Universal lentivirus titer detection method based on flow cytometry and application of universal lentivirus titer detection method

Abstract

The invention belongs to the technical field of biology, and particularly relates to a universal type lentivirus titer detection method based on flow cytometry and application of the universal type lentivirus titer detection method. The detection method comprises the following steps: adding a transfection medium and lentivirus diluted by the transfection medium into 293T cells, and culturing; adding pancreatin for digestion to form a cell dispersion liquid; performing centrifugation and PBS resuspension on the cell dispersion liquid, adding a protein L antibody, performing incubation at 2-8 DEG C in a dark place for 30-60 minutes, then performing washing, and performing flow type detection on a CAR positive expression rate to obtain the lentivirus titer. The flow cytometry-based universal type lentivirus titer detection method provided by the invention is simple, convenient and rapid, has a relatively good linear range, and can accurately evaluate the titer of a lentivirus containing a CAR (Chimeric Antigen Receptor) gene.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a universal lentivirus titer detection method based on flow cytometry and its application. Background Art

[0002] Lentiviruses have been widely used as gene delivery vectors. Compared to other viral vectors, lentiviruses possess the ability to stably integrate into the host cell genome, can infect both dividing and non-dividing cells, and possess a large packaging capacity. Viral titer determination is a fundamental quality assessment of lentivirus-based drugs, and its accuracy is crucial for patient therapeutic dosing. Currently, there are three main methods for lentivirus titer determination: ELISA for capsid protein p24, RT-qPCR for lentiviral genome copy number, and qPCR for lentiviral functional titer.

[0003] The ELISA method determines the number of viral particles by measuring the amount of p24 protein. For example, Chinese Invention Patent Publication No. CN117074654A discloses a lentiviral titer p24 ELISA test kit and its use method. The kit comprises at least: an HIV-1 p24-coated ELISA plate, a detection antibody conjugated to horseradish peroxidase, an HIV-1 p24 standard, a viral lysis buffer, a sample diluent, 10x PBST wash buffer, a TMB chromogenic substrate, and a stop solution. The kit contains pre-assembled reagents and can be used directly in experiments, significantly reducing testing time, providing high specificity, and reliable performance. However, the titer data obtained by this method includes not only the expected infectious lentiviral particles, but also free p24 protein and genome-defective viruses. Therefore, the viral titer measured by the ELISA method is often much higher than the actual viral titer.

[0004] The RT-qPCR method reverse-transcribes the lentiviral RNA genome into cDNA, which is then used as a template for qPCR to detect the copy number of the lentiviral genome cDNA. For example, Chinese Invention Patent Publication No. CN119287078A discloses a method for detecting lentiviral titers using a double-standard curve RT-qPCR method. This invention uses a test sample containing a recombinant lentivirus to infect the target cells, collects and extracts the genomic DNA of the infected target cells; uses gradient-diluted original virus standards and internal reference standards, and the extracted target cell genomic DNA as templates to perform viral genome RT-qPCR amplification reactions and internal reference genome RT-qPCR amplification reactions; and calculates the lentiviral titer corresponding to the sample based on the data obtained from the RT-qPCR amplification reactions. Compared with the ELISA method, although the RT-qPCR method avoids the interference of empty shell viruses on titer detection, the presence of genome-defective viruses still has a significant impact on the titer detection results, which is up to 20 times higher than the actual viral titer, and the fluctuation range between different groups is very large.

[0005] The qPCR method uses the cell genome extracted after virus transduction into cells as a template for qPCR. The data obtained can intuitively reflect the copy number of the viral genome integrated into the host genome, but cannot evaluate whether it is expressed.

[0006] In view of this, there is an urgent need in the art to provide a simpler, faster, and more accurate detection method for evaluating the titer of lentivirus containing the CAR gene. Summary of the Invention

[0007] In view of the problems existing in the prior art, the present invention provides a universal lentivirus titer detection method based on flow cytometry and its application.

[0008] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0009] A universal lentiviral titer detection method based on flow cytometry includes the following steps:

[0010] (1) Adding transfection medium and lentivirus diluted in the transfection medium to 293T cells and culturing them; then adding trypsin for digestion to form a cell dispersion;

[0011] (2) The cell dispersion was centrifuged and resuspended in PBS, and protein L antibody was added. The cells were incubated in the dark at 2-8°C for 30-60 min, then washed. The CAR positive expression rate was detected by flow cytometry to obtain the lentiviral titer.

[0012] Preferably, the transfection medium is DMEM medium containing 7-9 μg / ml polybrene and 10% FBS.

[0013] More preferably, the transfection medium is DMEM medium containing 8 μg / ml polybrene and 10% FBS.

[0014] Preferably, the 293T cells in step (1) need to be pretreated, and the pretreatment process includes: taking 450-550 μl of a 1.5×10 5 -2.5×10 5 293T cells at a concentration of 100 cells / ml were seeded into a well plate, cultured at 37°C and 5% CO2 for 16-24 hours, and the supernatant was discarded.

[0015] Preferably, the volume ratio of the 293T cells, the transfection medium, and the lentivirus diluted with the transfection medium is 4-6:4.5-5.5:1.

[0016] Preferably, the dilution multiple in step (1) is 10 times, 20 times, 40 times, 80 times or 160 times.

[0017] Preferably, the culture conditions in step (1) include: culturing at 37° C. and 5% CO 2 for 48-72 hours.

[0018] Preferably, the mass concentration of the pancreatic enzyme in step (1) is 0.2-0.3%.

[0019] Preferably, the density of the cell dispersion in step (2) is 1.0×10 6 -2.0×10 6 pcs / ml.

[0020] Preferably, the centrifugation process in step (2) includes: taking 2×10 5 The cells were centrifuged at 300-500 × g for 4-8 min, the supernatant was discarded, and the cells were washed with PBS, and then centrifuged at 300-500 × g for 4-8 min, and the supernatant was discarded.

[0021] Preferably, the PBS contains 1% FBS.

[0022] Preferably, the volume ratio of the cell dispersion, PBS and protein L antibody is 250-300:45-50:2-5.

[0023] More preferably, the volume ratio of the cell dispersion, PBS and protein L antibody is 270-280:46-48:2-4.

[0024] Further preferably, the volume ratio of the cell dispersion, PBS and protein L antibody is 275:48:2.

[0025] The present invention also provides the use of the above detection method in preparing a product for detecting the titer of a universal lentivirus.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention provides a universal flow cytometry-based lentivirus titer detection method. By screening conditions such as the amount of protein L antibody added, incubation time, and culture medium, the method is simple and rapid, has a good linear range, and can accurately evaluate the titer of lentivirus containing the CAR gene. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The CAR positive expression rate when the amount of protein L antibody added in test example 1 was 10 μl.

[0029] Figure 2 This is the CAR positive expression rate when the amount of protein L antibody added in Test Example 1 is 8 μl.

[0030] Figure 3 This is the CAR positive expression rate when the amount of protein L antibody added in Test Example 1 is 4 μl.

[0031] Figure 4 This is the CAR positive expression rate when the amount of protein L antibody added in Test Example 1 is 2 μl.

[0032] Figure 5 This is the CAR positive expression rate when the amount of protein L antibody added in test example 1 is 1 μl.

[0033] Figure 6 This is the CAR positive expression rate when the amount of protein L antibody added in Test Example 1 is 0.5 μl.

[0034] Figure 7 It is the CAR positive expression rate of the blank control group in the specific experiment in Test Example 5.

[0035] Figure 8 It is the CAR positive expression rate of the NC group in the specific experiment in Test Example 5.

[0036] Figure 9 This is the CAR positive expression rate when the lentivirus is diluted 10 times in the specific experiment in Test Example 5.

[0037] Figure 10 This is the CAR positive expression rate when the lentivirus is diluted 20 times in the specific experiment in Test Example 5.

[0038] Figure 11This is the CAR positive expression rate when the lentivirus is diluted 40 times in the specific experiment in Test Example 5.

[0039] Figure 12 This is the CAR positive expression rate when the lentivirus is diluted 80 times in the specific experiment in Test Example 5.

[0040] Figure 13 This is the CAR positive expression rate when the lentivirus is diluted 160 times in the specific experiment in Test Example 5.

[0041] Figure 14 It is a linear graph. DETAILED DESCRIPTION

[0042] It is worth noting that the raw materials used in this invention are all common commercially available products. Among them, 293T cells (catalog number CRL-3216) were purchased from ATCC; DMEM medium and PBS were purchased from Gibco; 1640 medium, trypsin, and double-antibody were purchased from Shanghai Yuanpei Biotechnology Co., Ltd.; FBS was purchased from Suzhou Yikesai Biotechnology Co., Ltd.; polybrene was purchased from Yisheng Biotechnology; and protein L antibody was purchased from Sino Biological.

[0043] Example

[0044] The universal lentiviral titer detection method based on flow cytometry is as follows:

[0045] (1) Take 293T cells in the logarithmic growth phase and prepare them to a density of 2×10 5 500 μl of the cell suspension was inoculated into a 24-well plate and cultured at 37°C and 5% CO2 for 16 h. The supernatant was discarded.

[0046] (2) Add 500 μl of DMEM medium containing 8 μg / ml polybrene and 10% FBS to each well, and 100 μl of lentivirus gradiently diluted with DMEM medium containing 8 μg / ml polybrene and 10% FBS (dilution multiples 10 times, 20 times, 40 times, 80 times and 160 times). Take another well and add 600 μl of DMEM medium containing 8 μg / ml polybrene and 10% FBS as NC (blank control group). Place the 24-well plate in 37°C and 5% CO2 and continue to culture for 3 days. After the culture is completed, the cell supernatant is aspirated and 150 μl of 0.25% trypsin is added to each well. Gently shake the 24-well plate until the cells fall off the plate. Add 400 μl of DMEM medium containing 10% FBS to each well to terminate the digestion. Gently blow to form a cell dispersion and transfer all to a 1.5 ml EP tube.

[0047] (3) Take the cell dispersion from the NC well and count it. According to the counting results, take 2×10 5 Cells were centrifuged at 500 × g for 5 minutes, the supernatant discarded, and the cells were washed with 1000 μl of PBS containing 1% FBS. The cells were centrifuged at 500 × g for 5 minutes, the supernatant discarded, and the cells were resuspended in 96 μl of PBS containing 1% FBS per tube. Finally, 4 μl of protein L antibody was added. The cells were incubated at 4°C in the dark for 30 minutes, then washed and the CAR positive expression rate was determined by flow cytometry. The results of the NC wells were used as a control for gating, and the results with a positive rate between 5% and 30% were used for calculation to obtain the lentiviral titer.

[0048] Test Example 1 Screening for the optimal addition amount of protein L antibody

[0049] 1. Experimental method: (1) 293T cells in the logarithmic growth phase were taken and prepared with DMEM medium containing 10% FBS and 1% PBS to a density of 2×10 5 500 μl of the cell suspension was inoculated into a 24-well plate and cultured at 37°C and 5% CO2 for 16 h. The supernatant was discarded.

[0050] (2) Add 500 μl of DMEM medium containing 8 μg / ml polybrene and 10% FBS to each well, and 100 μl of lentivirus diluted 5 times with DMEM medium containing 8 μg / ml polybrene and 10% FBS. Place the 24-well plate in 37°C, 5% CO2 and continue to culture for 3 days. After the culture is completed, aspirate the cell supernatant, add 150 μl of 0.25% trypsin to each well, gently shake the 24-well plate, wait for the cells to fall off the well plate, add 400 μl of DMEM medium containing 10% FBS to each well to terminate the digestion, gently blow to form a cell dispersion, and transfer all to a 1.5 ml EP tube.

[0051] (3) Count the cell dispersion obtained in step (2), taking 2×10 5 Cells were centrifuged at 500 × g for 5 min, the supernatant was discarded, and the cells were washed with 1000 μl of PBS containing 1% FBS and centrifuged at 500 × g for 5 min. The supernatant was discarded, and 96 μl of PBS containing 1% FBS was added to each tube for resuspending. Finally, 10 μl, 8 μl, 4 μl, 2 μl, 1 μl or 0.5 μl of protein L antibody were added, and the cells were incubated in the dark at 4°C for 30 min and then washed. The CAR positive expression rate was detected by flow cytometry, and the optimal antibody concentration was screened according to the test results.

[0052] 2. Experimental results: As shown in Table 1 and Figure 1-6As shown, Figure 1 It is the CAR positive expression rate when the amount of protein L antibody added is 10 μl; Figure 2 The CAR positive expression rate when the amount of protein L antibody added was 8 μl; Figure 3 The CAR positive expression rate when the amount of protein L antibody added was 4 μl; Figure 4 is the CAR positive expression rate when the amount of protein L antibody added is 2 μl; Figure 5 is the CAR positive expression rate when the amount of protein L antibody added is 1 μl; Figure 6 The CAR positive expression rate is when the amount of protein L antibody added is 0.5 μl. It can be seen that the optimal amount of protein L antibody added is 4-10 μl.

[0053] Table 1 Effects of different amounts of protein L antibody added on CAR positive expression rate

[0054]

[0055]

[0056] Test Example 2: Effect of adding polybrene on test results

[0057] 1. Experimental method: Referring to the example, 293T cells were inoculated in a 24-well plate, cultured for 16 hours, and then infected with lentivirus. The cells were divided into two experimental groups. One group used DMEM medium containing 8 μg / ml polybrene and 10% FBS to gradiently dilute the lentivirus (10 times, 20 times, 40 times, 80 times, and 160 times); the other group used DMEM medium containing 10% FBS to gradiently dilute the lentivirus (10 times, 20 times, 40 times, 80 times, and 160 times). 100 μl of each diluted gradient lentivirus was added to a 24-well plate, and the 24-well plate was placed in 37 ° C and 5% CO2 for continued culture. On the 4th day, the cells were digested and counted, and the CAR positive expression rate was detected by flow cytometry. The blank control group results were used as the control for the gate. The positive rate between 5-30% was calculated to obtain the lentiviral titer.

[0058] 2. Experimental Results: As shown in Table 2, during viral infection, the mean titer of the lentivirus with polybrene added was 5.16E+08TU / ml; the mean titer of the lentivirus without polybrene added was 4.24E+08TU / ml. The RSD of the two test results was less than 15%, so the addition of polybrene had no significant effect on the test results.

[0059] Table 2 Effect of adding polybrene to the diluent on the test results

[0060]

[0061] Test Example 3: Effect of different incubation times of protein L antibody on test results

[0062] 1. Experimental method: Referring to the example, 100 μl of each gradient diluted lentivirus (dilution factor 10x, 20x, 40x, 80x and 160x) was added to a 24-well plate. The 24-well plate was placed in 37°C, 5% CO2 and continued to be cultured. On the 4th day, the cells were digested and counted. Based on the counting results, three 2×10 5 Cells were washed and incubated with protein L antibody. The first aliquot was incubated at 4°C in the dark for 10 minutes, then washed. The second aliquot was incubated at 4°C in the dark for 30 minutes, then washed. The third aliquot was incubated at 4°C in the dark for 60 minutes, then washed. CAR positive expression was detected by flow cytometry. The blank control group was used as the reference for gating. The positive rate between 5% and 30% was used for calculation to determine the lentiviral titer.

[0063] 2. Experimental results: As shown in Table 3, it can be seen that when the protein L antibody incubation time is 10 minutes, the antibody and the sample are not fully bound, resulting in a low sample test value; while the two test results of the incubation time of 30 minutes and 60 minutes are close, with RSD less than 3%. In the future, the incubation time of 30 minutes can be selected as the incubation time, which saves time and does not affect the test results.

[0064] Table 3 Effects of different incubation times on test results

[0065]

[0066] Test Example 4: Effects of different culture media on test results

[0067] 1. Experimental method: Set up two well plates, one plate is cultured with 1640 culture medium (containing 10% FBS and 1% double antibody), and the other plate is cultured with DMEM culture medium (containing 10% FBS and 1% double antibody). Referring to the example, 293T cells were inoculated in a 24-well plate. After culturing for 16 hours, 100 μl of the same lentivirus (10 times, 20 times, 40 times and 80 times) diluted in 1640 culture medium and DMEM culture medium were taken respectively. The 24-well plate was placed in 37 ° C and 5% CO2 for further culture. On the 4th day, the cells were digested and counted. According to the counting results, 2×10 5The cells were washed and incubated with protein L antibody. After incubation, the cells were washed and the CAR positive expression rate was detected by flow cytometry. The results of the blank control group were used as the reference for gating. The results with a positive rate between 5% and 30% were used for calculation to obtain the lentiviral titer.

[0068] 2. Experimental results: As shown in Table 4, it can be seen that the two culture media have little effect on the test results of the same sample, but DMEM culture medium is recommended.

[0069] Table 4 Effects of different culture media on test results

[0070]

[0071] Test Case 5 Methodology Verification

[0072] 1. Specificity

[0073] 1. Experimental method: Refer to the example, take a 24-well plate, inoculate 293T cells, and inoculate 1×10 5 The experimental group (5 wells), blank control group (1 well) and NC group (1 well) were set up for a total of 7 wells. After 16 hours of culture, 0.1 ml of lentivirus with different dilutions (10 times, 20 times, 40 times, 80 times and 160 times) was added to each well of the experimental group, and 0.1 ml of DMEM medium containing 8 μg / ml polybrene and 10% FBS was added to the other groups and cultured continuously. On the 4th day, the cells were digested and counted, and 2×10 cells were added to each tube. 5 4 μl of protein L antibody was added to the experimental group and the NC group respectively, while the blank control group was not treated. After incubation at 4°C in the dark for 30 minutes, the CAR positive expression rate was detected by flow cytometry.

[0074] 2. Experimental results: Figure 7-13 As shown, Figure 7 is the CAR positive expression rate of the blank control group; Figure 8 is the CAR positive expression rate of the NC group; Figure 9 The CAR positive expression rate is 10 times the lentivirus dilution; Figure 10 The CAR positive expression rate is 20 times diluted lentivirus; Figure 11 The CAR positive expression rate when the lentivirus was diluted 40 times; Figure 12 The CAR positive expression rate when the lentivirus was diluted 80 times; Figure 13The CAR positive expression rate after lentivirus was diluted 160 times. It can be seen that after staining with protein L antibody, the CAR positive expression rate of the NC group was 0.32%, while the CAR positive expression rates of the experimental group samples at each gradient (10x, 20x, 40x, 80x, and 160x) were all greater than 2%, indicating that the detection method has good specificity.

[0075] 2. Linearity

[0076] 1. Experimental method: Referring to the example, 293T cells were seeded in a 24-well plate, with 1×10 5 Cells were plated in 6 wells. After 16 hours of culture, the lentivirus was diluted gradiently (10 times, 20 times, 40 times, 80 times, and 160 times) in DMEM containing 8 μg / ml polybrene and 10% FBS. 100 μl of each diluted gradient lentivirus was added to a 24-well plate for a total of 5 wells. 600 μl of DMEM medium containing 8 μg / ml polybrene and 10% FBS was added to the other well as NC. The 24-well plate was placed in 37 ° C, 5% CO2 and continued to be cultured for 48 hours. On the 4th day, the cells were removed and the CAR positive expression rate was detected by flow cytometry.

[0077] 2. Experimental results: As shown in Table 5, the theoretical sample CAR positive expression rate is the X-axis and the actual detection CAR positive expression rate is the Y-axis, and a linear graph analysis is made ( Figure 14 ), R 2 =0.998, good linearity.

[0078] Table 5 Theoretical CAR positive expression rate and actual CAR positive expression rate at different dilution factors

[0079] Dilution multiple Theoretical CAR positive expression rate (%) Actual CAR positive expression rate (%) 10 38.07 38.07 20 19.04 18.06 40 9.52 9.53 80 4.76 5.67 160 2.38 2.22

[0080] 3. Intermediate Precision

[0081] 1. Experimental method: Different experimenters conducted the “two-line” experiment on different days.

[0082] 2. Experimental results: As shown in Table 6, different personnel tested the same sample on different days. The RSD of CAR positive rate at the same dilution factor was within 15%, and the intermediate precision was good.

[0083] Table 6 Intermediate precision test results

[0084] Dilution multiple Experimenter 1 / day 1 Experimenter 2 / day 2 Experimenter 3 / day 3 RSD% 10 38.07％ 39.90％ 32.77％ 10.03 20 18.06％ 19.89％ 20.90％ 7.34 40 9.53％ 10.23％ 12.10％ 12.51 80 5.67％ 6.03％ 5.95％ 3.21 160 2.22％ 2.39％ 2.04％ 7.90

[0085] 4. Durability

[0086] 1. Experimental method: Referring to the example, after lentivirus infection of 293T cells, the culture time was shortened to 40 hours, and the test was performed to examine whether the CAR positive expression rate of each dilution gradient sample was affected.

[0087] 2. Experimental Results: As shown in Table 7, flow cytometry was performed after 293T cells were infected with the lentivirus and cultured for approximately 40 hours. The test results showed that after shortening the culture time, the RSD of the CAR positivity test results of each dilution gradient sample and the test results of the original planned culture time were less than 10%, indicating good durability of the method.

[0088] Table 7 CAR positive expression rate at different culture times

[0089] Dilution multiple Cultivation time (60h) Cultivation time (40h) RSD% 10 38.07％ 37.78％ 0.54 20 18.06％ 18.70％ 2.46 40 9.53％ 10.02％ 3.54 80 5.67％ 5.52％ 1.90 160 2.22％ 2.02％ 6.67

[0090] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A universal lentivirus titer detection method based on flow cytometry, characterized in that: The steps include: (1) Adding transfection medium and lentivirus diluted in the transfection medium to 293T cells and culturing them; then adding trypsin for digestion to form a cell dispersion; (2) The cell dispersion was centrifuged and resuspended in PBS, protein L antibody was added, and the cells were incubated in the dark at 2-8°C for 30-60 min, then washed, and the CAR positive expression rate was detected by flow cytometry to obtain the lentiviral titer. Wherein, the transfection medium is a DMEM medium containing 7-9 μg / ml polybrene and 10% FBS.

2. The detection method according to claim 1, characterized in that The 293T cells in step (1) need to be pretreated. The pretreatment process includes: taking 450-550 μl of a 1.5×10 5 -2.5×10 5 293T cells at a concentration of 100 cells / ml were seeded into a well plate, cultured at 37°C and 5% CO2 for 16-24 hours, and the supernatant was discarded.

3. The detection method according to claim 2, characterized in that The volume ratio of the 293T cells, the transfection medium, and the lentivirus diluted with the transfection medium is 4-6:4.5-5.5:

1.

4. The detection method according to claim 1, wherein The dilution multiple in step (1) is 10 times, 20 times, 40 times, 80 times or 160 times.

5. The detection method according to claim 1, wherein The culture conditions in step (1) include: culturing at 37° C. and 5% CO 2 for 48-72 hours.

6. The detection method according to claim 1, characterized in that The mass concentration of the pancreatic enzyme in step (1) is 0.2-0.3%.

7. The detection method according to claim 1, characterized in that The density of the cell dispersion in step (2) is 1.0×10 6 -2.0×10 6 pcs / ml.

8. The detection method according to claim 7, characterized in that The centrifugation process in step (2) includes: taking 2×10 5 The cells were centrifuged at 300-500×g for 4-8 minutes, the supernatant was discarded, and the cells were washed with PBS, and then centrifuged at 300-500×g for 4-8 minutes, and the supernatant was discarded; the PBS contained 1% FBS.

9. The detection method according to claim 8, characterized in that The volume ratio of the cell dispersion, PBS and protein L antibody is 250-300:45-50:2-5.

10. Use of the detection method according to any one of claims 1 to 9 in preparing a product for detecting universal lentivirus titers.

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