-11 were diluted 2.5 times, with 11 concentration gradients. The LZM003 sample was diluted with DPBS solution containing 0.05% CHAPS.

[0312] 2. The inoculation amount of HEK293 / LHCGR recombinant cells is 50,000 cells / well, and the 187.5 μL system is used. The starting concentration of LZM003 is 250 ng / mL (corresponding to the concentration of 50 ng / mL in the first well). Columns 2-4 are diluted 4 times, columns 5-8 are diluted 2 times, columns 9-11 are diluted 4 times, and there are 11 concentration gradients. The LZM003 sample is diluted with DPBS solution containing 0.05% CHAPS. The starting concentration and dilution gradient of LZM003 can be selected according to the curve.

[0313] Results: See Figure 3 (Method 1) and Figure 4 (Method 2).

[0314] Conclusion: Comparison Figure 3 and Figure 4 It can be seen that the LZM003 sample was diluted with DPBS solution containing 0.05% CHAPS, the initial concentration of LZM003 was 250 ng / mL, and the gradient dilution was 4 times in columns 2-4, 2 times in columns 5-8, and 4 times in columns 9-11. The four-parameter curve had 3 points on the upper platform, 3 points on the lower platform, 3 points in the middle, and 1 point each at the inflection point of the upper and lower platforms, which met the four-parameter fitting requirements.

[0315] Example 3. Method validation

[0316] 3.1. Exclusivity

[0317] 3.1.1.LZM003 drug specificity

[0318] Objective: To investigate the specificity of r-hCG binding to the surface receptors of HEK293 / LHCGR recombinant cells using r-hCG, r-hCG Buffer (preparation buffer), AT132 (anti-TNFα monoclonal antibody), LZM002C (anti-CD20 monoclonal antibody), LZM005 (anti-HER2 monoclonal antibody), and LZM009 (anti-PD-1 monoclonal antibody) as samples.

[0319] Procedure: According to the determined reaction conditions, dilute the above samples with DPBS solution containing 0.0.5% CHAPS.

[0320] Results: See Figure 5 .

[0321] Conclusion: The results of the action of different samples showed that r-hCG had good binding specificity with the surface receptors of HEK293 / LHCGR recombinant cells.

[0322] 3.1.2. Cell specificity

[0323] Objective: To investigate the specificity of r-hCG binding to surface receptors of HEK293 / LHCGR recombinant cells using several different cell experiments.

[0324] Procedure: LHCGR recombinant HEK293 cells (HEK293 recombinant cells genetically modified to express G protein-coupled receptors and cyclic nucleotide-gated channels, from Codex Biosolutions), Raji cells (human lymphoma cells, from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences), Jurkat cells (human T lymphocytoma cells, from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences), PD-1effector cell cells (Jurkat cell line overexpressing human PD-1, from Promega), TF-1 cells (human blood leukemia cells, from ATCC), and HEK293 (IL-6R) cells (HEK293 cell line overexpressing human IL-6R, from Wuhan Pujian Biotechnology) were inoculated at a density of 50,000 cells per well.

[0325] Results: See Figure 6 .

[0326] Conclusion: The results of the action on different cells in this case showed that r-hCG had the best binding specificity to the surface receptor of HEK293 / LHCGR recombinant cells.

[0327] 3.2. Stability-indicating properties

[0328] Purpose: To verify that the method can effectively indicate changes in sample activity.

[0329] Procedure: Select the analytical standard (ARM-LZM003-003) and treat it in a 70℃ water bath for 2min, 5min, 10min, 20min, and 30min, respectively. Then, perform activity determination under the same conditions as the untreated analytical standard. Take the untreated analytical standard as the reference and calculate the changes in relative activity of samples under different treatment conditions.

[0330] Results: See Figure 7 and Table 6.

[0331] Table 6. Changes in sample activity under different treatment conditions

[0332] Processing conditions Relative activity ARM-LZM003-003 100％ 70℃2min 39.1％ 70℃5min 32.9％ 70℃10min 29.3％ 70℃20min 23.4％ 70℃30min 18.7％

[0333] Conclusion: The results show that the activity gradually decreases with the increase of treatment time, indicating that the method can effectively detect the activity changes of LZM003, so the method has good stability.

[0334] 3.3. Accuracy

[0335] Purpose: To determine the accuracy by using addition and recovery experiments, i.e. adding a known amount of LZM003 to the blank for determination and comparing the ratio of the measured value to the true value (recovery rate).

[0336] Procedure: According to the determined operating conditions and procedures, three analysts measured 50%, 75%, 100%, 125% and 200% concentration working samples on different days, and calculated the coefficient of variation (CV%) and range of the recovery rate.

[0337] The recovery rate was calculated as: measured concentration / theoretical concentration × 100%.

[0338] Set acceptance criteria: The recovery rate of each experiment should be between 70% and 130%, the CV% between each repeated experiment should be ≤30%, and the average recovery rate should be between 80% and 120%.

[0339] Results: See Table 7.

[0340] Table 7. Statistical results of recovery rate at different detection concentrations

[0341]

[0342] Conclusion: The experimental recovery rate ranges from 89% to 116%; the repeated experimental CV% ≤30%; the average recovery rate ranges from 98% to 107%; the total recovery rate is 100%, the total CV% ≤30%, and the accuracy is high.

[0343] 3.4. Repeatability

[0344] Objective: To investigate the differences in relative activity of 100% concentration assays between different analysts on different experimental days.

[0345] Procedure: According to the determined operating procedures, three analysts will test the 100% concentration of analytical standards on different days. Each analyst will perform two experiments and calculate the coefficient of variation (CV%) of the relative activity.

[0346] Acceptability criteria were set as follows: the coefficient of variation (CV%) of relative activity should be less than 30%.

[0347] Results: See Table 8.

[0348] Table 8. Repeatability test results

[0349]

[0350] Conclusion: Different analysts measured 100% concentration samples on different days, and the CV% of 6 experiments was ≤30%, which met the acceptable standard requirements for repeatability.

[0351] 3.5. Intermediate precision

[0352] Purpose: To compare the precision of different assay concentrations (50%, 71%, 100%, 141% and 200%) on different days under different experimental conditions, including different analysts, different experimental dates, etc. The aforementioned concentrations can correspond to the theoretical concentrations of the analytical standards.

[0353] Procedure: Three analysts tested samples at 50%, 75%, 100%, 125% and 200% of the test concentration on different days. Each analyst performed 2 experiments at each concentration and the coefficient of variation (CV%) of the relative activity was calculated.

[0354] Set acceptance criteria: The coefficient of variation of each concentration result measured by different analysts on different days should be less than 30%.

[0355] Results: See Table 9.

[0356] Table 9. Intermediate precision statistics

[0357]

[0358] Conclusion: The coefficient of variation of the results of different concentrations measured by different analysts on different days was less than 30%, which met the acceptable standard requirements.

[0359] 3.6. Linear

[0360] Linearity purpose: To determine the linear correlation between the test sample at each concentration and the measured value.

[0361] Experimental procedure: According to the determined operating procedure, 50%, 75%, 100%, 125% and 200% concentrations of working standards were measured on different days. Three experiments were performed for each concentration. The average relative activity of each concentration was calculated, and linear fitting was performed with the theoretical relative activity as the horizontal axis and the measured relative activity as the vertical axis.

[0362] Set the acceptance criteria: Linear correlation coefficient R of the fitted curve 2 Should not be less than 0.99.

[0363] Results: See Table 9 and Figure 8 .

[0364] Conclusion: The linear correlation coefficient R of the fitting curve 2 It is equal to 0.9995, which meets the acceptance standard requirements.

[0365] 3.7. Scope

[0366] Purpose: To determine the detection range of the analytical method.

[0367] Procedure: Referring to the accuracy experiment, 50%, 75%, 100%, 125% and 200% concentrations of analytical standards were measured respectively, and the variation of the results and the range of recovery at different concentrations were statistically analyzed.

[0368] Setting acceptance criteria: Same as accuracy experiment.

[0369] Results: Same as accuracy experiment.

[0370] Conclusion: Within the test range of 50% to 200%, the CV% of the determination results of each concentration was ≤30%, and the average recovery rate ranged from 98% to 107%, which met the acceptable standard requirements. 3.8.

[0372] According to the aforementioned embodiments, the specificity, repeatability, intermediate precision, accuracy, linearity, range, and stability-indicating properties have been verified.

[0373] The experimental results show that the detection method provided by the present invention has good specificity, good stability indication property, intermediate precision coefficient of variation (CV%) is less than 30%, the recovery rate of each measurement result ranges from 89% to 116%, and the linear correlation coefficient is R 2 It can reach 0.9995, and the detection range of relative activity is 50% to 200%. It can be used to determine the biological activity of LZM003.

[0374] Furthermore, a set of preferred main parameters is provided: the concentration of LZM003 in the first well is 50 ng / mL (concentration after sample addition), 4-fold gradient dilution is performed in columns 2-4, 2-fold gradient dilution is performed in columns 5-8, and 4-fold gradient dilution is performed in columns 9-11, with a total of 11 concentration gradients (concentration gradient before sample addition is 250 ng / mL, 62.5 ng / mL, 15.63 ng / mL, 3.91 ng / mL, 1.95 ng / mL, 0.98 ng / mL, 0.49 ng / mL, 0.24 ng / mL, 0.061 ng / mL, 0.015 ng / mL and 0.0038 ng / mL), the LZM003 sample is diluted with DPBS solution containing 0.05% CHAPS, the sample volume is 37.5 μL, and the total volume after sample addition is 187.5 μL.

[0375] The technical features of the above-mentioned embodiments and examples can be combined in any appropriate manner. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0376] The above embodiments only express several implementation methods of the present invention, which is convenient for understanding the technical solution of the present invention in detail, but it cannot be understood as limiting the scope of protection of the present invention. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present invention, several variations and improvements can be made, which all belong to the protection scope of the present invention. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and the equivalent forms obtained also fall within the protection scope of the present invention. It should also be understood that the technical solutions obtained by those skilled in the art on the basis of the technical solution provided by the present invention through logical analysis, reasoning or limited experiments are all within the protection scope of the claims attached to the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the content of the attached claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. A method for detecting the activity of human chorionic gonadotropin (hCG), characterized in that: The steps include: Obtaining hCG analysis standards, hCG test products and tool cells, wherein the tool cells are cells capable of expressing G protein-coupled receptors and cyclic nucleotide-gated channels; Prepare an analytical standard solution, wherein the analytical standard solution is prepared from a CHAPS diluent and the hCG analytical standard; the CHAPS diluent is a mixture of CHAPS and DPBS solution, and the concentration of CHAPS relative to the DPBS solution is 0.04% to 0.06% (w / v); preparing a test solution, wherein the test solution is prepared from a CHAPS diluent and the hCG test solution; Incubating the tool cells with Ro20-ACTOne dye in the dark to obtain a cell dye sample; wherein the Ro20-ACTOne dye is an ACTOne membrane potential dye containing the phosphodiesterase inhibitor Ro20-1724; and The analytical standard solution and the test solution are respectively mixed with the cell dye sample, incubated, and the relative activity of the hCG test sample is determined based on the staining results of the cell sample.

2. The detection method according to claim 1, characterized in that: The steps include: Obtaining the hCG analysis standard, the hCG test product and the tool cell; The tool cells are incubated with the Ro20-ACTOne dye in a dark environment and incubated in multiple culture wells under the same conditions to obtain multiple cell dye samples, and initial fluorescence signal values ​​F0 of the multiple cell dye samples are collected; Adding the same volume of analytical standard solution with different hCG concentrations to the plurality of cell dye samples to obtain a plurality of cell sample solutions with a first hCG concentration series; Adding the same volume of the test solution at different hCG concentrations to the other multiple cell dye samples to obtain multiple cell sample solutions with a second hCG concentration series; incubating each cell sample solution in the dark under the same conditions, and testing the endpoint fluorescence signal value Ft after the incubation; wherein the analytical standard solution of each concentration is prepared by the CHAPS diluent and the hCG analytical standard, and the test solution of each concentration is prepared by the CHAPS diluent and the hCG test solution; According to the F0 and Ft of the multiple cell sample solutions of the first hCG concentration series, the half effective concentration EC of the hCG analysis standard is obtained by analysis. 50(s) According to the F0 and Ft of the multiple cell sample solutions of the second hCG concentration series, the half effective concentration EC of the hCG test sample is obtained by analysis. 50(t) According to EC 50(s) and EC 50(t) The relative activity of the hCG test sample is calculated by the ratio of 50(s) and the EC 50(t) All the results were obtained by using four-parameter logistic fitting analysis method.

3. The detection method according to claim 2, characterized in that: Meet one or more of the following characteristics: (ta1) the highest hCG concentration in the first hCG concentration series and the highest hCG concentration in the second hCG concentration series are each independently greater than or equal to 50 ng / mL; (ta2) the lowest hCG concentration in the first hCG concentration series and the lowest hCG concentration in the second hCG concentration series are each independently less than or equal to 0.0008 ng / mL; (ta3) the dilution factor between any two adjacent hCG concentrations in the first hCG concentration series and the dilution factor between any two adjacent hCG concentrations in the second hCG concentration series are each independently selected from 1.5 to 5; (ta4) the numbers of hCG concentration distributions in the first hCG concentration series and the second hCG concentration series are each independently 8, 9, 10, 11, 12, 13, 14 or 15; (ta5) the hCG concentration distribution range of the first hCG concentration series and the second hCG concentration series each independently includes 0.00076-50 ng / mL, and the dilution factor each independently is 2 or 4; (ta6) the first hCG concentration series and the second hCG concentration series each independently include the following hCG concentration values: the dilution factors of the concentration values ​​ranked from high to low relative to the first well concentration are 1, 4, 4, 4, 2, 2, 2, 2, 4, 4 and 4, respectively, wherein the first well concentration is 50 ng / mL; (ta7) the hCG concentration distributions of the first hCG concentration series and the second hCG concentration series are set to be the same; and (ta8) In the step of incubating each cell sample solution in the dark under the same conditions, the incubation temperature is 25-30°C and the incubation time is 30-60 min.

4. The detection method according to claim 2, characterized in that: Meet one or more of the following characteristics: (tb1) The tool cells are HEK293 / LHCGR recombinant cells; (tb2) In the step of incubating the tool cells with the Ro20-ACTOne dye in the dark, the inoculation density of the tool cells in each culture chamber is 1.4×10 3 ~ 1.6×10 3 Pieces / mm 2 ; and (tb3) In the step of incubating the tool cells with the Ro20-ACTOne dye in the dark, the incubation temperature is 25-30° C. and the incubation time is 120±15 min.

5. The detection method according to any one of claims 1 to 4, characterized in that The Ro20-ACTOne dye is prepared by a method comprising the following steps: adding 0.04-0.06 μmol of Ro20-1724 to each milliliter of 1×ACTOne membrane potential dye.

6. The detection method according to claim 5, characterized in that: The Ro20-ACTOne dye is prepared by a method comprising the following steps: mixing 40-60 mM Ro20-1724 solution and 1×ACTOne membrane potential dye at a volume ratio of 1:(1000±200), wherein the Ro20-1724 solution is a DMSO solution of Ro20-1724.

7. The detection method according to claim 2, characterized in that: In the step of incubating the tool cells and the Ro20-ACTOne dye in the dark, the amount of the Ro20-ACTOne dye added is 1 volume; In the step of adding the same volume of the analytical standard solution with different hCG concentrations to the plurality of cell dye samples, the amount of the analytical standard solution added to each cell dye sample is 0.25 times the volume; In the step of adding the same volume of the test solution with different hCG concentrations to the plurality of cell dye samples, the amount of the test solution added to each cell dye sample is 0.25 times the volume.

8. The detection method according to claim 4, characterized in that: Meet one or more of the following characteristics: (tc1) F0 and F t The acquisition conditions included: excitation wavelength (535±5) nm, emission wavelength (595±5) nm; (tc2) performing the four-parameter logistic fitting analysis based on the curve of the Ft / F0 ratio changing with the logarithmic value of hCG concentration; (tc3) performing the four-parameter logistic fitting analysis including the step of eliminating abnormal data; and (tc4) The detection method described in the invention also includes testing the half effective concentration EC of hCG quality control product. 50(z) , wherein the method of obtaining the hCG quality control product is the same as that of the hCG analysis standard product; the detection method also includes a step of judging the system adaptability, and when the following two conditions are met at the same time, the system adaptability is judged to be passed: condition A, analyzing the EC of the hCG analysis standard product, the hCG quality control product and the hCG test product in duplicate wells 50 The repeatability coefficient of variation CV% should not be greater than 30%; and condition B, the relative activity of the quality control product should be between 70% and 130%.

9. The detection method according to any one of claims 1 to 4, characterized in that: The relative activity test results of the hCG test product meet one or more of the following characteristics: The intermediate precision coefficients of variation for the tests of 50%, 75%, 100% and 125% theoretical concentration values ​​were all less than 30%; The average recovery of relative activity was 98% to 107%; The recovery rate of each determination result of relative activity was selected from 89% to 116%; The linear fitting correlation coefficient between the measured relative activity and the theoretical relative activity is R 2 ≥0.9995; and The relative activity detection range includes 50%~200%.

10. The detection method according to claim 9, characterized in that: The relative activity test results of the hCG test product meet the following characteristics: the intermediate precision coefficient of variation of the tests of 50%, 75%, 100%, 125% and 200% theoretical concentration values ​​are all less than 30%.

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