CN114107557A - Method for determining titer of reovirus type 3 by tissue median infection staining - Google Patents
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Abstract
The invention discloses a method for determining the titer of reovirus type 3 by staining with a tissue median infection method, which comprises the steps of taking rhesus monkey kidney cells (LLC-MK 2 cells) as indicator cells for detecting reovirus type 3 (Reo-3 virus), titrating reovirus type 3 by using the tissue median infection method, judging cytopathic effect by combining with a crystal violet staining method, and finally calculating the titer of the virus. The determination method provided by the invention judges the cytopathic effect by combining the tissue median infection method and the crystal violet staining method, can simply and visually observe the cell condition, effectively reduces the subjectivity influence of observation under the microscope of experimenters, and has good stability, good repeatability and no influence on the experimental result by different operators at different time.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a method for determining the titer of reovirus type 3 by staining with a tissue median infection method.
Background
With the development of biotechnology, many biological medicines have entered the drug market, and besides blood products, biological products such as monoclonal antibodies and recombinant proteins are also important for the development of the biomedical industry due to the characteristics of strong targeting property, small side effect, significant curative effect and the like. The biological products are generally organisms derived from microorganisms, cells, animal or human tissues and body fluids, for example, animal cells are used as a protein expression production system, the biological products have the risk of virus contamination in various links, and the biological products are generally directly injected for administration, so that the products can cause serious harm to patients if the products are contaminated by viruses. Therefore, all national drug administration organizations need to provide the information of virus removal/inactivation verification research of the purification process in declaration materials before clinical test and production stage, so as to ensure that no matter the purification process is used for clinical test patients or products brought to the market, the virus pollution can not occur.
Virus removal/inactivation validation studies evaluate the ability of various steps in the purification process to remove/inactivate virus by adding indicator virus. Virus removal/inactivation verification cannot be done once for every contaminating virus that may occur due to costs in time, labor, and capital, and so forth, officials of drug administration in the united states, european union, and so forth, and scientists in the field of virology have proposed the use of indicator viruses whose characteristics include the following four points: "Single and double stranded RNA and DNA, lipid-coated and non-lipid-coated, strong and weak resistance, large and small particles, etc.".
Reovirus Type 3 (Reovirus Type 3, Reo-3) is a commonly used indicator virus, is a representative strain of the genus orthoreovirus of the reoviridae family, is a non-enveloped double-stranded RNA, has a virus size of 80-110nm, and is a model virus for endogenous virus-like particles in CHO cells as well as other enveloped viruses. Quantitative determination methods for Reo-3 virus include plaque method, fluorescent quantitative PCR method and other optical detection methods. The plaque method is a quantitative process of infecting cells by virus to cause cytopathy to form plaques, observing the plaque morphology, recording the number of the plaques, and finally performing corresponding calculation on the virus titer. In the process of applying the plaque method, some differences of plaque numbers can be generated due to subjective factors of experimenters, and some viruses are not easy to form plaques. Fluorescence quantitative PCR is also a commonly used virus quantitative method, and is used for indirectly quantifying viruses by extracting RNA in a sample and detecting a specific gene of Reo-3 by using a reverse transcription fluorescence quantitative PCR (RT-qPCR) method. The method has the advantages of rapid detection, high sensitivity and the like, but the PCR method is easy to generate false positive, and the detection result can not reflect the infection activity of the virus.
The tissue median infectious dose endpoint titration method is widely used as a classical virus titer determination method, and the basic method is as follows: the virus is diluted according to a certain proportion and is inoculated with cells, the cells are cultured for several days, the cytopathic effect (CPE) condition is observed under a microscope, and whether each hole is infected or not is judged. Then, the virus concentration at which half of the tissues were infected was calculated from the positive rate of each dilution, and the virus was quantified. The method needs to search specific indicator cells with obvious CPE for different viruses, and is generally influenced by experience and subjective factors of scientific researchers when CPE is judged. Therefore, some methods are needed to reduce the effect of subjective factors on the detection result, for example, dyes can be used to stain cells to make the result more intuitive, thereby reducing the subjective factors.
Through the search of Chinese and English patents, a patent for determining Reo-3 titer by using a tissue median infection method is not found at all. Reo-3 infects LLC-MK2 cells and has obvious CPE effect, so that the TCID50 is used in detecting Reo-3 in virus eliminating/inactivating research.
The invention is expected to accurately determine the Reo-3 virus with infection activity by combining a tissue median infection method with a crystal violet staining method so as to meet the requirement of virus elimination/inactivation research. The tissue half infection method is used as a virus titer determination method at a cell level, has higher requirements on cell strains, for example, viruses need to have strong susceptibility on indicator cells and can cause obvious CPE (CPE), so that pathological cells and normal cells can be distinguished conveniently; in addition, the method also needs to meet the following requirements: within a proper virus titer range, the titer of the virus and the dilution multiple should have a good linear relationship; the measuring result should have stability, different operators can not influence the experimental result at different time, the repeatability is good, and the detection sensitivity is high; various parameters of the method, including linear standard curve, linear correlation coefficient, measuring range, detection limit, precision, sensitivity and the like, are required to meet the requirements of relevant regulations.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for determining the titer of reovirus type 3 by tissue median infection staining, which comprises the following steps of taking rhesus monkey kidney cells (LLC-MK 2 cells) as indicator cells for detecting reovirus type 3 (Reo-3 virus), titrating reovirus type 3 by adopting a tissue median infection method, judging cytopathic effect by combining a crystal violet staining method, and finally calculating the titer of the virus, wherein the method comprises the following steps:
1) inoculating LLC-MK2 cells into a cell culture pore plate containing a cell culture medium, and culturing until the confluency of LLC-MK2 cells is 70-100%;
2) diluting the Reo-3 virus, respectively adding the diluted Reo-3 viruses with different dilutions into culture holes containing LLC-MK2 cells, transferring the cell culture pore plate inoculated with the Reo-3 virus into a cell culture box, and culturing for 6-7 days;
3) stopping culturing after LLC-MK2 cells have obvious cytopathic effect, adding a histiocyte fixing solution into each hole of cells to fix LLC-MK2 cells, and removing the histiocyte fixing solution after fixing is finished;
4) and adding a staining solution to stain LLC-MK2 cells, washing and airing a cell culture pore plate after staining is finished, reading the number of cell pores with cytopathic effect under each dilution, and calculating the titer of the Reo-3 virus according to a Spearman-Kaerber method.
Specifically, in step 1), each culture well on the cell culture well plate is inoculated with 4 × 104And (4) cells.
Specifically, in the step 2), the dilution of the Reo-3 virus refers to a serial dilution of the Reo-3 virus, wherein the serial dilution is one of 10 times, 3.2 times and 2.5 times.
Specifically, in the step 2), the same volume of inoculum size is added into each culture well on the cell culture well plate, and 8 duplicate wells are made for Reo-3 virus solution under each dilution.
Specifically, in step 1), the number of culture days of the cells in the cell culture well plate before inoculating Reo-3 virus does not exceed 2 days, and in step 2), the cell culture medium in the cell culture well plate needs to be replaced by fresh culture medium before inoculating Reo-3 virus.
Specifically, the cell culture medium is an MEM culture medium with fetal bovine serum content of 8%, and the fresh culture medium is an MEM culture medium with fetal bovine serum content of 2%.
Specifically, in the step 2), when culturing is performed after the Reo-3 virus is inoculated, the cell culture pore plate is sealed by using a sealing film.
Specifically, in step 2), the dilution solution used for diluting the Reo-3 virus was EMEM medium using HEPES as a buffer, the concentration of which was 0.25 mol/L.
Specifically, in step 3), the fixed time is not less than 15 minutes.
Specifically, in the step 4), the dyeing solution is a crystal violet solution with a concentration of 0.5%, and the dyeing time is 3 minutes.
Compared with the prior art, the invention has the beneficial effects that:
1. after the indicator cell LLC-MK2 is infected by Reo-3 virus, the LLC-MK2 cell shows obvious CPE under a microscope, and the stained crystal violet shows a staining form which is obviously different from that of an uninfected cell, so that compared with microscopic observation, the staining is more beneficial to unifying judgment standards, and errors caused by subjective factors of personnel are reduced;
2. the stability and repeatability of the determination of the tissue half infection method of Reo-3 infected LLC-MK2 cells are verified through controlling variables, and the result shows that under the conditions of different time and different operators, the Reo-3 virus titer determined by the method has no obvious difference, a drawn standard curve has a good linear relation, the result stability is good, and the repeatability is strong;
3. various parameters of the method include: the method meets the requirements of relevant regulations in the aspects of linear standard curve, linear correlation coefficient, measuring range, detection limit, precision, sensitivity and the like, and shows that the method can be applied to the measurement of the 3-type titer of the reovirus in virus removal/inactivation verification research;
4. the method describes the method for determining the virus titer by using LLC-MK2 cells as indicator cells and using the tissue median infection method staining of Reo-3 virus in detail, provides a good reference basis for other organizations needing virus removal/inactivation verification research, and also provides a referable experimental method for determining the virus titer of other subtypes of Reo-3.
Drawings
FIG. 1 is a diagram of a typical half-infected cell of Reo-3 virus tissue of the present invention;
FIG. 2 is a linear relationship of LLC-MK2 cell tissue half infection method to determine Reo-3 virus titer in example two of the present invention;
FIG. 3 is a linear relationship of Reo-3 virus titer measured by the same operator at different times in the third example of the present invention;
FIG. 4 shows the linear relationship between Reo-3 virus titer measured by different operators in the fourth example of the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely below, and it should be apparent that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention. Materials, instruments, reagents and the like used in the following examples are commercially available unless otherwise specified. The technical means used in the examples are conventional means well known to those skilled in the art, unless otherwise specified.
EXAMPLE detailed implementation of a tissue median infection staining method for determining Reo-3 Virus Titers
Firstly, after thawing Reo-3 virus, carrying out ultrasonic treatment and filtration treatment, and carrying out 10-time serial dilution according to actual conditions, wherein the virus diluent adopts an EMEM culture medium added with HEPES as a buffer solution, and the concentration of the HEPES is 0.25 mol/L;
next, a MEM medium having a fetal bovine serum content of 8% was added to the cell culture 96-well plate, and LLC-MK2 cells were seeded into the cell culture 96-well plate at about 4X 10 cells/well4Individual cells, 5% CO at 37 ℃2Performing cell culture under the culture condition, observing cells until the next day, performing subsequent operation until the cell confluency is 70-100%, and adjusting the number of inoculated LLC-MK2 cells within a reasonable range to make the cell confluency reach 70-100% within two days;
again, LLC-MK2 cells were infected with prepared Reo-3 virus at different dilutions: sucking out the cell culture medium, adding 50 mu LLLC-MK2 virus liquid, adding 200 mu L of fresh culture medium to each cell well, wherein the fresh culture medium is MEM culture medium, the fetal bovine serum content is 2%, making 8 duplicate wells for each dilution virus liquid, immediately transferring the cell plate inoculated with the virus into a cell culture box, and culturing for 6-7 days;
and finally, after the cells have obvious cytopathic effect, terminating the culture, adding a proper amount of tissue cell fixing solution into each hole of the cells to ensure that the tissue cell fixing solution fully covers the cell culture holes, wherein the fixing time is at least 15 minutes, then slightly removing the tissue cell fixing solution, adding a proper amount of 0.5 percent crystal violet solution into the cells to dye the cells, removing the crystal violet solution after dyeing for 3 minutes, and washing the cells with clear water.
The cell culture 96-well plates were air-dried in a fume hood and the wells where CPE appeared were recorded were observed under a white light transilluminator and after recording the positive proportion, the virus titer was calculated. FIG. 1 is a photograph showing an example of a Reo-3 tissue half infection method according to the present invention. Example two linearity and Range of Reo-3 tissue median infection
According to the operation flow of the first embodiment, two experiment operators perform 3 Run experiments, each Run performs independent serial dilution of 6 samples, each sample performs 3 repetitions, each serial dilution contains 8 different dilution factors, samples with virus concentrations lower than the detection limit are excluded, four samples are selected to calculate the virus titer of each dilution series, including drawing a standard curve, calculating a linear correlation coefficient and determining a virus detection range, and the results of the standard curve and the linear correlation coefficient are shown in fig. 2.
As can be seen from FIG. 2, within the range of the measured virus titer, the standard curves of 3 Run-independent serial dilutions have good linear relationship, good stability of the result and strong repeatability.
EXAMPLE three comparison of experiments with the same operator at different times on LLC-MK2 cells infected with Reo-3 Virus
According to the operation flow of the first embodiment, an Operator1 performs Reo-3 virus titer determination experiments Run1 and Run3 on the first day and the second day respectively, each Run comprises 6 sample experiments, each sample is subjected to three repetitions, after serial dilution of the samples, each dilution is subjected to 8 repeated-well experiments, samples with virus concentration lower than the detection limit are excluded, four samples are selected for result analysis, and the result is shown in FIG. 3.
As can be seen from FIG. 3, at two different time points, the LLC-MK2 cells infected by Reo-3 virus showed good linear relationship under different dilution factors, and the results were stable and reproducible.
Example comparison of four different laboratory operators
According to the operation flow of the first embodiment, two experiment operators, Operator1 and Operator 2, respectively and independently perform experiments, Run1 and Run2, each Run comprises experiments of 6 samples, each sample is subjected to three repetitions, after the samples are serially diluted, each dilution is subjected to 8 repeated-hole experiments, samples with virus concentration lower than the detection limit are excluded, four samples are selected for result analysis, and the results are shown in fig. 4.
As can be seen from fig. 4, Operator1 and Operator 2 have no significant difference, and both operators exhibit good linear relationship under different dilution factors.
Example statistical analysis of the Virus Titers of five Reo-3
According to the operation flow of the first embodiment, two experimental operators Operator1 and Operator 2 participate in the experiment, 18 independent serial dilution experiments are performed, the Operator1 completes 12 independent serial dilution experiments, the Operator 2 completes 6 independent serial dilution experiments, and the 18 groups of experiment results are subjected to statistical analysis, which specifically comprises: accuracy, linearity, assay range, quantitation limit, reproducibility, intermediate precision, and the like, the results are shown in the following table:
| analysis of parameters | Statistical parameters, units | Numerical value |
| Accuracy of | ％ | 98％-102％ |
| Linearity | Coefficient of Determination，R2 | 0.9948 |
| Range | TCID50/mL | Infection with viral infectionRate: 0% to 100% |
| Limit of quantification | TCID50/mL | 7.5 |
| Repeatability of | 95% confidence interval (Log10 titer) | 0.08 |
| Intermediate precision | 95% confidence interval (Log10 titer) | 0.11 |
The above results indicate that the LLC-MK2 cells are used as indicator cells for TCID50The method for determining Reo-3 virus titer by Assay staining has the advantages that Reo-3 virus has strong susceptibility on an indicator cell LLC-MK2, can generate obvious cytopathic effect, can obviously distinguish diseased cells from normal cells, and the titer of the Reo-3 virus and the dilution multiple should have good linear relation in a proper virus titer range; in addition, the experimental results cannot be influenced by different operators at different times, which shows that the method has high stability, good repeatability and high detection sensitivity, and various parameters of the method comprise: the linear standard curve, the linear correlation coefficient, the measurement range, the detection limit, the precision, the sensitivity and the like all meet the requirements of GLP regulations related to FDA, ICH and USP, and can be used for virus removal/inactivation verification research.
In summary, the above embodiments are merely preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalents, improvements, etc. made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (10)
1. A method for determining the titer of reovirus type 3 through tissue median infection staining is characterized in that rhesus monkey kidney cells (LLC-MK 2 cells) are used as indicator cells for detecting reovirus type 3 (Reo-3 virus), the reovirus type 3 is titrated through a tissue median infection method, cytopathic effect is judged through combination with a crystal violet staining method, and the titer of the Reo-3 virus is finally calculated, wherein the method comprises the following steps:
1) inoculating LLC-MK2 cells into a cell culture pore plate containing a cell culture medium, and culturing until the confluency of LLC-MK2 cells is 70-100%;
2) diluting the Reo-3 virus, respectively adding the diluted Reo-3 viruses with different dilutions into culture holes containing LLC-MK2 cells, transferring the cell culture pore plate inoculated with the Reo-3 virus into a cell culture box, and culturing for 6-7 days;
3) stopping culturing after LLC-MK2 cells have obvious cytopathic effect, adding a histiocyte fixing solution into each hole of cells to fix LLC-MK2 cells, and removing the histiocyte fixing solution after fixing is finished;
4) and adding a staining solution to stain LLC-MK2 cells, washing and airing a cell culture pore plate after staining is finished, reading the number of cell pores with cytopathic effect under each dilution, and calculating the titer of the Reo-3 virus according to a Spearman-Kaerber method.
2. The method according to claim 1, wherein in step 1), each culture well of the cell culture well plate is seeded with 4 x 104And (4) cells.
3. The method according to claim 1, wherein in step 2), the dilution of Reo-3 virus is performed by performing serial dilution on Reo-3 virus, and the serial dilution is one of 10-fold, 3.2-fold and 2.5-fold.
4. The method of claim 1, wherein in step 2), the same volume of inoculum is added to each culture well in the cell culture well plate, and 8 replicate wells are made for Reo-3 virus fluid at each dilution.
5. The method according to claim 1, wherein in step 1), the number of days of culturing the cells in the cell culture well plate before inoculating the Reo-3 virus is not more than 2 days, and in step 2), the cell culture medium in the cell culture well plate needs to be replaced by fresh culture medium before inoculating the Reo-3 virus.
6. The method according to claim 5, wherein the cell culture medium is MEM medium containing 8% fetal bovine serum, and the fresh medium is MEM medium containing 2% fetal bovine serum.
7. The method according to claim 1, wherein in step 2), the cell culture well plate is sealed with a sealing film when culturing is performed after inoculating Reo-3 virus.
8. The method according to claim 1, wherein the dilution solution used for diluting Reo-3 virus in step 2) is EMEM medium using HEPES with a concentration of 0.25mol/L as a buffer.
9. The method of claim 1, wherein in step 3), the fixed time is not less than 15 minutes.
10. The method according to claim 1, wherein in the step 4), the dyeing solution is a crystal violet solution with a concentration of 0.5%, and the dyeing time is 3 minutes.
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| 孟淑芳等: "SV40 感染滴度测定方法的建立及高滴度病毒的制备", 《中国生物制品学杂志》 * |
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