Lentivirus titer detection kit and detection method

Abstract

The invention discloses a lentivirus titer detection kit and a detection method, the kit comprises a capture plate and a detection antibody solution, the capture plate is prepared by coating an HIV-1p24 antibody and sealing with a sealing liquid; the detection antibody solution is prepared by labeling an HIV-1p24 antibody with biotin to obtain a biotin-labeled HIV-1p24 antibody and then using a sample diluent. According to the kit, a p24 protein concentration determination method suitable for rapidly detecting any HIV-1-based lentivirus is established, and a simple and rapid detection tool is provided for lentivirus titer detection and replicated lentivirus (RCL) detection. Through verification, the kit disclosed by the invention has the characteristics of good linearity, specificity, durability, precision, accuracy, detection limit, hook effect and stability, simplicity and convenience in operation, short detection period (single detection time is within 2 hours) and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological analysis and detection, and in particular relates to a lentivirus titer detection kit and a detection method, which are suitable for quantitatively detecting HIV-1p24 protein concentration in a sample. Background Art

[0002] Outside the human body, the modification system constructed by genetic engineering technology can effectively transfer genetic material into specific target cells, which is used to modify the genetic material of target cells, change the gene expression mode or regulate the biological characteristics of cells. At present, lentivirus (LV) vectors, γ-retroviral vectors, etc. are commonly used to introduce chimeric antigen receptor (CAR) genes into T cells to achieve targeted killing of tumors by CAR-T cells. Lentivirus vectors play a role by mediating the integration of target genes into the cell genome. Lentivirus vectors can infect cells with active and inactive mitosis. At present, human lentivirus, non-human primate and non-primate lentivirus are common lentivirus vectors for in vitro gene modification. Among them, lentivirus vectors based on human immunodeficiency virus type I (HIV-1) are the most commonly used. Titer detection in the preparation process of lentivirus vectors plays an important role in the research and control of vector quality. At present, the main methods for detecting lentivirus titers are physical titer, genomic titer and transduction titer. The main risks in the preparation and clinical use of lentiviral vectors include the production of replication competent lentivirus (RCL), in vivo recombination, and the insertion of proviral DNA in or near related genes, which may cause or promote tumorigenesis and other cytopathic conditions.

[0003] p24 is the main structural protein of HIV virus particles (the most abundant signature protein in the virus shell), and plays a key role in the packaging and maturation of the virus. The p24 protein has a highly conserved amino acid sequence. If p24 is missing, HIV virus cannot be assembled normally. Studies have shown that each lentivirus contains about 2,000 p24 protein molecules, and 1 ng of p24 is equivalent to 1.25×10 7 The physical titer of the lentivirus can be obtained by detecting the content of HIV-1p24 protein.

[0004] Lentiviral titer detection is a key step to ensure the quality of lentiviral vectors. By accurately calculating the lentiviral titer, we can ensure that the number of viral particles used in each experiment is consistent, improving the repeatability of the experiment. Accurate lentiviral titer is crucial for designing gene transduction experiments. The appropriate infection dose can ensure sufficient gene expression without causing excessive cell damage.

[0005] In order to ensure the safety of the product, the lentiviral vectors currently used in gene therapy products are all non-replicating. However, homologous or non-homologous recombination may occur between the components of the vector system or between the vector components and the cell genome, leading to the production of replicative lentivirus (RCL). Once a replicative lentivirus (RCL) is produced, it may integrate with the cell genome, leading to the risk of secondary cancer. In response to this risk, domestic and foreign regulatory agencies have required the establishment of a detection method for replicative lentivirus (RCL) and the control of RCL at different stages of product production and application to ensure the safety of patients' medication.

[0006] The lentivirus titer (HIV-1p24) detection kit is suitable for rapid detection of p24 protein concentration of any HIV-1-based lentivirus, providing a simple and rapid detection tool for lentivirus titer detection and replication-competent lentivirus (RCL) detection. Summary of the invention

[0007] The purpose of the present invention is to provide a lentivirus titer detection kit and a detection method, which is suitable for rapid detection of p24 protein concentration of any HIV-1-based lentivirus, has good linearity, specificity, durability, precision, accuracy, detection limit, hook effect, stability, and has the advantages of simple operation, short detection cycle, etc.

[0008] In order to achieve the above purpose, this application adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a lentivirus titer detection kit, the kit comprising a capture plate and a detection antibody solution,

[0010] The capture plate is prepared by coating with HIV-1p24 antibody and blocking with a blocking solution;

[0011] The detection antibody solution is prepared by labeling HIV-1p24 antibody with biotin to obtain biotin-labeled antibody, and then using sample diluent to prepare the antibody solution.

[0012] In the above technical solution, the method for preparing the capture plate comprises the following steps:

[0013] S11, dilute HIV-1p24 antibody to a concentration of 2 μg / mL using coating solution 2#, add 100 μL / well evenly to the ELISA plate, seal with sealing film, and incubate at 2-8°C for 16-20 hours;

[0014] S12, discarding the coating solution in the ELISA plate and washing the plate twice;

[0015] S13, add 2# blocking solution, and block at 25-37°C for 1-4 hours;

[0016] S14, discarding the 2# blocking solution in the ELISA plate, and placing the ELISA plate at 25-37° C. to dry for 1-4 hours;

[0017] S15, after drying is completed, the coated ELISA plate is vacuum packed to obtain the capture plate.

[0018] In the above technical solution, the preparation method of the detection antibody solution is:

[0019] Biotin-LC-LC-NHS can react with primary amino groups to form stable, irreversible amide bonds, thereby achieving the effect of labeling antibodies containing primary amino groups;

[0020] According to the ratio of 1 mg of antibody, 15 μL of 10 mM Biotin-LC-LC-NHS solution was added, and the antibody was labeled in an ultrafiltration tube with a molecular weight cutoff of 50 kDa, free biotin was removed, the concentration of biotin labeled protein was determined, and an equal volume of label protection solution was added to obtain the biotin labeled antibody;

[0021] The biotin-labeled antibody is diluted to a working solution of 0.1 to 0.5 μg / mL using the sample diluent to obtain the detection antibody solution.

[0022] In the above technical solution, the kit also includes a lysate, a calibrator, a 10× washing solution, the sample diluent, an HRP labeling solution, a TMB substrate color developing solution and a stop solution.

[0023] As one embodiment, the kit includes a capture plate coated with HIV-1p24 antibodies, a lysate, a calibrator (containing HIV-1p24 protein solutions of different concentrations), a 10× wash solution, a sample diluent, a detection antibody solution, an HRP labeling solution, a TMB substrate color development solution, a stop solution, a sealing film, an aluminum foil bag, and a product instruction manual.

[0024] In the above technical solution, the sample diluent is prepared by accurately weighing 2.27 g Na 2 HPO 4 , 0.54gKH 2 PO 4 , 16.01g NaCl, 0.40g KCl, 40.00g sucrose, and 20.00g BSA are placed in a preparation container, and appropriate amount of ultrapure water is added, and stirred to dissolve. After dissolution, 1.00mL Tween-20 and 1.00mL ProClin 300 are added, and stirred to mix. The volume is made up to 2.00L with ultrapure water, and filtered through a filter with a membrane pore size of 0.22μm.

[0025] In the above technical solution, the preparation method of the 2# coating solution is: accurately weigh 1.14g Na 2 HPO 4 , 0.27gKH 2 PO 4 , 8.01g NaCl, 0.20g KCl into a preparation container, add appropriate amount of ultrapure water, stir to dissolve, dilute to 1.00L, and filter through a filter with a membrane pore size of 0.22μm;

[0026] The preparation method of the 2# blocking solution is as follows: weigh 1.14 g Na 2 HPO 4 , 0.27g KH 2 PO 4 , 8.01g NaCl, 0.20g KCl, 10.00g BSA into a preparation container, add appropriate amount of ultrapure water, stir to dissolve, add 0.50mL Tween-20 and 0.50mL ProClin 300 after dissolution, stir to mix, make up to 1.00L with ultrapure water, and filter through a filter with a membrane pore size of 0.22μm.

[0027] In the above technical solution, the preparation method of the 10× washing solution is: accurately weigh 11.36g Na 2 HPO 4 , 2.72gKH 2 PO 4 , 80.06g NaCl, 2.01g KCl to a preparation container, add appropriate amount of ultrapure water, stir to dissolve, add 5.00mL Tween-20, 5.00mL ProClin 300 after dissolution, stir to mix, make up to 1.00L with ultrapure water, and filter through a filter with a membrane pore size of 0.22μm;

[0028] The preparation method of the calibrator is as follows: thawing Recombinant HIV p24 protein at 2-8°C, returning to room temperature after thawing, oscillating and mixing, centrifuging instantaneously, and then using a sample diluent for gradient dilution to prepare Standard 1 of 200 pg / mL, using Standard 1 to prepare Standard 2 of 100 pg / mL, and so on, 2-fold gradient dilution to prepare Standard 7 of 3.125 pg / mL, and the sample diluent is Standard 8 of 0 pg / mL;

[0029] The HRP labeling solution is prepared by accurately measuring 300 mL of sample diluent into a preparation container, adding 7.5 μL of HRP-labeled streptavidin to prepare a working solution with a concentration of 25 ng / mL;

[0030] The preparation method of the stop solution is: accurately measure 270 mL of ultrapure water into a preparation container, add 30 mL of 10 mol / L hydrochloric acid solution to prepare a working solution with a concentration of 1 mol / L, and filter it using a filter with a membrane pore size of 0.22 μm.

[0031] In a second aspect, the present invention provides a method for detecting a lentivirus titer, wherein the detection method is performed using the above-mentioned kit.

[0032] In the above technical solution, the detection method comprises the following steps:

[0033] S21, return all reagents to room temperature and dilute 10× wash solution to 1× wash solution;

[0034] S22, returning all samples to room temperature;

[0035] S23, experimental steps:

[0036] Add 10 μL of lysate to the corresponding wells of the kit, then add 90 μL of standard and sample to be tested, seal the plate with a sealing film, and incubate at 37°C for 20-90 minutes;

[0037] Discard the solution, add 260 μL of 1× wash solution to each well, wash the plate, and pat dry on absorbent paper;

[0038] Add 100 μL of detection antibody solution to each well, seal the plate with a new sealing film, and incubate at 37°C for 30 minutes;

[0039] Discard the solution, add 260 μL of 1× wash solution to each well, wash the plate, and pat dry on absorbent paper;

[0040] Add 100 μL of HRP labeling solution to each well, seal the plate with a new sealing film, and incubate at 37°C for 30 minutes;

[0041] Discard the solution, add 260 μL of 1× wash solution to each well, wash the plate, and pat dry on absorbent paper;

[0042] Add 100 μL of TMB substrate colorimetric solution to each well, protect from light, and incubate at room temperature for 5-25 minutes;

[0043] Add 100 μL of stop solution to each well to terminate the reaction;

[0044] Complete OD in 15 minutes 450nm reading;

[0045] S24, absorbance calculation:

[0046] Calculate the average absorbance of the calibrator and sample duplicate wells;

[0047] With the concentration of the calibrator as the horizontal axis and the OD value as the vertical axis, the calibration curve was generated by regression fitting using relevant software;

[0048] The mean absorbance value corresponding to the sample was inserted into the calibration curve, and the lentivirus titer was calculated.

[0049] The beneficial effects of the present invention are:

[0050] 1. The kit of the present invention comprises a capture plate coated with HIV-1p24 antibody, so that the capture plate can specifically recognize HIV-1p24 protein without interference from foreign proteins.

[0051] 2. The kit of the present invention can specifically identify HIV-1p24 protein by using a capture plate coated with HIV-1p24 antibody and a biotin-labeled HIV-1p24 antibody in combination.

[0052] 3. The kit of the present invention is not limited by sample type and can be used to detect the p24 protein concentration of any HIV-1-based lentivirus.

[0053] 4. The kit of the present invention establishes a method for determining the concentration of p24 protein suitable for rapid detection of any HIV-1-based lentivirus, and provides a simple and rapid detection tool for lentivirus titer detection and replicating lentivirus (RCL) detection. It has been verified that the kit of the present invention has good linearity, specificity, durability, precision, accuracy, detection limit, hook effect, stability, and is easy to operate and has a short detection cycle (single detection time within 2 hours). BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 The signal-to-noise ratio results of the antibody pairing test (15×15);

[0055] Figure 2 The signal-to-noise ratio results of the antibody pairing test (12×12);

[0056] Figure 3 It is the working principle diagram of the kit of the present invention;

[0057] Figure 4 Determine the experimental results for the antibody pairs in Example 2;

[0058] Figure 5 The results of the antibody solution concentration optimization experiment in Example 2 are as follows;

[0059] Figure 6 This is the experimental result of optimizing the reaction time of the detection method in Example 2. DETAILED DESCRIPTION

[0060] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. The present invention can be implemented in many different forms and should not be construed as being limited to the embodiments set forth herein. On the contrary, these embodiments are provided so that this disclosure will be thorough and complete, and the concept of the present invention will be fully conveyed to those skilled in the art, and the present invention will only be limited by the claims.

[0061] In the present invention, unless otherwise specified, all parts and percentages are by weight, and the equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following embodiments are all conventional methods in the art unless otherwise specified.

[0062] Unless otherwise specified, the reagents used in the following examples can be obtained from commercial sources.

[0063] Ultra-micro-volume UV-visible spectrophotometer, Hangzhou Miou Instrument Co., Ltd., item number ND-100F.

[0064] The following solutions were prepared as follows:

[0065] Preparation of 1# coating solution: Accurately weigh 1.59g Na 2 CO 3 , 2.94 g NaHCO 3 Add an appropriate amount of ultrapure water to the preparation container, stir to dissolve, dilute to 1.00L with ultrapure water, and filter with a filter with a membrane pore size of 0.22μm. The solution can be stored stably at 2-8℃ for 1 year.

[0066] Preparation of 2# coating solution: Accurately weigh 1.14g Na 2 HPO 4 , 0.27g KH 2 PO 4 , 8.01g NaCl, 0.20g KCl into a preparation container, add appropriate amount of ultrapure water, stir to dissolve, dilute to 1.00L, and filter with a filter with a membrane pore size of 0.22μm. The solution can be stored stably at 2-8℃ for 1 year.

[0067] Preparation of 2# blocking solution: weigh 1.14g Na 2 HPO 4 , 0.27g KH 2 PO 4, 8.01g NaCl, 0.20g KCl, 10.00g BSA into a preparation container, add appropriate amount of ultrapure water, stir to dissolve, add 0.50mL Tween-20, 0.50mL ProClin 300 after dissolution, stir to mix, make up to 1.00L with ultrapure water, and filter with a filter with a membrane pore size of 0.22μm. The solution can be stably stored at 2-8℃ for 1 year.

[0068] Preparation of 3# blocking solution: weigh 1.14g Na 2 HPO 4 , 0.27g KH 2 PO 4 , 8.01g NaCl, 0.20g KCl, 10.00g gelatin into a preparation container, add appropriate amount of ultrapure water, stir to dissolve, add 0.50mL Tween-20, 0.50mL ProClin 300 after dissolution, stir to mix, dilute to 1.00L with ultrapure water, and filter with a filter with a membrane pore size of 0.22μm. The solution can be stored stably at 2-8℃ for 1 year.

[0069] 1# Sample diluent: weigh 1.14g Na 2 HPO 4 , 0.27g KH 2 PO 4 , 8.01g NaCl, 0.20g KCl, 20.00g sucrose, 10.00g BSA into a preparation container, add appropriate amount of ultrapure water, stir to dissolve, add 0.50mL Tween-20, 0.50mL ProClin 300 after dissolution, stir to mix, make up to 1.00L with ultrapure water, and filter with a filter with a membrane pore size of 0.22μm. The solution can be stably stored at 2-8℃ for 2 years.

[0070] 2# Sample diluent: weigh 1.14g Na 2 HPO 4 , 0.27g KH 2 PO 4 , 8.01g NaCl, 0.20g KCl, 20.00g trehalose, 10.00g BSA to the preparation container, add appropriate amount of ultrapure water, stir to dissolve, add 0.50mL Tween-20, 0.50mL ProClin 300 after dissolution, stir to mix, make up to 1.00L with ultrapure water, and filter with a filter with a membrane pore size of 0.22μm. The solution can be stably stored at 2-8℃ for 2 years.

[0071] 3# Sample diluent: weigh 1.14g Na 2 HPO 4, 0.27g KH 2 PO 4 , 8.01g NaCl, 0.20g KCl, 20.00g 2-hydroxypropyl-β-cyclodextrin, 10.00g BSA to the preparation container, add appropriate amount of ultrapure water, stir to dissolve, add 0.50mL Tween-20, 0.50mL ProClin 300 after dissolution, stir to mix, make up to 1.00L with ultrapure water, and filter with a filter with a membrane pore size of 0.22μm. The solution can be stably stored at 2-8℃ for 2 years.

[0072] Preparation of 10× washing solution: Accurately weigh 11.36g Na 2 HPO 4 , 2.72g KH 2 PO 4 , 80.06g NaCl, 2.01g KCl into a preparation container, add appropriate amount of ultrapure water, stir to dissolve, add 5.00mL Tween-20, 5.00mL ProClin300 after dissolution, stir to mix, make up to 1.00L with ultrapure water, and filter with a filter with a membrane pore size of 0.22μm. The solution can be stored stably at 2-8℃ for 2 years.

[0073] Lysis buffer: The lysate used in this example was purchased from Sigma-Aldrich, model number C2978. Other lysates may also be used.

[0074] Preparation of detection antibody solution: Accurately measure 300mL of sample diluent into the preparation container, add a certain volume of biotin-labeled HIV-1p24 antibody (Hengyu Biotechnology, homemade), and prepare a working solution with a concentration of 0.1-1μg / mL. The solution can be stably stored at 2-8℃ for 1 year.

[0075] Preparation of HRP labeled solution: Accurately measure 300 mL of sample dilution solution into the preparation container, add a certain volume of HRP labeled streptavidin (Thermo Fisher Scientific, 21130), and prepare a working solution with a concentration of 25-100 ng / mL. The solution can be stored stably at 2-8°C for 1 year.

[0076] Preparation of TMB substrate colorimetric solution: TMB Super Sensitive One Component HRP Microwell Substrate (Surmodics, Inc., TMBS-1000-01) is directly available for aliquoting. The solution can be stored stably at 2-8°C for a period of time that is consistent with the original manufacturer's expiration date.

[0077] Preparation of stop solution: Accurately measure 270 mL of ultrapure water into the preparation container, add 30 mL of 10 mol / L hydrochloric acid solution (Sinopharm Chemical Reagent Co., Ltd., 53100258), prepare a working solution with a concentration of 1 mol / L, and filter it with a filter with a membrane pore size of 0.22 μm. The solution can be stored stably at 2-8°C for 3 years.

[0078] Ag1: Recombinant HIV p24 protein (His Tag), Suzhou Nearshore Protein Technology Co., Ltd., catalog number DRA19;

[0079] Ag2: P24 antigen quality control product, Guangdong Feipeng Biological Co., Ltd., catalog number P24-Ag;

[0080] Ag3: recombinant HIV1 p24 protein, Abcam (Shanghai) Trading Co., Ltd., catalog number AB43037;

[0081] Ag4: HIV (HIV1-HXB2) Capsid protein p24, His Tag (MALS verified), Beijing Biopsies Biotechnology Co., Ltd., Catalog No. CP4-H52H3;

[0082] Ab1: P24 antibody, Guangdong Feipeng Biological Co., Ltd., catalog number HIV-REAB-C4-015;

[0083] Ab2: P24 antibody, Guangdong Feipeng Biological Co., Ltd., catalog number HIV-REAB-C4-017;

[0084] Ab3: P24 antibody, Guangdong Feipeng Biological Co., Ltd., catalog number HIV-REAB-C4-018;

[0085] Ab4: P24 antibody, Guangdong Feipeng Biological Co., Ltd., catalog number P24-Ab2#;

[0086] Ab5: P24 antibody, Guangdong Feipeng Biological Co., Ltd., catalog number P24-Ab55#;

[0087] Ab6: P24 monoclonal antibody, Guangdong Feipeng Biological Co., Ltd., catalog number HIV-REAB-C4-014;

[0088] Ab7: P24 monoclonal antibody, Guangdong Feipeng Biological Co., Ltd., catalog number P24-Ab3#;

[0089] Ab8: HIV-1p24 Antibody (M564), mAb, Mouse, Nanjing GenScript Biotechnology Co., Ltd., Catalog No. V10902;

[0090] Ab9: HIV-1p24 Antibody (5A4), mAb, Mouse, Nanjing GenScript Biotechnology Co., Ltd., Catalog No. V10903;

[0091] Ab10: HIV-1p24 Antibody (P152), mAb, Mouse, Nanjing GenScript Biotechnology Co., Ltd., Catalog No. V10901;

[0092] Ab11: Human Immunodeficiency Virus type 1 (HIV-1) p24 / Capsid Protein p24 Antibody, Rabbit Mab, Beijing Sino Biological Science and Technology Co., Ltd., Catalog No. 11695-R002;

[0093] Ab12: HIV Gag-p24Antibody, Mouse Mab, Beijing Sino Biological Science and Technology Co., Ltd., Catalog No. 11695-MM04;

[0094] Ab13: HIV-1p24 (group M, subtype B, strain 92418) Antibody, Mouse Mab (11695-MM08T), Beijing Sino Biological Science and Technology Co., Ltd., Catalog No. 11695-MM08T;

[0095] Ab14: HIV-1p24 (group M, subtype B, strain 92418) Antibody, Mouse Mab (11695-MM15), Beijing Sino Biological Science and Technology Co., Ltd., Catalog No. 11695-MM15;

[0096] Ab15: Human Immunodeficiency Virus type 1 (HIV-1) Gag-p24 Antibody, Mouse MAb, Beijing Sino Biological Science and Technology Co., Ltd., Catalog No. 11695-MM13.

[0097] The preparation method of the capture plate in the following examples is:

[0098] S1, coating

[0099] HIV-1p24 antibody was diluted to a concentration of 0.5-20 μg / mL using coating solution, and 100 μL / well was evenly added to the ELISA plate, and the plate was sealed with sealing film and incubated at 2-8°C for 16-20 hours;

[0100] S2, Washing plate

[0101] Discard the coating solution in the ELISA plate and wash the plate twice (260 μL / well of washing solution, 30 to 60 seconds for each wash);

[0102] S3, closed

[0103] Add blocking solution evenly at 200 μL / well and block at 25-37°C for 1-4 hours;

[0104] S4. Drying

[0105] Discard the blocking solution in the ELISA plate, pat it dry on absorbent paper, and place the ELISA plate at 25-37℃ to dry for 1-8 hours;

[0106] S5. Packaging

[0107] After drying, the coated ELISA plate is vacuum packed in an aluminum foil bag (a desiccant needs to be placed in the aluminum foil bag) to form a capture plate.

[0108] Example 1 HIV-1p24 Antibody Biotin Labeling

[0109] Biotin-LC-LC-NHS can be used to bind to primary amino groups (NH 2 -) reaction to form a stable, irreversible amide bond, thereby achieving the 2 -) HIV-1p24 antibody labeling effect.

[0110] 1. Preparation of biotin labeling solution

[0111] Preparation of 10mM Biotin-LC-LC-NHS: Accurately weigh 10.0mg Biotin-LC-LC-NHS into a brown sample tube, add 1.761mL DMSO, tighten the tube cap, gently mix and continue to dissolve at room temperature for 30 minutes. The solution can be stored stably at -80℃ for up to 6 months (store in aliquots and use a new one each time).

[0112] Preparation of labeling buffer: Accurately weigh 1.14 g Na 2 HPO 4 , 0.27g KH 2 PO 4 , 8.01g NaCl, 0.20g KCl into a preparation container, add appropriate amount of ultrapure water, stir to dissolve, and then dilute to 1.00L with ultrapure water, and filter with a filter with a membrane pore size of 0.22μm. The solution can be stored stably at 2-8℃ for 1 year.

[0113] Preparation of marker protection solution: accurately weigh 0.2g BSA, 0.8g ultrapure water, and 9g glycerol into a 15mL reagent bottle (or 15mL centrifuge tube), tighten the bottle (or tube) cap, place on a rotary mixer and mix thoroughly, and filter with a filter with a membrane pore size of 0.22μm after complete dissolution. The solution can be stably stored at -20℃ for 2 years.

[0114] 2. Calculation of biotin dosage

[0115] Add 15 μL of 10 mM Biotin-LC-LC-NHS solution to 1 mg of antibody, and calculate the required volume of 10 mM Biotin-LC-LC-NHS solution based on the amount of antibody to be labeled (not less than 0.1 mg).

[0116] 3. Biotin labeling

[0117] Transfer the substance to be labeled to an ultrafiltration tube with a molecular weight cutoff of 50 kDa, add labeling buffer (10 mM PBS, pH 7.2 ± 0.2) that does not exceed the maximum volume of the ultrafiltration tube, and centrifuge at 12000 × g for 10 minutes (this step can be repeated 2 to 3 times). After the last ultrafiltration is completed, add an appropriate amount of labeling buffer to adjust the concentration of the substance to be labeled to about 2 mg / mL.

[0118] Add an appropriate volume of Biotin-LC-LC-NHS solution to the above ultrafiltration tube and gently pipette to mix. Incubate in a 37°C incubator in the dark for 30 minutes or incubate in the dark at room temperature for 1 hour.

[0119] 4. Removal of free biotin

[0120] The labeled biotin marker was placed in a centrifuge and centrifuged at 12000×g for 10 minutes at room temperature.

[0121] Add an appropriate amount of labeling buffer to the above ultrafiltration tube, gently pipette to mix, centrifuge at 12000×g for 10 minutes at room temperature, and repeat this step 3 times.

[0122] Add an appropriate amount of labeling buffer to the ultrafiltration tube again, mix gently by pipetting, and centrifuge at 4000×g for 5 minutes.

[0123] The ultrafiltration tube membrane was placed upside down in the collection tube and centrifuged at 12000×g for 2 minutes at room temperature.

[0124] 5. Determination of biotin-labeled protein concentration

[0125] The protein concentration of the biotinylated protein was measured at 280 nm using an ultra-micro-volume UV-visible spectrophotometer (repeated twice, average value calculated).

[0126] 6. Storage of biotin markers

[0127] Collect the solution in the ultrafiltration tube into a brown sample tube, add an equal volume of marker protection solution, and store at -20°C for later use.

[0128] Example 2 Establishment of a double antibody sandwich ELISA detection method based on the biotin-streptavidin system

[0129] 1. Operation steps of the double antibody sandwich ELISA detection method based on the biotin-streptavidin system

[0130] 1) Sample addition: Add 10 μL of lysis solution to the corresponding wells, then add 90 μL of calibrators (Standard 1 to Standard 8) and the sample to be tested, seal the plate with a sealing film, and incubate at 37°C for 30 to 60 minutes. Discard the solution, add 260 μL of 1× washing solution to each well, wash the plate, and wash 5 times (pat dry on absorbent paper each time).

[0131] 2) Add detection antibody solution (biotin-labeled HIV-1p24 antibody solution): Add 100 μL of detection antibody solution of appropriate concentration to each well, seal the plate with a new sealing film, and incubate at 37°C for 30 to 60 minutes. Discard the solution, add 260 μL of 1× washing solution to each well, wash the plate, and wash 5 times (pat dry on absorbent paper each time).

[0132] 3) Add HRP labeling solution (HRP-labeled streptavidin solution): Add 100 μL of HRP labeling solution of appropriate concentration to each well, seal the plate with a new sealing film, and incubate at 37°C for 30 minutes. Discard the solution, add 260 μL of 1× washing solution to each well, wash the plate, and wash 5 times (pat dry on absorbent paper each time).

[0133] 4) Color development: Add 100 μL TMB substrate color development solution to each well, protect from light, and incubate at room temperature for 15 minutes.

[0134] 5) Stop reaction: Add 100 μL of stop solution to each well to stop the reaction.

[0135] 6) Reading: Complete OD within 15 minutes 450nm reading.

[0136] 2. Optimization of various conditions during the establishment of the double antibody sandwich ELISA detection method based on the biotin-streptavidin system

[0137] 1) Antibody pairing test (chessboard method, 15×15)

[0138] according to Figure 1An antibody pairing test (chessboard method, 15×15) was set up for preliminary screening of suitable antibody pairs, and the antigen concentration was set at three gradient levels (10000 pg / mL, 1000 pg / mL, 100 pg / mL).

[0139] The signal-to-noise ratio (S / N) results at each concentration level showed that Ab11, Ab14, and Ab15 antibodies could be preliminarily excluded, and the remaining antibodies could be used for subsequent optimization experiments.

[0140] 2) Antibody pairing test (chessboard method, 12×12)

[0141] according to Figure 2 An antibody pairing test (chessboard method, 12×12) was set up for preliminary screening of suitable antibody pairs, and three gradient levels of antigen concentration were set (1000 pg / mL, 100 pg / mL, 10 pg / mL).

[0142] The signal-to-noise ratio (S / N) results at each concentration level showed that the antibody pair Ab3&Biotin-Ab2 and the antibody pair Ab5&Biotin-Ab2 could be used for subsequent optimization experiments.

[0143] 3) Antibody pair determination

[0144] Ab3 (P24 antibody, Guangdong Feipeng Biological Co., Ltd., catalog number HIV-REAB-C4-018) and Ab5 (P24 antibody, Guangdong Feipeng Biological Co., Ltd., catalog number P24-Ab55#) were prepared into solutions with a concentration of 2 μg / mL using 2# coating solution, and blocked using 2# blocking solution to prepare capture plates. During the experiment, 1 μg / mL of biotin-labeled Ab2 (P24 antibody, Guangdong Feipeng Biological Co., Ltd., catalog number HIV-REAB-C4-017) was added.

[0145] The results are as follows Figure 4 As shown, a higher signal-to-noise ratio can be obtained by using the antibody pair Ab3 & Biotin-Ab2, and this antibody pair was selected for subsequent optimization experiments.

[0146] 4) Optimization of coating solution, blocking solution and calibration material

[0147] An orthogonal test (16 different conditions) was set up according to Table 1 for preliminary screening of suitable coating solution, suitable blocking solution and suitable calibrator. The calibrator had 8 concentrations (200 pg / mL, 100 pg / mL, 50 pg / mL, 25 pg / mL, 12.5 pg / mL, 6.25 pg / mL, 3.125 pg / mL, 0 pg / mL).

[0148] The signal-to-noise ratio (S / N) results at each concentration level showed that different calibrators had a great influence on the reaction signal-to-noise ratio, while coating solution and blocking solution had little influence on the reaction signal-to-noise ratio. 2# coating solution, 2# blocking solution and Ag1 can be selected for subsequent optimization experiments.

[0149] Table 1 Results of orthogonal test for optimization of coating solution, blocking solution and calibration substance

[0150]

[0151]

[0152] 5) Optimization of detection antibody solution concentration

[0153] Ab3 (P24 antibody, Guangdong Feipeng Biological Co., Ltd., catalog number HIV-REAB-C4-018) was prepared into a solution with a concentration of 2 μg / mL using 2# coating solution, and blocked using 2# blocking solution to prepare a capture plate. During the experiment, 1 μg / mL, 0.5 μg / mL, and 0.25 μg / mL of biotin-labeled Ab2 (P24 antibody, Guangdong Feipeng Biological Co., Ltd., catalog number HIV-REAB-C4-017) were added respectively.

[0154] Test results such as Figure 5 As shown, with the decrease in the concentration of the detection antibody solution (biotin-labeled HIV-1p24 antibody), the signal-to-noise ratio gradually increased, and it was determined to use 0.25 μg / mL of biotin-labeled Ab2 (P24 antibody, Guangdong Feipeng Biological Co., Ltd., catalog number HIV-REAB-C4-017) for subsequent optimization experiments.

[0155] 6) Calibrator dilution optimization

[0156] Ab3 (P24 antibody, Guangdong Feipeng Biological Co., Ltd., catalog number HIV-REAB-C4-018) was prepared into a solution with a concentration of 2 μg / mL using 2# coating solution, and blocked using 2# blocking solution to prepare a capture plate. During the experiment, 1 μg / mL, 0.5 μg / mL, and 0.25 μg / mL of biotin-labeled Ab2 (P24 antibody, Guangdong Feipeng Biological Co., Ltd., catalog number HIV-REAB-C4-017) were added respectively. Ag1 (Recombinant HIV p24 protein (His Tag), Suzhou Nearshore Protein Technology Co., Ltd., catalog number DRA19) was prepared into solutions with concentrations of 200 pg / mL, 100 pg / mL, 50 pg / mL, 25 pg / mL, 12.5 pg / mL, 6.25 pg / mL, 3.125 pg / mL, and 0 pg / mL using 1# sample diluent, 2# sample diluent, and 3# sample diluent, respectively. The solution under each condition was divided into 4 portions and stored at 37°C for 0 day, 1 day, 3 days, and 5 days, respectively.

[0157] The test results are shown in Table 2. The 37°C accelerated stability results of the calibrator prepared using 1# sample diluent are better than those using 2# sample diluent and 3# sample diluent (the signal-to-noise ratio is reduced the least). 1# sample diluent is selected for the preparation of the calibrator.

[0158] Table 2 Optimization results of calibrator dilution

[0159]

[0160]

[0161] 7) Optimization of detection method reaction time

[0162] Ab3 (P24 antibody, Guangdong Feipeng Biological Co., Ltd., catalog number HIV-REAB-C4-018) was prepared into a solution with a concentration of 2μg / mL using 2# coating solution, and blocked with 2# blocking solution to prepare a capture plate. Ag1 (RecombinantHIV p24 protein (His Tag), Suzhou Nearshore Protein Technology Co., Ltd., catalog number DRA19) was prepared into samples with concentrations of 200pg / mL, 100pg / mL, 50pg / mL, 25pg / mL, 12.5pg / mL, 6.25pg / mL, 3.125pg / mL, and 0pg / mL using 1# sample diluent. During the experiment, the concentration of the detection antibody solution was set to 0.25μg / mL, and the concentration of the HRP-labeled antibody solution was set to 50ng / mL. The effects of the two reaction steps of adding the sample and adding the detection antibody solution on the reaction results were studied at 37°C for 60 minutes and 30 minutes, respectively.

[0163] The absorbance test results are shown in Table 3. The reaction signal background of the two reaction steps of adding sample and adding detection antibody solution at 37°C with an incubation time of 30 minutes is lower than that of the incubation time of 60 minutes. Figure 6 As shown in the figure, the two reaction steps of adding sample and adding detection antibody solution at 37°C have a better signal-to-noise ratio when the incubation time is 30 minutes compared with 60 minutes. Considering the time-consuming nature of the detection method, the 30-minute incubation time was selected for the development of the detection kit.

[0164] Table 3 Detection method reaction time optimization absorbance (OD 450nm )result

[0165]

[0166]

[0167] Example 3 Lentivirus Titer (HIV-1p24) Detection Kit

[0168] Figure 3 The working principle diagram of the kit of the present invention is shown in FIG. The kit of the present invention is used for rapid detection of p24 protein concentration of any HIV-1-based lentivirus. The kit components listed in Table 4 are assembled into a kit, and the kit is stored at 2-8° C. after assembly.

[0169] Table 4 Lentivirus titer (HIV-1p24) detection kit components, quantities and main ingredients

[0170]

[0171] The method for detecting the lentivirus titer in a sample using the lentivirus titer (HIV-1p24) detection kit of the present invention comprises the following specific steps:

[0172] S1. Preparation of experimental reagents

[0173] Return all reagents to room temperature before testing;

[0174] If the concentrated reagent crystallizes, it needs to be warmed at 37°C until all the crystals are dissolved;

[0175] Dilute 10× washing solution to 1× washing solution with purified water (or distilled water, deionized water) (e.g., add 60 mL of 10× washing solution to 540 mL of purified water). Gently mix the 1× washing solution and set aside (1× washing solution can be stored in a refrigerator at 2-8°C for 2 weeks);

[0176] S2. Preparation of experimental samples

[0177] All samples were returned to room temperature before testing;

[0178] It is recommended to use fresh samples;

[0179] S3. Experimental steps

[0180] Prepare the reagents within 30 minutes before the experiment;

[0181] 15 minutes before the experiment, equilibrate all materials and prepared reagents to room temperature;

[0182] It is recommended that all quality controls and samples be repeated.

[0183] Remove the unneeded slats, put them back into the foil bag containing the desiccant, and seal it tightly;

[0184] Add 10 μL of lysate to the corresponding wells, then add 90 μL of standard (Standard 1 to Standard 8) and the sample to be tested, seal the plate with a sealing film, and incubate at 37°C for 30 minutes;

[0185] Discard the solution, add 260 μL of 1× washing solution to each well, wash the plate 5 times (pat dry on absorbent paper each time);

[0186] Add 100 μL of detection antibody solution to each well, seal the plate with a new sealing film, and incubate at 37°C for 30 minutes;

[0187] Discard the solution, add 260 μL of 1× washing solution to each well, wash the plate 5 times (pat dry on absorbent paper each time);

[0188] Add 100 μL of HRP labeling solution to each well, seal the plate with a new sealing film, and incubate at 37°C for 30 minutes;

[0189] Discard the solution, add 260 μL of 1× washing solution to each well, wash the plate 5 times (pat dry on absorbent paper each time);

[0190] Add 100 μL of TMB substrate colorimetric solution to each well, protect from light, and incubate at room temperature for 15 minutes;

[0191] Add 100 μL of stop solution to each well to terminate the reaction;

[0192] Complete OD in 15 minutes 450nm reading;

[0193] S4. Absorbance calculation

[0194] Calculate the average absorbance of the calibrator and sample duplicate wells;

[0195] With the concentration of the calibrator as the horizontal axis and the OD value as the vertical axis, use relevant software for regression fitting to generate a calibration curve (it is recommended to use a four-parameter logistic equation);

[0196] Substitute the mean absorbance value corresponding to the sample into the calibration curve and back-calculate the corresponding concentration.

[0197] S5. Result determination

[0198] Experimental Validity: Calibration Curve: R 2 >0.99; Standard 1 absorbance (Mean OD 450nm ):>1.000;Standard 8 absorbance (Mean OD 450nm ):<0.150; the precision (coefficient of variation, CV) of the calibration standard back-calculated concentration should be <20% (LLOQ<25%), and the accuracy (relative error, RE) should be <±20% (LLOQ<±25%);

[0199] Sample result calculation: Substitute the sample absorbance mean into the calibration curve, and the result is the p24 protein concentration of the sample (if the sample absorbance mean is higher than the Standard 1 absorbance mean, the sample needs to be diluted and retested). The lentiviral titer of the sample can be calculated based on the p24 protein concentration of the sample (1 ng of p24 is equivalent to 1.25×10 7 lentiviral physical particles).

[0200] Example 4 Kit Performance Verification

[0201] 1. Linearity & Range

[0202] The kit and detection method of Example 3 were used to measure 6 independent analysis batches on different days, and the coefficient of variation (CV) and relative error (RE) of the back-calculated concentration of the calibration standard were calculated. The results were reported in a tabular form to determine the overall robustness of the calibration curve regression model, as shown in Table 5.

[0203] Table 5 Kit linearity & range

[0204]

[0205]

[0206] The test results show that the linearity is: within the quantitative range, the calibration curve R 2>0.99; Range: The concentration interval between the lower limit of quantification (LLOQ) and the upper limit of quantification (ULOQ) is the quantitative range of the calibration curve. Within this interval, the precision of the back-calculated concentration of the calibration standard should be <20% (LLOQ <25%), and the accuracy should be <±20% (LLOQ <±25%). The linearity & range verification of the kit has passed.

[0207] 2. Exclusivity

[0208] The kit and detection method of Example 3 were used to detect different concentrations of BSA, C8166 cell protein, Vero cell protein, HEK293 cell protein, HEK293T cell protein, and BHK-21 cell protein, respectively. Each sample was tested twice, and the back-calculated concentration of each sample was calculated according to the calibration curve. See Table 6 for details.

[0209] Table 6 Kit specificity

[0210]

[0211]

[0212] The test results showed that the detection results of 10 ng / mL BSA, C8166 cell protein, Vero cell protein, HEK293 cell protein, HEK293T cell protein, and BHK-21 cell protein samples were all below the quantitative limit, and the specificity of the kit was verified.

[0213] 3. Durability

[0214] The kit and detection method of Example 3 were used, and the reaction time (+20% and -20%), reaction temperature (+20% and -20%), and reaction volume (+20% and -20%) were changed under standard experimental parameters, and 8 calibration standards (Standard 1→Standard 8) were detected respectively, and each sample was detected twice. See Table 7 for details.

[0215] Table 7 Kit durability

[0216]

[0217] The results showed that when there were small changes in the experimental parameters, the experiment was still valid, meeting the experimental validity standards, and the durability of the kit was verified.

[0218] 4. Precision

[0219] Using the kit and detection method of Example 3, at least 3 independent analysis batches of quality control samples (HQC, MQC, LQC) were determined within a few days. Each quality control sample in each batch was tested no less than 10 times. The mean (Mean), standard deviation (SD), and coefficient of variation (CV) of the concentration of quality control samples in each batch and between batches were calculated, as shown in Table 8.

[0220] Table 8 Kit precision

[0221]

[0222]

[0223] The test results showed that the intra-batch precision and inter-batch precision of the kit were both less than 10%, and the precision verification of the kit passed.

[0224] 5. Accuracy

[0225] Using the kit and the detection method of Example 3, at least 3 independent analysis batches of quality control samples (HQC, MQC, LQC) were determined within a few days. Each quality control sample in each batch was tested no less than 10 times, and the mean (Mean), standard deviation (SD), and relative error (RE) of the concentration of quality control samples in each batch and between batches were calculated, as shown in Table 9.

[0226] Table 9 Kit Accuracy

[0227]

[0228]

[0229] The results showed that the intra-batch accuracy and inter-batch accuracy of the kit were both less than 20%, and the accuracy of the kit passed the verification.

[0230] 6. Detection limit

[0231] Detect the blank calibration standard (Standard 8, repeated no less than 20 times), calculate the absorbance mean (Mean) and standard deviation (SD), substitute the absorbance corresponding to Mean+2SD into the calibration curve (or substitute the blank calibration standard and the lower limit of quantitation for a linear equation obtained by two-point regression fitting), and convert the corresponding concentration to the detection limit (LOD) of the kit. The detection limit should be investigated by at least 3 independent analytical batches (the final detection limit is the mean of 3 tests). See Table 10 for details.

[0232] Table 10 Detection limit of the kit

[0233]

[0234] The results showed that the detection limit of the kit was 0.861pg / mL.

[0235] 7. Hook Effect

[0236] The upper limit of quantitation, 10 times, 100 times, 1000 times, 10000 times, 100000 times, and 1000000 times of the upper limit of quantitation were tested respectively, and each sample was tested twice, and the average absorbance value (Mean) of each sample was calculated. The performance of the detection signal of each sample with the increase of concentration was observed, and the first concentration point when the detection signal decreased with the increase of concentration was the hook effect concentration point, as shown in Table 11.

[0237] Table 11 Kit Hook Effect

[0238]

[0239] The results showed that the hook effect would not occur when the sample concentration was not higher than 20 μg / mL.

[0240] 8. Stability

[0241] The kit and the detection method of Example 3 were used, and the key components of the kit were stored in a 37°C environment. Quality inspections were performed at regular intervals to examine their stability under accelerated conditions. See Table 12 for details.

[0242] Table 12 Kit stability

[0243]

[0244] The results showed that the accelerated stability of the key components of the kit at 37°C was no less than 10 days.

[0245] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A lentivirus titer detection kit, comprising a capture plate and a detection antibody solution, characterized in that: The capture plate is prepared by coating with HIV-1p24 antibody and blocking with a blocking solution; The detection antibody solution is prepared by labeling HIV-1p24 antibody with biotin to obtain biotin-labeled antibody, and then using sample diluent to prepare the antibody solution.

2. The kit according to claim 1, characterized in that: The method for preparing the capture plate comprises the following steps: S11, dilute HIV-1p24 antibody to a concentration of 2 μg / mL using coating solution 2#, add 100 μL / well evenly to the ELISA plate, seal with sealing film, and incubate at 2-8°C for 16-20 hours; S12, discarding the coating solution in the ELISA plate and washing the plate twice; S13, add 2# blocking solution, and block at 25-37°C for 1-4 hours; S14, discarding the 2# blocking solution in the ELISA plate, and placing the ELISA plate at 25-37° C. to dry for 1-4 hours; S15, after drying is completed, the coated ELISA plate is vacuum packed to obtain the capture plate.

3. The kit according to claim 1, characterized in that: The preparation method of the detection antibody solution is: Biotin-LC-LC-NHS can react with primary amino groups to form stable, irreversible amide bonds, thereby achieving the effect of labeling antibodies containing primary amino groups; According to the ratio of 1 mg of antibody, 15 μL of 10 mM Biotin-LC-LC-NHS solution was added, and the antibody was labeled in an ultrafiltration tube with a molecular weight cutoff of 50 kDa, free biotin was removed, the concentration of biotin labeled protein was determined, and an equal volume of label protection solution was added to obtain the biotin labeled antibody; The biotin-labeled antibody is diluted to a working solution of 0.25 μg / mL using the sample diluent to obtain the detection antibody solution.

4. The kit according to claim 1, characterized in that: The kit also includes a lysate, a calibrator, a 10× washing solution, the sample diluent, an HRP labeling solution, a TMB substrate color developing solution and a stop solution.

5. The kit according to claim 1, 3 or 4, characterized in that: The preparation method of the sample diluent is as follows: accurately weigh 2.27g Na2HPO4, 0.54g KH2PO4, 16.01g NaCl, 0.40g KCl, 40.00g sucrose, and 20.00g BSA into a preparation container, add an appropriate amount of ultrapure water, stir to dissolve, add 1.00mL Tween-20 and 1.00mL ProClin 300 after dissolution, stir to mix, make up to 2.00L with ultrapure water, and filter using a filter with a membrane pore size of 0.22μm.

6. The kit according to claim 2, characterized in that: The preparation method of the 2# coating solution is as follows: accurately weigh 1.14g Na2HPO4, 0.27g KH2PO4, 8.01g NaCl, and 0.20g KCl into a preparation container, add an appropriate amount of ultrapure water, stir to dissolve, dilute to 1.00L, and filter using a filter with a membrane pore size of 0.22μm to obtain; The preparation method of the 2# blocking solution is as follows: weigh 1.14g Na2HPO4, 0.27g KH2PO4, 8.01g NaCl, 0.20g KCl, and 10.00g BSA into a preparation container, add an appropriate amount of ultrapure water, stir to dissolve, add 0.50mL Tween-20 and 0.50mL ProClin 300 after dissolution, stir to mix, make up to 1.00L with ultrapure water, and filter using a filter with a membrane pore size of 0.22μm.

7. The kit according to claim 4, characterized in that: The preparation method of the 10× washing solution is as follows: accurately weigh 11.36 g Na2HPO4, 2.72 g KH2PO4, 80.06 g NaCl, and 2.01 g KCl into a preparation container, add an appropriate amount of ultrapure water, stir to dissolve, add 5.00 mL Tween-20 and 5.00 mL ProClin 300 after dissolution, stir to mix, make up to 1.00 L with ultrapure water, and filter through a filter with a membrane pore size of 0.22 μm to obtain the solution; The preparation method of the calibrator is as follows: thawing Recombinant HIV p24 protein at 2-8°C, returning to room temperature after thawing, oscillating and mixing, centrifuging instantaneously, and then using a sample diluent for gradient dilution to prepare Standard 1 of 200 pg / mL, using Standard 1 to prepare Standard 2 of 100 pg / mL, and so on, 2-fold gradient dilution to prepare Standard 7 of 3.125 pg / mL, and the sample diluent is Standard 8 of 0 pg / mL; The HRP labeled solution is prepared by accurately measuring 300 mL of sample diluent into a preparation container, adding 7.5 μL of HRP labeled streptavidin to prepare a working solution with a concentration of 25 ng / mL; The preparation method of the stop solution is: accurately measure 270 mL of ultrapure water into a preparation container, add 30 mL of 10 mol / L hydrochloric acid solution to prepare a working solution with a concentration of 1 mol / L, and filter it using a filter with a membrane pore size of 0.22 μm.

8. A method for detecting lentivirus titer, characterized in that: The detection method is carried out using the kit according to any one of claims 1 to 7.

9. The detection method according to claim 8, characterized in that: The detection method comprises the following steps: S21, return all reagents to room temperature and dilute 10× wash solution to 1× wash solution; S22, returning all samples to room temperature; S23, experimental steps: Add 10 μL of lysate to the corresponding wells of the kit, then add 90 μL of standard and sample to be tested, seal the plate with a sealing film, and incubate at 37°C for 20-90 minutes; Discard the solution, add 260 μL of 1× wash solution to each well, wash the plate, and pat dry on absorbent paper; Add 100 μL of detection antibody solution to each well, seal the plate with a new sealing film, and incubate at 37°C for 30 minutes; Discard the solution, add 260 μL of 1× wash solution to each well, wash the plate, and pat dry on absorbent paper; Add 100 μL of HRP labeling solution to each well, seal the plate with a new sealing film, and incubate at 37°C for 30 minutes; Discard the solution, add 260 μL of 1× wash solution to each well, wash the plate, and pat dry on absorbent paper; Add 100 μL of TMB substrate colorimetric solution to each well, protect from light, and incubate at room temperature for 5-25 minutes; Add 100 μL of stop solution to each well to terminate the reaction; Complete OD in 15 minutes 450nm reading; S24, absorbance calculation: Calculate the average absorbance of the calibrator and sample duplicate wells; With the concentration of the calibrator as the horizontal axis and the OD value as the vertical axis, the calibration curve was generated by regression fitting using relevant software; The mean absorbance value corresponding to the sample was inserted into the calibration curve, and the lentivirus titer was calculated.

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