CN116219078B - IL-29 biological activity determination method - Google Patents
IL-29 biological activity determination method Download PDFInfo
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- CN116219078B CN116219078B CN202310505271.1A CN202310505271A CN116219078B CN 116219078 B CN116219078 B CN 116219078B CN 202310505271 A CN202310505271 A CN 202310505271A CN 116219078 B CN116219078 B CN 116219078B
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Abstract
The invention discloses a method for measuring the biological activity of IL-29. Specifically, the method of the invention selects human embryonic lung epithelial cells (L132 cells) as detection cells, vesicular Stomatitis Virus (VSV) as detection viruses, and can protect the human embryonic lung epithelial cells (L132) from being damaged by Vesicular Stomatitis Virus (VSV) according to IL-29, stains the surviving L132 cells, and measures the absorbance of the surviving L132 cells at a certain wavelength to obtain a protective effect curve of the IL-29 on the L132 cells, thereby measuring the biological activity of the IL-29. The method adopts a four-parameter regression calculation method to process data, and the obtained four-parameter curve fitting degree R 2 High accuracy and repeatability, and is very suitable for detecting the biological activity of IL-29.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a method for measuring IL-29 biological activity.
Background
Bioassays are one method of assessing the biological activity of drugs using organisms including whole animals, ex vivo tissues, organs, cells, and microorganisms. Based on pharmacological action of medicine and biological statistics, the specific reaction between the test sample and the corresponding standard or reference sample is compared under certain condition by means of specific experiment design, and the potency, bioactivity or impurity caused toxicity of the test sample is determined through calculation of the ratio between the equal reaction doses or the biological reaction degree caused by limiting dose.
Bioassays are those wherein the effects of a test substance and its standard substance or reference substance on an organism or its isolated organ tissue, etc. are simultaneously compared under the same experimental conditions, and the equivalent dose ratio (R) of these substances is calculated by comparison. Bioassays have a certain experimental error, the main source of which is biological variability, so bioassays must be careful to control biological variation, or to reduce biological variation itself, or to reduce the influence of biological variation on experimental results with a proper experimental design, to reduce experimental error. The reliability test of biological detection requires that the dose or the response (or the function of the response) of the logarithmic dose accords with the requirement of a specific model in the dose range used in the experiment, and the linearity of the standard substance and the test substance meets the requirement of a calculation principle, namely meets the requirements of system applicability and sample applicability, and the potency and the credibility limit of the test substance can be calculated according to related formulas. The four parameter model requires that the logarithmic dose and response (or function of response) are S-curve over the dose range used, with the sigmoid curves of the test and standard being parallel.
IL-29 is type III interferon IFN- λ1, the antiviral effect of which firstly needs to form IFNLR1-IFN- λIL10R2 ternary complex by binding with a type III interferon receptor, and then activates the same Jak-STAT (Janus kinase-signal transducer of transcription) signaling pathway as that of type I interferon to trigger the expression of a plurality of ISGs (interferon stimulating genes) with antiviral function, thereby generating antiviral effect.
Although IL-29 and type I interferon (including IFN alpha and IFN beta) receptor different, but in the signal transduction pathway of the high similarity, so its biological activity detection method can refer to type I interferon. A method for inhibiting cytopathy of three general rules 3523 of Chinese pharmacopoeia includes such steps as staining the surviving WISH cells with crystal violet, measuring absorbance at 570nm to obtain the protecting effect curve of interferon to WISH cells, and measuring the biological activity of I-type interferon.
Accordingly, there remains a need in the art to develop an assay that can accurately measure IL-29 biological activity.
Disclosure of Invention
The invention aims to provide a determination method capable of accurately evaluating the biological activity of IL-29. The testing principle of the IL-29 biological activity testing method of the invention is as follows: IL-29 can be used for protecting human embryo lung epithelial cells (L132) from being destroyed by Vesicular Stomatitis Virus (VSV), and the living L132 cells are stained, and the absorbance of the living L132 cells is measured at a certain wavelength to obtain a protective effect curve of the IL-29 on the L132 cells, so that the biological activity of the IL-29 can be measured.
In a first aspect of the invention, there is provided a method for determining the biological activity of IL-29, said method comprising the steps of:
(1) Virus amplification: inoculating Vesicular Stomatitis Virus (VSV) into a single layer of cultured L132 cells for virus amplification, and collecting a culture supernatant when the L132 cell lesions reach 75% -100%, wherein the supernatant contains amplified vesicular stomatitis virus;
(2) Virus titration: half cell culture infection of amplified vesicular stomatitis virus obtained in step (1) using L132 cell titration (50% cell culture infectious dose, CCID) 50 ) The dilution factor of virus at half cell culture infection was recorded as 1: x is the virus titer;
(3) Reference and test solution preparation: serial dilution is carried out on the reference stock solution and the test stock solution to obtain m1 dose groups of reference stock solution and m2 dose groups of test stock solution, wherein m1=m2;
(4) Test article determination: the L132 cells and the amplified vesicular stomatitis virus are used for measuring a test sample, and the method specifically comprises the following substeps:
(4-a) inoculating L132 cells into detection wells of a culture plate, and culturing for T1 hour;
(4-b) adding the reference solution and the test solution prepared in the step (3) to the test wells of the L132 cell-inoculated culture plate, respectively, and culturing for T2 hours, and setting n complex wells corresponding to each dose group, wherein n is 2 or 3;
(4-c) amplifying vesicular stomatitis virus obtained in step (1) by 1: diluting the dilution factor of Y, adding the diluted dilution factor into a detection hole of a culture plate inoculated with L132 cells, and culturing for T3 hours, wherein the value of X/Y is 5-20;
(4-d) after the completion of the culture, discarding the culture supernatant, staining the cells in the detection wells with a crystal violet staining solution, and measuring the absorbance value of each detection well;
(5) Data processing and result calculation: processing data by four-parameter regression calculation method, taking the concentrations of each dosage group of serially diluted reference solution and test solution as abscissa, taking absorbance average value calculated based on compound holes of each concentration as ordinate, taking four-parameter curves of reference and test, and calculating half-Effective Concentration (EC) of reference and test 50 ) Thereby calculating the relative biological activity of the IL-29 of the test sample;
meanwhile, the reliability test is carried out on the measurement result, and the curve fitting degree R of the four-parameter curve of the reference and the test sample is included 2 And off-parallel.
In another preferred embodiment, in step (1), the collected culture supernatant is cryopreserved for subsequent use, the cryopreservation conditions being-80 ℃ and below.
In another preferred embodiment, in step (1), the L132 cells are cultured using a complete culture broth comprising: DMEM broth, 10% fetal bovine serum, based on the total volume of the complete broth.
In another preferred embodiment, in step (1), a stock solution (seed solution) of vesicular stomatitis virus is diluted 1:100 in DMEM medium containing 10% pancreatin, and then inoculated into a single layer of cultured L132 cells for virus amplification.
In another preferred embodiment, in step (2), the virus titration specifically includes the steps of:
(2-a) inoculating L132 cells into detection wells of a culture plate, and culturing for Ta hours;
(2-b) serial dilution of the amplified vesicular stomatitis virus obtained in step (1);
(2-c) adding serial dilutions of vesicular stomatitis virus to the detection wells inoculated with L132 cells, and culturing for Tb hours;
(2-d) after completion of the culture, discarding the culture supernatant, staining the cells in the detection wells with a crystal violet staining solution, and measuring the absorbance value of each detection well to calculate the half cell culture infection amount (50% cell culture infectious dose, CCID) of the amplified vesicular stomatitis virus obtained in the step (1) 50 ) The dilution factor of virus at half cell culture infection was recorded as 1: x is the virus titer.
In another preferred embodiment, in step (2-a), the L132 cells are seeded at a density of 1.0' 10 5 -2.0´10 5 And each ml.
In another preferred embodiment, in step (2-a), L132 cells are seeded with an assay medium comprising: DMEM broth, 7% fetal bovine serum, based on the total volume of the assay broth.
In another preferred embodiment, in step (2-a), ta is 24.+ -. 2.
In another preferred embodiment, in step (2-b), serial dilutions are performed using a measurement broth comprising: DMEM broth, 7% fetal bovine serum, based on the total volume of the assay broth.
In another preferred embodiment, in step (2-b), the serial dilution is an equal dilution, the equal dilution being 2-10 times, preferably 5 times.
In another preferred embodiment, in step (2-c), tb is 24.+ -. 2.
In another preferred example, in the step (2-d), "staining cells in the detection well with crystal violet staining solution" includes the steps of: after 50. Mu.l/well of the staining solution is added thereto, the mixture is left at room temperature for 20 to 40 minutes (preferably 30 minutes), the staining solution and dead cells are washed off with running water, and after the residual moisture is sucked dry, 100. Mu.l/well of the decoloring solution is added thereto, and the mixture is left at room temperature for 3 to 5 minutes.
In another preferred example, the crystal violet staining solution has the following formula: 50mg of crystal violet was weighed, 20ml of absolute ethanol was added thereto for dissolution, and then water was added thereto for dilution to 100ml.
In another preferred embodiment, the decolorization solution is prepared as follows: 50ml of absolute ethyl alcohol and 0.1ml of acetic acid are measured, and water is added for dilution to 100ml.
In another preferred embodiment, in the step (2-d), absorbance is measured with reference wavelength of 630nm and detection wavelength of 570 nm.
In another preferred embodiment, in step (3), the serial dilution is an equal dilution, the equal dilution being 2-5 times, preferably 3-4 times.
In another preferred embodiment, in step (3), the first dose group of the serial dilutions is suitably at a concentration of 100-300ng/ml, preferably 200ng/ml.
In another preferred embodiment, in step (3), m1 (m 2) is from 8 to 12, preferably 10.
In another preferred embodiment, in step (3), serial dilutions are performed using a measurement broth comprising: DMEM broth, 7% fetal bovine serum, based on the total volume of the assay broth.
In another preferred embodiment, in step (4-a), the L132 cells are seeded at a density of 1.0' 10 5 -2.0´10 5 cells/ml。
In another preferred embodiment, in step (4-a), L132 cells are seeded with an assay medium comprising: DMEM broth, 7% fetal bovine serum, based on the total volume of the assay broth.
In another preferred embodiment, in step (4-a), T1 is 4-6.
In another preferred embodiment, in step (4-b), T2 is 18-24.
In another preferred embodiment, in step (4-b), the serial dilutions of the reference and test solutions are added to each detection well in a volume of 50-200. Mu.l/well, preferably 100. Mu.l/well.
In another preferred embodiment, in step (4-c), the amplified vesicular stomatitis virus obtained in step (1) is diluted with a challenge culture medium comprising: DMEM broth, 3% fetal bovine serum, based on the total volume of the challenge broth.
In another preferred embodiment, in step (4-c), T3 is 24.+ -. 2.
In another preferred embodiment, in step (4-c), the value of X/Y is 10, i.e., the challenge amount of amplified vesicular stomatitis virus is 10 CCID 50 。
In another preferred example, in the step (4-d), "staining cells in the detection well with crystal violet staining solution" includes the steps of: after 50. Mu.l/well of the staining solution is added thereto, the mixture is left at room temperature for 20 to 40 minutes (preferably 30 minutes), the staining solution and dead cells are washed off with running water, and after the residual moisture is sucked dry, 100. Mu.l/well of the decoloring solution is added thereto, and the mixture is left at room temperature for 3 to 5 minutes.
In another preferred example, the crystal violet staining solution has the following formula: 50mg of crystal violet was weighed, 20ml of absolute ethanol was added thereto for dissolution, and then water was added thereto for dilution to 100ml.
In another preferred embodiment, the decolorization solution is prepared as follows: 50ml of absolute ethyl alcohol and 0.1ml of acetic acid are measured, and water is added for dilution to 100ml.
In another preferred embodiment, in the step (4-d), absorbance is measured with reference wavelength of 630nm and detection wavelength of 570 nm.
In another preferred embodiment, in step (5), a four-parameter regression algorithm is selected, using the reference EC of the fitted curve in the constraint model 50 And test sample EC 50 Dividing to calculate the relative biological activity of the test sample IL-29, namely
Test sample IL-29 biological relative activity (%) = (reference EC) 50 Test article EC 50 ）×100%。
In another preferred embodiment, in step (5), the reliability criteria for the measurement result are as follows:
curve fitting degree R of free model and constraint model of four-parameter curve of reference and test sample 2 Not less than 0.98;
the deviation from parallelism should not be significant, i.e. P.gtoreq.0.01.
In another preferred embodiment, the reference is an IL-29 standard for which biological activity is determined.
In another preferred embodiment, the culture plate is a 96 well cell culture plate.
In another preferred embodiment, the method is carried out under conditions of 37℃and 5% CO 2 。
In a second aspect of the invention, there is provided an IL-29 biological activity assay reagent combination comprising:
(Z1) L132 cells;
(Z2) vesicular stomatitis virus seed solution;
(Z3) a reference that is an IL-29 standard for biological activity determination.
In another preferred embodiment, the reagent combination further comprises:
(Z4) DMEM broth and fetal bovine serum for use in formulating complete broth, assay broth and challenge broth, wherein the complete broth comprises: DMEM broth, 10% fetal bovine serum, based on the total volume of the complete broth; the assay medium comprises: DMEM broth, 7% fetal bovine serum, based on the total volume of the assay broth; the toxicity counteracting culture solution comprises the following components: DMEM broth, 3% fetal bovine serum, based on the total volume of the challenge broth; and/or
(Z5) crystal violet staining solution and decolorizing solution.
In a third aspect of the invention, there is provided an IL-29 biological activity assay kit comprising:
(C1) A first container, L132 cells located within the first container;
(C2) A second container, a vesicular stomatitis virus seed solution within the second container;
(C3) A third container, and a reference positioned within the third container, the reference being a biologically active, defined IL-29 standard;
And, the kit comprises a description which describes the method for measuring IL-29 biological activity according to the first aspect of the present invention.
In another preferred embodiment, the kit further comprises:
(C4) Fourth and fifth containers, DMEM culture solution in the fourth container and fetal bovine serum in the fifth container; and/or
(C5) A sixth container and a seventh container, and a crystal violet staining solution in the sixth container and a decolorizing solution in the seventh container.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
FIG. 1 shows WISH-VSV detection of IL-29 and IFN alpha 1b biological activity dose response curve; wherein, plot1 is IFN alpha 1B, plot2 is self-made batch IL-29 sample A, and Plot3 is self-made batch IL-29 sample B.
FIG. 2 shows a graph of the dose response of HepG2-VSV to detect IL-29; wherein, plot1 is self-made batch IL-29 sample C, plot2 is self-made batch IL-29 sample D.
FIG. 3 shows a graph of the dose response of L132-VSV to detect the biological activity of three IL-29 samples.
FIG. 4 shows a reference and test free model fitting curve; wherein Plot2 is the free model fitting curve of the reference, and Plot3 is the free model fitting curve of the test sample.
FIG. 5 shows a reference and test constraint model fitting curve; wherein, plot3 is the fitting curve of the reference constraint model, and Plot4 is the fitting curve of the test constraint model.
FIG. 6 shows four parameter fitting graphs of a reference and test article of the present invention; wherein, plot1 is the fitting curve of the reference constraint model, and Plot3 is the fitting curve of the test constraint model.
FIG. 7 shows a linear regression equation for the log and log values of the theoretical biological activity of the IL-29 bioassay.
Detailed Description
The present inventors have conducted extensive and intensive studies and, for the first time, have unexpectedly developed an assay that enables accurate assessment of IL-29 biological activity. The inventor selects L132 cells (a human embryo lung epithelial cell) which have good response to detection of viruses VSV and IL-29 from a plurality of cells through a large number of screening experiments and are easy to fall off from a cell culture plate after being infected by the VSV viruses as detection cells, so that the background level of subsequent cell staining is reduced to the greatest extent, and the cell staining detection result is more accurate. Meanwhile, reasonable detection steps and data analysis methods are formulated, and parameters in the steps of virus amplification, virus titration, reference and test solution preparation and test measurement are further determined, so that the measurement result is accurate and reliable and the method is applied to extensive IL-29 biological activity evaluation.
On this basis, the present invention has been completed.
Terminology
In order that the invention may be more readily understood, certain technical and scientific terms are defined below. Unless defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
As used herein, when used in reference to a specifically recited value, the term "about" means that the value can vary no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).
As used herein, the term "comprising" or "including" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….
The method of the invention
The invention provides a method for measuring the biological activity of IL-29, which comprises the following steps:
(1) Virus amplification: inoculating Vesicular Stomatitis Virus (VSV) into a single layer of cultured L132 cells for virus amplification, and collecting a culture supernatant when the L132 cell lesions reach 75% -100%, wherein the supernatant contains amplified vesicular stomatitis virus;
(2) Virus titration: make the following stepsTitration of half cell culture infectious amount of amplified vesicular stomatitis virus obtained in step (1) with L132 cells (50% cell culture infectious dose, CCID) 50 ) The dilution factor of virus at half cell culture infection was recorded as 1: x is the virus titer, the step comprises the following substeps:
(2-a) inoculating L132 cells into detection wells of a culture plate, and culturing for 24+ -2 hours;
(2-b) serial dilution of the amplified vesicular stomatitis virus obtained in step (1);
(2-c) adding serial dilutions of vesicular stomatitis virus to the detection wells inoculated with L132 cells, and culturing for 24±2 hours;
(2-d) after completion of the culture, discarding the culture supernatant, staining the cells in the detection wells with a crystal violet staining solution, and measuring the absorbance value of each detection well to calculate the half cell culture infection amount (50% cell culture infectious dose, CCID) of the amplified vesicular stomatitis virus obtained in the step (1) 50 ）；
(3) Reference and test solution preparation: serial dilution is carried out on the reference stock solution and the test stock solution to obtain m1 dose groups of reference stock solution and m2 dose groups of test stock solution, wherein m1=m2, and m1 (m 2) is 8-12, preferably 10;
(4) Test article determination: the L132 cells and the amplified vesicular stomatitis virus are used for measuring a test sample, and the method specifically comprises the following substeps:
(4-a) inoculating L132 cells into detection wells of a culture plate, and culturing for 4-6 hours;
(4-b) adding the reference solution and the test solution prepared in the step (3) to the detection wells of the L132 cell-inoculated culture plate, respectively, and culturing for 18-24 hours, wherein n complex wells are arranged corresponding to each dose group, wherein n is 2 or 3;
(4-c) amplifying vesicular stomatitis virus obtained in step (1) by 1: dilution of Y is followed by addition to the detection wells of the L132-inoculated plates for 24.+ -. 2 hours, wherein X/Y has a value of 5-20, preferably 10;
(4-d) after the completion of the culture, discarding the culture supernatant, staining the cells in the detection wells with a crystal violet staining solution, and measuring the absorbance value of each detection well;
(5) Data processing and result calculation: processing data by four-parameter regression calculation method, taking the concentrations of each dosage group of serially diluted reference solution and test solution as abscissa, taking absorbance average value calculated based on compound holes of each concentration as ordinate, taking four-parameter curves of reference and test, and calculating half-Effective Concentration (EC) of reference and test 50 ) Thereby calculating the relative biological activity of the IL-29 of the test sample;
meanwhile, the reliability test is carried out on the measurement result, and the curve fitting degree R of the four-parameter curve of the reference and the test sample is included 2 And off-parallel.
Selection for detecting cells and detecting viruses:
the detected cells are human embryo lung epithelial cells (L132 cells), and compared with other cells, the cells have good response to the detection viruses VSV and IL-29, can fall off from a cell culture plate after being infected and dead by the viruses, and can not influence the subsequent staining of crystal violet so as to cause high background.
For the preparation of the reagents:
complete culture solution: DMEM medium containing 10% FBS for normal culture of L132 cells; measuring the culture solution: DMEM medium containing 7% FBS for pre-inoculation dilution of L132 cells in the virus titration step and serial dilution of vesicular stomatitis virus, serial dilution in the reference and test sample solution preparation step, and pre-inoculation dilution of L132 cells in the test sample measurement step; toxin-counteracting culture solution: DMEM medium containing 3% FBS was used for serial dilutions of vesicular stomatitis virus in the test item assay step. The FBS concentrations of the measured culture solution and the toxicity-attacking culture solution are not adjusted at will, and the adjusted FBS concentrations can influence the adding concentrations of the IL-29 reference substance and the sample, the toxicity-attacking dosage and the upper and lower platforms of the four-parameter fitting curve.
In virus titration:
the manner of virus titration is consistent with the conditions for the determination of IL-29 biological activity, including the number of cells inoculated, the culture medium, the staining method, data processing, etc., so that the virus titer can be matched with the virus titer suggested for the determination of IL-29.
For the preparation of reference and test solutions:
the number of reference and test solutions required in the experimental design should be equal, as should the number of response values per dose group. Each group of dose intervals is typically serially diluted at equal ratios. The series of concentrations in the plates of the reference and test solutions should ensure that the final four parameter curves are both approximately S-shaped and that a distinct plateau appears. In view of the peripheral well rejection with edge effects, the reference and test solutions are typically of 10 series of concentrations.
According to the comparison of the invention, the serial dilution multiple is selected to be 4 times, so that the upper and lower platforms can be ensured to have 2-3 concentration points respectively, and the requirement of 'having obvious upper and lower platforms' in reliability test is met. If experimental conditions and test samples are stable and the operation is more skilled, serial dilution multiple can be adjusted to 3 times, the detection accuracy can be further improved, the multiple dilution is not recommended, and the simultaneous appearance of an obvious upper platform and a lower platform is difficult to ensure.
For test article determination:
the titres used for VSV challenge should ensure that the dose-response fit curve for IL-29 is S-shaped, too high a viral titre may affect the appearance of the upper plateau, while too low a viral titre may affect the appearance of the lower plateau.
After the cells are stained, the staining solution needs to be carefully flushed by running water, residual moisture is absorbed, and if the running water is relatively rapid, the adherent living cell layers can be flushed down, so that the difference of multiple holes is large. Meanwhile, residual moisture needs to be sucked up, and the residual moisture can also cause large difference of multiple holes.
In the four-parameter regression calculation method in the data processing and calculation results:
data processing and result calculation are required according to four-parameter regression calculation method in the three general rules 1431 biological verification statistical method of Chinese pharmacopoeia 2020 edition. The method requires that the logarithmic doses x of a standard substance or a reference substance (S) and a test substance (T) are in an S-shaped or S-shaped relation with a reaction value or a specific function y of the reaction value in a certain dose range, a four-parameter logistic regression equation can be fitted, the fitted curve is symmetrical to an inflection point, an gradual-in line is respectively arranged at the upper part and the lower part, and when the active components of S and T are basically the same, the two fitted curves are parallel.
Four-parameter logistic curve equation:
Wherein y is a reaction value or a specific function of the reaction value
d is the dosage of the standard substance or the test substance
A is y (S shape: lower asymptote; inverse S shape: upper asymptote) when d.fwdarw.0
D is y (S shape: lower asymptote; inverse S shape: upper asymptote) at D → infinity
C is y= (a+d)/2, i.e. 50% Effect Concentration (EC) 50 Or ED 50 ）
B is a slope factor (and EC 50 Or ED 50 Slope correlation of the plot
And adopting a four-parameter logistic free model and a constraint model in computer software, and carrying out S and T free fitting and constrained fitting of the estimated values of A, B, C, D parameters in a curve according to the principle of a nonlinear least square method, wherein the constraint model is a parallel curve model, wherein the estimated values of the three parameters of A, B, D of an S fitting equation and the estimated values of the three parameters of T fitting equation are respectively the same, and only the estimated values of the parameter C are different.
For the experimental result of the establishment of reliability, the S and T fitting curve EC in the constraint model can be adopted according to the principle of equal response dose ratio 50 Calculating the relative biological activity (R) of the test sample
R= (standard EC 50 Test article EC 50 ）×100%
In the reliability test of the four-parameter regression calculation method in the data processing and calculation results:
The four parameter curves of the reference and test article are required to be similar to an S shape, and obvious upper and lower platforms appear. The estimated values of A, B, C, D four parameters of the four-parameter curve free fitting and constraint fitting are more accurate.
R 2 The measurement is the fitting degree of the four-parameter regression equation, which is the overall relation between the expression dependent variable and all independent variables, R 2 The closer to 1 the value of (c) is, the better the fitting degree of the four-parameter regression curve to the detection value is.
The deviation from parallelism reflects the degree of parallelism of the two fitted curves, which are parallel when the active components of S and T are substantially identical. The less significant the deviation from parallelism, the higher the degree of parallelism of the two fitted curves. Parameters C estimated in constraint model (EC representing S and T
50 ) The more accurate.
The reagent combination and the kit of the invention
The invention also provides a reagent combination for measuring the biological activity of IL-29, which comprises: (Z1) L132 cells; (Z2) vesicular stomatitis virus seed solution; (Z3) a reference that is a biologically active defined IL-29 standard; alternatively, the reagent combination may further comprise: (Z4) DMEM medium and fetal bovine serum for preparing complete medium, assay medium and challenge medium; and/or (Z5) crystal violet staining solution and decolorizing solution.
Further, the components of the above reagent combination may be placed in separate containers and the combination may be placed in a kit, thereby obtaining an IL-29 biological activity assay kit. Further, a description is provided in the kit, wherein the description describes the method for measuring IL-29 biological activity according to the first aspect of the present invention.
Cells for use in the present invention
L132 cells: a commercially available human embryonic lung epithelial cell, ATCC number CCL-5, available from Ningbo Biotechnology Co., ltd
WISH cells: a commercially available human amniotic cell, ATCC number CCL-25, available from Shanghai Orthosiphon Biotechnology Co., ltd
HepG2 cells: a commercially available human hepatoma cell, CCTCC library number GDC0024, is available from China center for type culture Collection
A549 cells: a commercially available human non-small cell lung cancer cell, ATCC number CCL-185, available from Shanghai, qiao Xin boat Biotechnology Co., ltd
The beneficial effects of the invention include:
the study of the invention shows that: even with the addition of high concentrations of IL-29, the protective effect on WISH cells was not achieved. The common human lung adenocarcinoma cells (A549) and human hepatoma cells (HepG 2) in the literature respond to IL-29 and VSV viruses, but the cells are difficult to fall off from a cell culture plate after being infected by the VSV viruses, so that a very high background is caused, and the problems of high platform value, less steep dose-response curve, larger experimental error and the like under a four-parameter fitting curve exist.
The L132 cells are sensitive to the detection of the virus VSV, have good response to the detection of the sample IL-29, and can fall off from the cell culture plate after being killed by the virus VSV, and can be washed out only by flowing water, so that high background caused by residues on the cell plate is avoided.
By determining the preferred serum concentration of each culture solution, the cell inoculation amount, the virus attack titer, the IL-29 sample concentration range, the IL-29 action time and the VSV virus action time, the cell staining mode can ensure that the logarithmic dosage x of the IL-29 and the absorbance y form an S-shaped relationship, and can be fitted into a four-parameter logistic regression equation. The method has the advantages of steep dose-response curve, high fitting degree of four-parameter curve, high passing rate of reliability test, small experimental error and high repeatability of detection results.
The invention will be further illustrated by the following examples. The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions such as Sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated. Unless otherwise specified, materials and reagents used in the examples of the present invention are commercially available products.
Example 1 cell Screen
To establish an assay for the biological activity of IL-29, attempts were first made to detect the biological activity of IL-29 using a combination of WISH cells and VSV virus, but even if IL-29 was added at a high concentration, it was not possible to protect WISH cells, and IFN alpha 1b was able to protect WISH cells from the destructive effects of VSV virus, and the results are shown in FIG. 1.
Since WISH cells do not respond significantly to IL-29, hepG2 (human hepatoma cell) commonly used in the literature was selected for testing. The results show that IL-29 can provide protection to HepG2 and that the results after crystal violet staining are shown in FIG. 2: the absorbance of the upper and lower platforms is about 0.8, and a dose response curve of IL-29 exists, however, the HepG2-VSV method still has the problems of high lower platform value, no steeper dose-response curve, larger experimental error and the like.
Human non-small cell lung cancer cells (A549 cells) were also tested according to the literature, and although the protection of A549 cells by IL-29 was observed by microscopy, there was little change in absorbance of the virus control group and IL-29 protected group after staining with crystal violet.
In view of the general high expression of type III interferon receptors by epithelial cells, human epithelial cell lines including HBE (human bronchial epithelial-like cells), HBEpiC (human bronchial epithelial cells), hACE2-HeLa (cervical cancer cells transformed with human ACE2 gene), HNEpC (nasal mucosa epithelial cells), vero (African green monkey kidney cells), L132 (human embryonic lung epithelial cells), calu-3 (human lung adenocarcinoma cells), IEC-6 (rat small intestine epithelial cells), INS-1 (rat islet cell tumor cells) and GL261 (mouse glioma cells) have been continuously screened. Research shows that L132 is not only sensitive to IL-29, but also has large absorbance difference after crystal violet dyeing, high fitting degree of four-parameter curve and R 2 Can reach more than 0.99 (see figure)3) The method was finally determined to be selected for the biological assay of IL-29.
EXAMPLE 2 method for determining the biological Activity of IL-29 of the invention
(1) Reagent, preparation for detecting virus and detecting cell
The following raw materials were mainly used: DMEM medium; fetal bovine serum; pancreatic enzymes; l132 cells.
Dyeing liquid: weighing 50mg of crystal violet, adding 20ml of absolute ethyl alcohol for dissolution, and then adding water for dilution to 100ml;
decolorization liquid: 50ml of absolute ethyl alcohol and 0.1ml of acetic acid are measured, and water is added for dilution to 100ml;
complete culture solution: 10ml of fetal bovine serum and 90ml of DMEM culture solution are added;
measuring the culture solution: 7ml of fetal bovine serum was added with 93ml of DMEM medium and the mixture was used.
Toxin-counteracting culture solution: 3ml of fetal bovine serum and 97ml of DMEM culture solution are added for preparation.
(2) Virus amplification, preservation and titration
a. Amplifying and preserving:
10% pancreatin diluted with DMEM medium, VSV diluted 1:100, was inoculated into well-grown monolayer L132 cells for proliferation, and cultured at 37℃with 5% CO 2 Culturing in an incubator until cytopathy reaches 75% -100%, centrifuging at 3000rpm for 20 min after blowing, collecting supernatant (containing amplified vesicular stomatitis virus), and freezing below-80deg.C;
b. Titration:
cell density was adjusted to 1.0' -10 5 ~2.0´10 5 cell/ml, inoculating cell suspension into 96-well plate, 100 μl/well, culturing at 37deg.C for 24 hr, serial diluting amplified vesicular stomatitis virus 5 times from original time, adding 100 μl/well into cells, and culturing at 37deg.C with 5% CO 2 After 24 hours of incubation in the incubator, the crystal violet staining was read and half the cell culture infection (50% cell culture infectious dose, CCID) was calculated 50 ) The viral titer used for challenge is typically about 10 CCID 50 。
(3) Reference and test solution preparation
The reference and test samples were diluted to appropriate concentrations, 4-fold serial dilutions were made in 96-well cell culture plates for a total of 10 dilutions, 2 wells per dilution. Operating under aseptic conditions.
(4) Test article measurement
L132 cell seeding: taking L132 cells in logarithmic growth phase, centrifuging, and adding DMEM culture solution for washing 3 times. The cells were resuspended in assay medium and the cell density was adjusted to 1.0' -10 5 ~2.0´10 5 cell/ml, cell suspension was inoculated in 96-well plate, 100. Mu.l/well, at 37℃with 5% CO 2 Culturing in an incubator for 4-6 hours.
b. Sample adding: the prepared reference and test solutions were transferred to L132 cell-inoculated plates at 100. Mu.l/well at 37℃with 5% CO 2 Culturing in an incubator for 18-24 hours. Adding the measurement culture solution into the outer ring of the 96-well plate, wherein the volume of the measurement culture solution is 100 mu l/well;
c. cell detoxification: the supernatant from the cell culture plate was discarded, and the preserved vesicular stomatitis virus ("supernatant" in (2) a) above was diluted to about 10 CCID with the challenge culture solution 50 100 μl/well at 37deg.C, 5% CO 2 Culturing in an incubator for 24 hours;
d. and (3) crystal violet detection: the supernatant in the cell culture plate is discarded, 50 mu l of staining solution is added to each hole, after the cell culture plate is placed at room temperature for 30 minutes, the staining solution is carefully flushed by using running water, residual moisture is absorbed, 100 mu l of decolorizing solution is added to each hole, and the cell culture plate is placed at room temperature for 3-5 minutes. Placing the 96-well plate into a multifunctional enzyme-labeled instrument, uniformly mixing, taking 630nm as a reference wavelength, measuring absorbance at 570nm wavelength, and recording the measurement result;
(5) Data processing and calculation of results
a. Adopting four-parameter regression calculation method of enzyme label instrument to process, taking reference and tested protein concentration (ng/ml) as abscissa and OD 570 Mean value is ordinate, four parameter curves are made and half effective concentration EC of reference and test sample is calculated respectively 50 (ng/ml), the relative biological activity of the test IL-29 (reference EC) was calculated as follows 50 And test sample EC 50 All are constraint models)
Test sample IL-29 biological relative activity (%) = (reference EC) 50 Test article EC 50 ）×100%
b. Acceptable standards for system applicability and sample applicability (reliability test)
The four parameter curves of the reference and the test sample are similar to an S shape, and obvious upper and lower platforms appear;
curve fitting degree R 2 Not less than 0.98;
the deviation from parallelism should not be significant (P.gtoreq.0.01).
The absorbance values of the series of concentration references and test samples obtained by the above test are shown in tables 2-1 and 2-2.
Table 2-1 reference line concentration and absorbance values
TABLE 2-2 test line concentration and absorbance values
Adopting a four-parameter logical Style free model and a constraint model in computer software, performing free fitting (figure 4) and constraint fitting of a reference and a test sample according to the principle of a nonlinear least square method, and estimating A, B, C, D four parameter values in a curve, R 2 Is the curve fitting degree; the constraint model is a parallel curve model, wherein the estimated values of the three parameters A, B, D of the fitting equation of the reference and the test sample are respectively the same, and only the estimated value of the parameter C is different (see fig. 5). The measured Ref.Plot value of Plot4 in the constraint model is the relative biological activity of the test sample, and the F-Prob value is the P value deviating from the parallel.
Example 3 four parameter fitness R of the method of the invention 2 Evaluation of the four-parameter fitting curve (FIG. 6) of the reference and test pieces obtained by the above test, the fitting degree R of the curve 2 Is the key of the reliability, accuracy and repeatability of the test result. Fitting degree R 2 Measured byThe fitting degree of the regression equation is the overall relation between the dependent variable and all independent variables. R is R 2 Equal to the sum of squares of the regression and the ratio of the sum of squares to the total sum of squares, i.e., the percentage of variability of the dependent variable that can be interpreted by the regression equation. R is R 2 The closer to 1 the value of (c) is, the better the fitting degree of the four-parameter regression curve to the detection value is.
Four-parameter curve fitting degree R in three general rule 3531 Nituozhu single antibiotic biological activity assay and general rule 3535 Kangbai cilp biological activity assay of Chinese pharmacopoeia 2 The requirements are "R 2 Should be greater than 0.92 "and" fitness R 2 Greater than 0.95%. The example is also a cell-based biological activity determination method, and four-parameter fitting curves of the reference are obtained through multiple times of the tests, and four-parameter curve fitting degrees R of the reference tested at 6 different dates are listed 2 The fitting degree results are all higher than 0.99 (see table 3-1), and the fitting degree results reach the requirement of the detection method based on the ELISA principle on the four-parameter curve fitting degree, which is far higher than the requirement of two cell-based biological activity detection methods listed in Chinese pharmacopoeia on the four-parameter curve fitting degree.
Table 3-1 reference four parameter Curve Fit R 2 Results summary of (2)
EXAMPLE 4 evaluation of relative accuracy and precision of the method of the invention
Relative accuracy: the degree to which the measured relative activity approaches the true or reference value is generally expressed in terms of relative bias (RB,%) within a specified range. The samples were diluted to different target activity levels, and 5 samples were evaluated for more reliable results, each activity level being measured at least 3 times independently. The log (ordinate) of the corresponding activity measurement value is subjected to linear regression with the log (abscissa) of the activity theoretical value. The relative accuracy was assessed using the relative bias of each activity level measurement and the trend of the change in the relative bias of the different activity levels, which can be assessed using the slope of the linear regression equation.
Rb= ((potency assay value +.titre theory) -1) ×100%
The invention selects and prepares 200%, 150%, 100%, 50% and 10% of test sample solutions corresponding to the biological activity of the reference sample, and the test sample solutions are used for examining the relative accuracy of the detection method, and the results are shown in Table 4-1. The relative bias of each concentration detected by the method is within a range of +/-25%, particularly more than 100%, and the relative bias is within a range of +/-10%. The logarithm (abscissa) of the activity theoretical value is used for carrying out linear regression on the logarithm (ordinate) of the corresponding activity measured value, the regression equation is y=1.05x+0.02, the slope of the regression equation is 1.05, the range requirement of 0.80-1.25 is met, and the slope is very close to the optimal value of 1.00. The results show that the relative accuracy of the method is high.
Table 4-1 IL-29 relative bias of biological Activity level measurements from different biological assays
Precision: the degree of closeness between the results of sampling and measuring the same uniform sample under a predetermined measurement condition is generally expressed by Geometric Standard Deviation (GSD) or geometric variation coefficient (GCV,%) for the relative activity measurement method. Within a specified range, the influence of random variation factors such as different dates, different analysts, different instruments, different key reagent batches and the like on precision is examined. At each activity level, the geometric standard deviation of the measured relative activity was calculated as follows:
GSD=antilog(SD)
where SD is the standard deviation of activity measurements for each activity level.
At each activity level, the geometric coefficient of variation of the measured relative activity was calculated as follows:
GCV=(GSD-1)×100%
the invention selects and prepares 200%, 150%, 100%, 50% and 10% of sample solution corresponding to the biological activity of the reference sample, and the sample solution is used for examining the precision of the detection method. The results are shown in Table 4-2, and the geometric variation coefficient GCV of the relative activity of each concentration measurement in the method is within + -25%, which shows that the method has high precision.
Table 4-2 IL-29 geometric standard deviation and coefficient of geometric variation for measured values of different levels of biological Activity
Linearity: generally refers to measuring the linear relationship between relative activity and a true or reference value, within the design envelope, as a dilution linearity related to relative accuracy. Typically, the reference is diluted to different target activity levels within a specified range, the logarithm of the activity theoretical value (abscissa) is plotted against the logarithm of the corresponding activity measurement value (ordinate), linear regression is performed by using a least square method, the linearity is generally represented by the correlation coefficient r of a linear regression equation, and as a result, as shown in fig. 7, the fitted linear regression equation is y=1.0535x+0.02, and the correlation coefficient is 0.995.
All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.
Claims (10)
1. A method for determining the biological activity of IL-29, said method comprising the steps of:
(1) Virus amplification: inoculating Vesicular Stomatitis Virus (VSV) into a single layer of cultured L132 cells for virus amplification, and collecting a culture supernatant when the L132 cell lesions reach 75% -100%, wherein the supernatant contains amplified vesicular stomatitis virus;
(2) Virus titration: half-finer of amplified vesicular stomatitis virus obtained in step (1) using L132 cell titrationCell culture infection (50% cell culture infectious dose, CCID) 50 ) The dilution factor of virus at half cell culture infection was recorded as 1: x is the virus titer;
(3) Reference and test solution preparation: serial dilution is carried out on the reference stock solution and the test stock solution to obtain m1 dose groups of reference stock solution and m2 dose groups of test stock solution, wherein m1=m2 and m1 (m 2) is 8-12;
(4) Test article determination: the L132 cells and the amplified vesicular stomatitis virus are used for measuring a test sample, and the method specifically comprises the following substeps:
(4-a) inoculating L132 cells into detection wells of a culture plate, and culturing for T1 hour;
(4-b) adding the reference solution and the test solution prepared in the step (3) to the test wells of the L132 cell-inoculated culture plate, respectively, and culturing for T2 hours, and setting n complex wells corresponding to each dose group, wherein n is 2 or 3;
(4-c) amplifying vesicular stomatitis virus obtained in step (1) by 1: diluting the dilution factor of Y, adding the diluted dilution factor into a detection hole of a culture plate inoculated with L132 cells, and culturing for T3 hours, wherein the value of X/Y is 5-20;
(4-d) after the completion of the culture, discarding the culture supernatant, staining the cells in the detection wells with a crystal violet staining solution, and measuring the absorbance value of each detection well;
(5) Data processing and result calculation: processing data by four-parameter regression calculation method, taking the concentrations of each dosage group of serially diluted reference solution and test solution as abscissa, taking absorbance average value calculated based on compound holes of each concentration as ordinate, taking four-parameter curves of reference and test, and calculating half-Effective Concentration (EC) of reference and test 50 ) Thereby calculating the relative biological activity of the IL-29 of the test sample;
meanwhile, the reliability test is carried out on the measurement result, and the curve fitting degree R of the four-parameter curve of the reference and the test sample is included 2 And off-parallel.
2. The method of claim 1, wherein in step (3), the serial dilutions are of an equal ratio dilution of 2 to 5 times.
3. The method of claim 1, wherein in step (3), serial dilutions are performed using an assay broth comprising: DMEM broth, 7% fetal bovine serum, based on the total volume of the assay broth.
4. The method of claim 1, wherein in step (4-a), the L132 cells are seeded at a density of 1.0' 10 5 -2.0´10 5 cells/ml。
5. The method of claim 1, wherein in step (4-a), the L132 cells are seeded with an assay broth comprising: DMEM broth, 7% fetal bovine serum, based on the total volume of the assay broth.
6. The method of claim 1, wherein T1 is 4-6, T2 is 18-24, and T3 is 24±2.
7. The method of claim 1, wherein in step (4-c), the amplified vesicular stomatitis virus obtained in step (1) is diluted with a challenge culture medium comprising: DMEM broth, 3% fetal bovine serum, based on the total volume of the challenge broth.
8. The method of claim 1, wherein in step (5), a four-parameter regression algorithm is selected, using the reference EC for fitting the curve in the constraint model 50 And test sample EC 50 Dividing to calculate the relative biological activity of the test sample IL-29, namely
Test sample IL-29 biological relative activity (%) = (reference EC) 50 Test article EC 50 ）×100%。
9. An IL-29 biological activity assay reagent combination, wherein the reagent combination comprises:
(Z1) L132 cells;
(Z2) vesicular stomatitis virus seed solution;
(Z3) a reference that is an IL-29 standard for biological activity determination.
10. An IL-29 biological activity assay kit, comprising:
(C1) A first container, L132 cells located within the first container;
(C2) A second container, a vesicular stomatitis virus seed solution within the second container;
(C3) A third container, and a reference positioned within the third container, the reference being a biologically active, defined IL-29 standard;
and, the kit comprises a description which describes the method for determining the biological activity of IL-29 according to claim 1.
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| RU2657808C1 (en) * | 2017-07-10 | 2018-06-15 | Татьяна Петровна Оспельникова | Method for determining production of interferons as congenital immunity parameters |
| CN111848814A (en) * | 2020-06-21 | 2020-10-30 | 北京伟杰信生物科技有限公司 | Recombinant porcine IL-29 fusion protein and preparation method and application thereof |
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