Detection method for detecting trophile murine retrovirus

Abstract

The detection method comprises the following steps: S1, selecting host cells, setting an amplification negative control group, an amplification positive control group, a sample detection group and an interference control group, culturing and taking supernatant; s2, inoculating each group of supernate collected in the S1 on indicator cells; s3, setting a negative control group and a positive control group; and S4, incubating and culturing each group of cells in the step S2 and the step S3, then carrying out an XC end-point experiment, observing lesion plaques and reading the plate to complete detection. The method is high in sensitivity and good in accuracy, and can be widely applied to the detection of the trophile murine retrovirus of biological products such as monoclonal antibodies and in-vitro diagnosis products.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and particularly relates to a detection method for detecting ecotropic mouse-derived retrovirus. Background Art

[0002] Currently, many biological (biological and biotechnology-derived) products and therapies for humans or animals are mostly derived from cell hybridoma cells, transgenic cell lines or primary cultured cells or cell lines. With the development of biological (biological and biotechnology-derived) products and therapies for humans or animals, the requirements for comprehensive virus detection methods in GMP production and biological product testing are increasing.

[0003] Ecotropic Murine Leukemia Virus (E-MuLV) is a type of virus commonly found in mice. The main methods for detecting retroviruses include reverse transcriptase activity PERT test, transmission electron microscopy, PCR, etc. Different methods have different detection characteristics. Reverse transcriptase activity indicates the possible presence of retroviruses. Transmission electron microscopy and specific PCR can prove the presence of virus particles and quantify them, but they cannot prove whether infectious retroviruses are present.

[0004] Infectivity tests for cell culture may have complementary sensitivity and specificity for viruses, and therefore, they can be used as limit tests for identification of cell banks and raw materials, as well as batch release tests for harvested fluids and harvested products during the production of biological products. In viral infectivity tests for cells, indicator (test) cells can be used as host cells for live viral particles present in the test article. In relevant regulations such as ICH Q5A (R2) and the Chinese Pharmacopoeia "Draft for Comments on Preparation and Quality Control of Animal Cell Substrates for the Production of Biological Products", it is proposed that ecotropic retroviruses need to be tested, and human cell lines are used for the detection of insect retroviruses.

[0005] Therefore, there is an urgent need in the art for a sensitive, accurate and comprehensive method for detecting ecotropic mouse-derived viruses, which can not only efficiently and sensitively perform infectivity assessment to reflect the infectivity of the virus, but also intuitively perform quantitative assessment of the virus titer, thereby achieving a more comprehensive detection of ecotropic mouse-derived retroviruses. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a method for detecting ecotropic mouse-derived retrovirus, characterized in that it comprises the following steps:

[0007] S1. Select host cells, set up amplification negative control group, amplification positive control group, sample detection group and interference control group, where:

[0008] The amplification negative control group is cultured alone with the host cells;

[0009] The amplification positive control group is amplified and cultured host cells with an ecotropic mouse-derived retrovirus;

[0010] The sample detection group is to amplify and culture the host cells and the test sample;

[0011] The interference control group is amplified and cultured host cells, test samples and ecotropic mouse-derived retrovirus;

[0012] The amplification negative control group, the amplification positive control group, the sample detection group and the interference control group are subcultured respectively, and the culture supernatant of each group is collected after the subculture is completed;

[0013] S2, selecting indicator cells, and inoculating the culture supernatant of the amplified negative control group on the indicator cells;

[0014] Inoculate the culture supernatant of the sample test group on the indicator cells;

[0015] Inoculate at least two dilutions of the culture supernatant of the amplified positive control group on the indicator cells;

[0016] Inoculate the indicator cells with at least two dilutions of the culture supernatant of the interference control group;

[0017] S3. Set up a negative control group and a positive control group, where:

[0018] The negative control group was inoculated with MEM in indicator cells;

[0019] The positive control group is inoculated with at least two dilutions of ecotropic mouse-derived retrovirus on indicator cells;

[0020] S4. Incubate and culture each group of cells in step S2 and step S3. After the culture is completed, the cells are subjected to ultraviolet irradiation, and then XC cells are added for culture. After the culture is completed, the cells are stained, the lesion plaques are observed, and the plate is read to complete the detection.

[0021] Specifically, the host cell is a SC-1 cell.

[0022] Specifically, the titer of the detectable ecotropic mouse-derived retrovirus is not less than 10 PFU.

[0023] Specifically, in S1, the subculture conditions are 36-38°C, CO2 concentration of 4%-6%, and subculture is performed twice or five times, with one subculture every 3-4 days.

[0024] Specifically, the indicator cells are SC-1 cells.

[0025] Specifically, the indicator cells were plated one day in advance.

[0026] Specifically, the two dilutions refer to 10 -1 and 10 -2 Dilution level.

[0027] Specifically, in S4, the incubation time is 60-90 minutes, and the culture conditions are incubation in fresh culture medium at 36-38° C. and a CO 2 concentration of 4%-6% for 3-4 days.

[0028] Specifically, the UV irradiation condition is (400-800) ±15% μW / cm 2 The culture time after adding XC cells is 3 to 4 days.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] 1. The detection method for detecting ecotropic mouse-derived retrovirus provided in the present application is based on the research after reasonable interpretation of relevant laws and regulations, and can be widely used in the detection of biological products such as monoclonal antibodies and in vitro diagnostic products for specific detection of ecotropic mouse-derived retrovirus.

[0031] 2. The detection method for detecting mouse-derived ecotropic viruses provided in the present application can directly and clearly show whether the sample being tested contains infectious retroviruses. When combined with reverse transcriptase activity PERT detection, transmission electron microscopy, and PCR, a more comprehensive mouse-derived ecotropic virus detection system can be achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a flow chart of the detection method for detecting ecotropic murine retrovirus provided by the present application in Example 1;

[0033] Figure 2 This is a result diagram of the absence of virus plaques when the detection method for detecting ecotropic murine retrovirus provided by the present application is used in Example 1;

[0034] Figure 3 This is a result diagram of virus plaques with different virus contamination degrees when the detection method for detecting ecotropic murine retrovirus provided by the present application is used in Example 1. DETAILED DESCRIPTION

[0035] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] Unless otherwise specified, the experimental materials involved in the present invention are all commercial reagents and can be obtained from commercial channels.

[0037] Example 1

[0038] Please refer to Figure 1 As shown in the flow chart, according to the detection method for detecting ecotropic murine retrovirus provided by the present invention, this embodiment shows a specific implementation process:

[0039] A: Selection of host cells. Cells with high sensitivity to ecotropic mouse-derived retroviruses are selected as host cells. The present invention selects (SC-1) as the detection host cell.

[0040] B: Design of experimental process.

[0041] Proliferation and culture of SC-1 cells

[0042] SC-1 cells were plated one day before inoculation, and the conditioned medium of SC-1 cells served as the negative control group for expansion.

[0043] SC-1 cells inoculated with the test articles were used as the sample detection group.

[0044] SC-1 cells were inoculated with 10 PFU of ecotropic mouse retrovirus to establish an amplification positive control group.

[0045] SC-1 cells were inoculated with 10 PFU of ecotropic mouse retrovirus, and the same volume of the test article as that of the sample test group was added as the interference control group.

[0046] After inoculation, incubate at 37±1°C and 5±1% CO2 for 60-90 minutes.

[0047] After the incubation, the incubation solution was discarded, and fresh culture medium was added and cultured at 37±1°C and 5±1% CO2.

[0048] The cells were subcultured every 3-4 days after inoculation for a total of two subcultures, during which the cells were cultured at 37±1°C and 5±1% CO2.

[0049] After the second passage, the cell culture supernatants of the amplification negative control group, amplification positive control group, sample detection group and interference control group were collected and retained.

[0050] XC Endpoint Trial

[0051] Indicator cells (SC-1) were plated in six-well plates one day before inoculation.

[0052] On the day of inoculation, SC-1 cells were inoculated with MEM as a negative control group.

[0053] All supernatant samples collected in the previous amplification stage were inoculated on SC-1 cells. Among them: the supernatant of the negative control and sample test group was inoculated; the supernatant of the positive control group and the interference control group after two generations of culture was inoculated after ten-fold dilution in MEM, containing at least 10 -1 ,10 -2 Dilution level.

[0054] Two dilutions of ecotropic murine retrovirus were inoculated on SC-1 cells as a positive control group.

[0055] After incubation for 60-90 minutes, discard the incubation solution, add fresh culture medium and culture at 37±1℃ and 5±1% CO2.

[0056] 3-4 days after inoculation, (400~800)±15%μW / cm 2 UV irradiation was performed at a certain UV intensity. After irradiation, diluted XC cells were added.

[0057] The medium was changed the next day after UV irradiation.

[0058] On the 4th day after UV irradiation, cells were fixed and stained, and air-dried after staining. Plates can be read at any time after air-drying.

[0059] The results show that the test results are valid. Figure 2 As shown in Figure 2, the negative control supernatant during the entire detection period showed no plaques in the XC endpoint test. Figure 3 As shown, the test is considered valid if the supernatant of the positive control of the amplification culture test inoculated with an ecotropic murine retrovirus shows plaques in the XC endpoint test.

[0060] In summary, the above embodiments are only preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting an ecotropic mouse-derived retrovirus, characterized in that: The steps include: S1. Select host cells, set up amplification negative control group, amplification positive control group, sample detection group and interference control group, where: The amplification negative control group is cultured alone with the host cells; The amplification positive control group is amplified and cultured host cells with an ecotropic mouse-derived retrovirus; The sample detection group is to amplify and culture the host cells and the test sample; The interference control group is amplified and cultured host cells, test samples and ecotropic mouse-derived retrovirus; The amplification negative control group, the amplification positive control group, the sample detection group and the interference control group are subcultured respectively, and the culture supernatant of each group is collected after the subculture is completed; S2, selecting indicator cells, and inoculating the culture supernatant of the amplified negative control group on the indicator cells; Inoculate the culture supernatant of the sample test group on the indicator cells; Inoculate at least two dilutions of the culture supernatant of the amplified positive control group on the indicator cells; Inoculate the indicator cells with at least two dilutions of the culture supernatant of the interference control group; S3. Set up a negative control group and a positive control group, where: The negative control group was inoculated with MEM in indicator cells; The positive control group is inoculated with at least two dilutions of ecotropic mouse-derived retrovirus on indicator cells; S4. Incubate and culture each group of cells in step S2 and step S3. After the culture is completed, the cells are subjected to ultraviolet irradiation, and then XC cells are added for culture. After the culture is completed, the cells are stained, the lesion plaques are observed, and the plate is read to complete the detection.

2. The method for detecting ecotropic murine retrovirus according to claim 1, characterized in that: The host cell is SC-1 cell.

3. The method for detecting ecotropic murine retrovirus according to claim 1, characterized in that: The titer of the detectable ecotropic mouse-derived retrovirus is not less than 10 PFU.

4. The method for detecting ecotropic murine retrovirus according to claim 1, characterized in that: In S1, the subculture conditions are 36-38°C, CO2 concentration of 4%-6%, and subculture is performed twice or five times, with one subculture every 3-4 days.

5. The method for detecting ecotropic murine retrovirus according to claim 1, characterized in that: The indicator cells are SC-1 cells.

6. The method for detecting ecotropic murine retrovirus according to claim 1, characterized in that: The indicator cells were plated one day in advance.

7. The method for detecting ecotropic murine retrovirus according to claim 1, characterized in that: The two dilutions refer to 10 -1 and 10 -2 Dilution level.

8. The method for detecting ecotropic murine retrovirus according to claim 1, characterized in that: In S4, the incubation time is 60-90 minutes, and the culture conditions are in fresh culture medium at 36-38°C and a CO2 concentration of 4%-6% for 3-4 days.

9. The method for detecting ecotropic murine retrovirus according to claim 1, characterized in that: UV irradiation conditions: (400~800)±15%μW / cm 2 The culture time after adding XC cells is 3 to 4 days.

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