A conjugate and use thereof

Abstract

The present application relates to the field of biotechnology and molecular detection, and specifically provides a conjugate and application thereof, the conjugate comprising a solid carrier; and a substance specifically binding to p24 protein coupled with the solid carrier. The conjugate provided by the present application can accurately evaluate the content of p24 protein of lentivirus particles, and is not affected by the content of free p24 protein in the solution, so that accurate lentivirus titer results are obtained, which is beneficial to improve the process stability and accuracy in the production process of lentivirus.

Description

[0001] Cross-reference information

[0002] This application claims priority to Chinese patent application No. 2023108730213, filed on July 14, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the fields of biotechnology and molecular detection, specifically to a conjugate and its applications. Background Technology

[0004] Lentiviral vectors (LVs) are viral vector systems developed based on human immunodeficiency virus (HIV-1). They have many advantages, including the ability to infect both dividing and non-dividing cells, the ability to stably express exogenous genes over a long period of time, and the integration site being far from the transcription start site. Therefore, they have become the mainstream viral tool for introducing exogenous genes in gene and cell therapy.

[0005] The p24 protein, a product of the lentiviral structural gene Gag, is the most abundant marker protein in the lentiviral coat and plays a crucial role in viral packaging and maturation. Therefore, assessing the p24 protein content in lentiviral samples is currently the most commonly used method for detecting lentiviral physical titers.

[0006] However, during lentivirus production, in addition to producing p24 protein on lentivirus particles, 293 cells also release free p24 protein into the supernatant during transfection. This portion of p24 protein can account for a large portion of the total p24 protein in the viral supernatant. Traditional p24 ELISA methods cannot distinguish between p24 protein on lentivirus particles and free p24 protein. Therefore, when detecting lentivirus titers, ELISA methods often overestimate the number of lentiviruses. Furthermore, the ratio of free p24 protein to p24 protein on lentivirus particles varies greatly across different batches, processes, and samples in the lentivirus vector production system, which can also lead to misjudgments of lentivirus titers. The "Technical Guidelines for In Vitro Gene-Modified Systemic Pharmaceutical Research and Evaluation (Trial)" issued by the National Medical Products Administration in May 2022 also emphasized the need to pay attention to the impact of free p24 protein on lentivirus titer determination results. Therefore, there is an urgent need to adopt specialized technical means or methods to remove free p24 protein in order to specifically detect p24 protein on lentiviral particles.

[0007] Currently, the main method for removing free p24 protein is to add a lentiviral precipitation reagent to cause lentivirus precipitation, while the free p24 protein does not precipitate and remains in the supernatant. After centrifugation to obtain lentivirus, it is then redissolved to obtain high-purity lentivirus. Subsequently, an ELISA kit can be used to detect only the p24 protein on the lentiviral particles. However, this method is time-consuming and complex. Furthermore, due to the variety of reagents and the large differences in conditions, other impurities are often introduced into the sample, interfering with the p24 protein detection results and further affecting the accuracy of lentiviral titer determination. Summary of the Invention

[0008] In order to address the aforementioned problems and shortcomings in the prior art, the first objective of this invention is to provide a conjugate comprising: a solid support; and a substance specifically binding to p24 protein conjugated to the solid support.

[0009] In some embodiments, the solid support is a plastic product, a microplate, magnetic beads, resin, or agarose.

[0010] In some preferred embodiments, the magnetic beads are modified magnetic beads. In some embodiments, 0.2-20 mg of a substance specifically binding to p24 protein is coupled to each 1 mL of modified magnetic beads. In some preferred embodiments, 1-10 mg of a substance specifically binding to p24 protein is coupled to each 1 mL of modified magnetic beads. In some more preferred embodiments, 2-5 mg of a substance specifically binding to p24 protein is coupled to each 1 mL of modified magnetic beads.

[0011] In some embodiments, the modified magnetic beads have a particle size of 50 nm to 200 μm. In some preferred embodiments, the modified magnetic beads have a particle size of 200 nm to 150 μm. In some more preferred embodiments, the modified magnetic beads have a particle size of 1 μm to 100 μm.

[0012] In some embodiments, the substance that specifically binds to the p24 protein is selected from antibodies or nucleic acid aptamers that specifically bind to the p24 protein.

[0013] In some preferred embodiments, the antibody that specifically binds to the p24 protein is one or more of the following: mouse antibody, rabbit antibody, sheep antibody, chicken antibody, camel antibody, chimeric antibody, humanized antibody, and fully human antibody.

[0014] In some preferred embodiments, the antibody that specifically binds to the p24 protein is one or more of the following: full-length antibody, single-chain antibody, antigen-binding fragment, single-domain antibody, bispecific antibody, and multispecific antibody.

[0015] In some embodiments, the modification includes at least one of chemical modification, activation modification, and biological modification. In some preferred embodiments, the chemical modification includes at least one of carboxyl modification, amino modification, and hydroxyl modification. In some preferred embodiments, the activation modification includes at least one of toluenesulfonyl modification, epoxy modification, chloromethyl modification, N-hydroxysuccinimide modification, and cyanogen bromide modification. In some preferred embodiments, the biological modification includes at least one of streptavidin, protein A, and protein G modification.

[0016] The second objective of this invention is to provide a method for preparing a conjugate, comprising the following steps: contacting and conjugating a solid support with a substance that specifically binds to p24 protein to obtain the conjugate.

[0017] In some embodiments, the solid support is a plastic product, a microplate, magnetic beads, resin, or agarose. In some preferred embodiments, the magnetic beads are modified.

[0018] In some implementations, the reaction conditions for the contact coupling are 2-4 hours at room temperature or overnight at 4°C.

[0019] The present invention also provides the use of the above-mentioned conjugate in removing free p24 protein from lentiviral supernatant.

[0020] The present invention also provides the application of the above-mentioned conjugate in the determination of lentivirus titers.

[0021] A third objective of this invention is to provide a method for removing free p24 protein from lentiviral supernatant, comprising contacting the above-mentioned conjugate with the sample to be tested for a certain period of time to remove free p24 protein from the lentiviral supernatant of the sample to be tested.

[0022] The present invention also provides a method for determining lentivirus titers, comprising the following steps:

[0023] 1) A substance that specifically binds to the p24 protein is coupled to a solid support.

[0024] 2) Contact the sample to be tested with the solid-phase support prepared in step 1) for a certain period of time to remove free p24 protein from the lentivirus supernatant, and

[0025] 3) Determine the titer of p24 protein in lentivirus particles in the lentivirus supernatant of the sample to be tested.

[0026] In some embodiments, the substance that specifically binds to the p24 protein is selected from antibodies or nucleic acid aptamers that specifically bind to the p24 protein.

[0027] In some embodiments, the solid support is a plastic product, a microplate, magnetic beads, resin, or agarose.

[0028] In some preferred embodiments, the magnetic beads are modified.

[0029] In some embodiments, the modification includes at least one of chemical modification, activation modification, and biological modification. In some preferred embodiments, the chemical modification includes at least one of carboxyl modification, amino modification, and hydroxyl modification. In some preferred embodiments, the activation modification includes at least one of toluenesulfonyl modification, epoxy modification, chloromethyl modification, N-hydroxysuccinimide modification, and cyanogen bromide modification. In some preferred embodiments, the biological modification includes at least one of streptavidin, protein A, and protein G modification.

[0030] In some implementations, the solid-phase carrier is coupled to an antibody or nucleic acid aptamer that specifically binds to the p24 protein.

[0031] In some implementations, the contact time in step 2) is 10-240 min, preferably 15-30 min; in step 3), the titer of p24 protein of lentivirus particles in the lentivirus supernatant is determined using an ELISA kit.

[0032] The present invention also provides a kit comprising the conjugate described in any of the above claims, and a buffer solution.

[0033] In the technical solution provided by this invention, a lentiviral supernatant free of free p24 protein is obtained by coupling a highly specific p24 protein-binding antibody or nucleic acid aptamer to a solid-phase support (e.g., plastic products, microplates, magnetic beads, resin, agarose, etc.) and then thoroughly mixing and reacting it with the sample to be tested for a certain period of time. The principle is that the p24 protein on lentiviral particles is a capsid protein, which is further encapsulated by matrix proteins and an envelope. Therefore, the p24 protein on the lentiviral vector will not bind to antibodies or nucleic acid aptamers that specifically bind to p24 protein. However, the free p24 protein in the viral supernatant has no envelope barrier and can specifically bind to the anti-p24 antibody or nucleic acid aptamer coupled to the solid-phase support. After removing the free p24 protein from the lentiviral supernatant, the p24 protein detected by the p24 protein detection kit is the p24 protein on the lentiviral particle. Through the above method, this invention can more accurately and quickly assess the titer of lentivirus, which is beneficial for monitoring and release during the lentiviral production process. Detailed Implementation

[0034] The present invention will now be described more fully with reference to specific embodiments, which show some, but not all, of the embodiments of the invention. In fact, the invention can be implemented in many different forms and should not be construed as limited to the embodiments listed herein. The following definitions and methods will better define the invention and guide those skilled in the art in implementing it. Those skilled in the art to which this invention pertains can propose many modifications and other embodiments of the invention based on the description herein and the teachings of related information. Therefore, it should be understood that the invention is not limited to the specific embodiments disclosed, and modifications and other embodiments based on the teachings of the invention are also included within the scope of the appended claims.

[0035] Unless otherwise stated, the terminology used herein should be understood in accordance with the usual usage of those skilled in the art. Generally, the nomenclature and techniques used in conjunction with these are well-known and commonly used in fields such as biochemistry, immunology, enzymology, molecular and cell biology, microbiology, and genetics.

[0036] Terminology Explanation:

[0037] As used in this article, "lentiviral titer" refers to the number of lentiviral particles, i.e., the number of viruses contained in each liter of solution. Generally, a p24 protein ELISA is used for detection, which can detect all p24 proteins, thus achieving quantitative determination of viral titer. Using an ELISA reader to read the data, at least pg-level lentiviral p24 proteins can be detected. Alternatively, real-time PCR can also be used to detect lentiviral titers.

[0038] As used in this article, the "p24 protein" is a product of the lentiviral structural gene Gag. It is the most abundant marker protein in the lentiviral coat and plays an important role in viral packaging and maturation. The physical titer of lentivirus is usually determined by assessing the content of p24 protein in a lentiviral sample.

[0039] The "antibody" mentioned in this invention refers to an immunoglobulin or polypeptide fragment that can specifically bind to a target antigen, target protein, or polypeptide fragment, including monoclonal antibodies, polyclonal antibodies, IgG, IgM, etc.

[0040] The "nucleic acid aptamer" mentioned in this invention usually refers to an oligonucleotide fragment obtained from a nucleic acid molecular library using in vitro screening technology. This nucleic acid aptamer can bind to the target substance with high specificity and high selectivity.

[0041] The "p24 protein of lentivirus particles in lentivirus supernatant" mentioned in this invention refers to the p24 protein present on lentivirus particles. It is a capsid protein, which is surrounded by matrix proteins and an envelope. Therefore, the p24 protein on lentivirus particles or lentivirus vectors will not bind to antibodies or nucleic acid aptamers that specifically bind to p24 protein.

[0042] The "free p24 protein in lentiviral supernatant" described in this invention refers to the free p24 protein released into the culture medium supernatant from cultured cells during transfection in the lentiviral production process. Because this portion of free p24 protein in the viral supernatant lacks an envelope barrier, it can specifically bind to anti-p24 antibodies or nucleic acid aptamers coupled to a solid-phase carrier. This portion of p24 protein typically does not precipitate, and its proportion of total p24 protein in lentiviral supernatants varies considerably across different batches, processes, and samples.

[0043] The present invention first provides a conjugate comprising: a solid support; and a substance specifically binding to p24 protein conjugated to the solid support.

[0044] In one embodiment, the solid support is a plastic product, a microplate, magnetic beads, resin, or agarose. Based on the findings of this invention, those skilled in the art can incorporate the above-mentioned solid supports into couplings, all of which can achieve the effects of this invention.

[0045] In one embodiment, the magnetic bead is a modified magnetic bead.

[0046] In one embodiment, 0.1-20 mg of a substance specifically binding to p24 protein is coupled to each 1 mL of modified magnetic beads; preferably, 1-10 mg of a substance specifically binding to p24 protein is coupled to each 1 mL of modified magnetic beads; more preferably, 2-5 mg of a substance specifically binding to p24 protein is coupled to each 1 mL of modified magnetic beads.

[0047] For example, in one specific implementation, each 1 mL of modified magnetic beads may be coupled with 0.1 mg, 0.5 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 7 mg, 10 mg, 15 mg or 20 mg of a substance that specifically binds to the p24 protein.

[0048] In one embodiment, the modified magnetic beads have a particle size of 50nm-200μm, preferably 200nm-150μm, and more preferably 1μm-100μm.

[0049] For example, in one specific implementation, the particle size of the modified magnetic beads can be 50nm, 100nm, 200nm, 400nm, 600nm, 800nm, 1μm, 5μm, 7μm, 10μm, 30μm, 50μm, 70μm, 90μm, 100μm, 125μm, 150μm, 175μm or 200μm.

[0050] In one embodiment, the substance specifically binding to the p24 protein described in this invention is selected from antibodies or nucleic acid aptamers of the p24 protein.

[0051] Preferably, the antibody that specifically binds to the p24 protein is one or more of the following: mouse antibody, rabbit antibody, sheep antibody, chicken antibody, camel antibody, chimeric antibody, humanized antibody, and fully human antibody.

[0052] Preferably, the antibody that specifically binds to the p24 protein is one or more of the following: full-length antibody, single-chain antibody (scFv), antigen-binding fragment (Fab, Fab', and F(ab)'2), single-domain antibody, bispecific antibody, and multispecific antibody.

[0053] In one embodiment, the modification includes at least one of chemical modification, activation modification, and biological modification.

[0054] Preferably, the chemical modification includes at least one of carboxyl modification, amino modification and hydroxyl modification.

[0055] Preferably, the activation modification includes at least one of toluenesulfonyl modification, epoxy modification, chloromethyl modification, N-hydroxysuccinimide modification and cyanogen bromide modification.

[0056] Preferably, the biomodification includes at least one of streptavidin, protein A, and protein G modification.

[0057] Those skilled in the art can use common sense to select one or more combinations of the above-mentioned chemical modification, activation modification and biological modification methods to enhance the coupling effect between magnetic beads and substances that specifically bind to p24 protein.

[0058] The present invention further provides a method for preparing a conjugate, comprising the following steps: contacting and conjugating a solid support with a substance that specifically binds to p24 protein to obtain a conjugate.

[0059] In one embodiment, the solid support is a plastic product, a microplate, magnetic beads, resin, or agarose; preferably, the magnetic beads are modified.

[0060] In one embodiment, the modification includes at least one of chemical modification, activation modification, and biological modification.

[0061] Preferably, the chemical modification includes at least one of carboxyl modification, amino modification and hydroxyl modification.

[0062] Preferably, the activation modification includes at least one of toluenesulfonyl modification, epoxy modification, chloromethyl modification, N-hydroxysuccinimide modification and cyanogen bromide modification.

[0063] Preferably, the biomodification includes at least one of streptavidin, protein A, and protein G modification.

[0064] In one embodiment, the reaction conditions for the contact coupling are 2-4 hours at room temperature or overnight at 4°C.

[0065] In one embodiment, after the contact coupling is completed, the coupling reaction product can be mixed with a blocking liquid for blocking, and then the coupling compound can be obtained.

[0066] The blocking solution mentioned therein can react with the active sites on the magnetic beads to block excess sites of substances that are not specifically bound to the p24 protein. The blocking solution does not bind to the p24 protein and does not affect the performance of the substances specifically bound to the p24 protein that are coupled to the magnetic beads.

[0067] In one embodiment, the sealing reaction conditions are specifically: sealing at room temperature for 2-4 hours or sealing at 4°C overnight.

[0068] In one embodiment, the preparation steps of the conjugate provided by the present invention include: dialysis to replace antibody buffer; thorough washing of modified magnetic beads; thorough mixing of the dialyzed antibody and the washed modified magnetic beads, reacting at room temperature for 2-4 hours or at 4°C overnight; then blocking with blocking solution at room temperature for 2-4 hours or at 4°C overnight; repeatedly washing the blocked magnetic beads and adding preservation solution for storage, finally obtaining the conjugate described in the present invention.

[0069] The present invention further provides the application of the above-mentioned conjugate in removing free p24 protein from lentiviral supernatant.

[0070] The present invention further provides the application of the above-mentioned conjugate in the determination of lentivirus titers.

[0071] In practical applications, those skilled in the art can choose to apply the finished coupling compound directly, or they can choose to prepare the coupling compound according to the method of this invention and then apply it subsequently. Correspondingly, this invention provides the following two application methods.

[0072] The present invention further provides a method for removing free p24 protein from lentiviral supernatant, comprising contacting the above-mentioned conjugate with the sample to be tested for a certain period of time to remove free p24 protein from the lentiviral supernatant of the sample to be tested.

[0073] The present invention further provides a method for determining lentivirus titers, comprising the following steps:

[0074] 1) A substance that specifically binds to the p24 protein is coupled to a solid support.

[0075] 2) Contact the sample to be tested with the solid-phase support prepared in step 1) for a certain period of time to remove free p24 protein from the lentivirus supernatant, and

[0076] 3) Determine the titer of p24 protein in lentivirus particles in the lentivirus supernatant of the sample to be tested.

[0077] Preferably, the substance that specifically binds to the p24 protein is selected from antibodies or nucleic acid aptamers that specifically bind to the p24 protein.

[0078] In one embodiment, the solid support is a plastic product, a microplate, magnetic beads, resin, or agarose.

[0079] In one embodiment, the magnetic bead is modified.

[0080] In one embodiment, the plastic product refers to a plastic substance made of materials such as polystyrene that can adsorb antibodies or nucleic acid aptamers; preferably, the plastic product of the present invention includes flat plates, 96-well plates, etc., made of materials such as polystyrene.

[0081] Preferably, the modification includes at least one of chemical modification, activation modification and biological modification.

[0082] More preferably, the chemical modification includes at least one of carboxyl modification, amino modification and hydroxyl modification.

[0083] More preferably, the pre-activation modification includes at least one of toluenesulfonyl modification, epoxy modification, chloromethyl modification, N-hydroxysuccinimide modification and cyanogen bromide modification.

[0084] More preferably, the biomodification includes at least one of streptavidin, protein A, and protein G modification.

[0085] In one embodiment, in the method for determining lentivirus titers according to the present invention, the contact time in step 2) is 10-240 min, preferably 15-30 min.

[0086] For example, in one implementation, the contact time in step 2) is 10 min, 15 min, 20 min, 25 min, 30 min, 60 min, 90 min, 120 min, 150 min, 180 min, 210 min, or 240 min.

[0087] In one embodiment, the mixing speed at room temperature in step 2) is 40-60 rpm, preferably 50 rpm, to prevent the coupling material from settling.

[0088] In one embodiment, step 3) uses an ELISA kit to determine the titer of p24 protein in lentivirus particles in the lentivirus supernatant. In a specific embodiment, the ELISA kit is a Lentivirus Titerp24 ELISA kit.

[0089] A fourth object of the present invention is to provide a kit comprising the conjugate described herein and a buffer solution.

[0090] In some implementations, the kit is used to remove free p24 protein from lentiviral supernatants.

[0091] In some implementations, the kit is used to determine lentivirus titers.

[0092] In some implementations, the kit also includes reagents and buffers for detecting p24 protein concentration.

[0093] Those in the art can combine the above-mentioned implementation schemes with common sense to obtain more preferred embodiments of the present invention.

[0094] Taking a conjugate containing a p24 antibody as an example, the invention can be exemplified in a preferred embodiment as follows:

[0095] 1. Couple an antibody that specifically binds to the p24 protein to NHS-activated magnetic beads;

[0096] 2. Remove free p24 protein from lentivirus supernatant;

[0097] 3. Detect the p24 protein of lentivirus particles in the lentivirus supernatant; the p24 protein detection kit used as an example is the Lentivirus Titer p24 ELISA kit (GenScript, L00938).

[0098] The specific plan is as follows:

[0099] 1. An antibody that specifically binds to the p24 protein is conjugated to NHS-activated magnetic beads to obtain a conjugate.

[0100] (1) The buffer solution prepared in this invention includes: ① washing buffer: 1mM HCl; ② coupling buffer: PBS, pH 7.0; ③ blocking buffer: 0.1M Tris-HCl, pH 8.0; ④ washing buffer A: 0.1M NaAc, 0.5M NaCl, pH 4.0; ⑤ washing buffer B: 0.1M Tris-HCl, 0.5M NaCl, pH 8.0; ⑥ storage buffer: PBS containing 1% BSA, pH 7.4.

[0101] (2) Remove the dialysis bag and dialysis clamp, clean them with pure water, add the antibody into the dialysis bag, then place the dialysis bag into a beaker containing 50-100 times the antibody volume of PBS, place the beaker on a magnetic stirrer, and dialyze at room temperature of 18-25℃ for at least 2 hours. Then remove the antibody from the dialysis bag and detect the concentration after dialysis using Nanodrop.

[0102] (3) Place the NHS-activated magnetic beads on a magnetic rack (the particle size of the NHS-activated magnetic beads is 7μm), discard the supernatant, and wash the NHS-activated magnetic beads three times with 10 times the volume of the magnetic beads using washing buffer. Invert the beads and mix for 1 minute each time to ensure that the NHS-activated magnetic beads are thoroughly washed. Then wash the NHS-activated magnetic beads three times with 10 times the volume of coupling buffer. Invert the beads and mix for 1 minute each time to ensure that the NHS-activated magnetic beads are thoroughly washed. After washing, set aside for use.

[0103] (4) Add the dialyzed antibody to the washed magnetic beads, mix thoroughly, place the reaction flask on a plasma mixer, and rotate the reaction vessel at 50 rpm for 2-4 hours at room temperature or overnight at 4°C. Ensure that the magnetic beads do not settle during the reaction to maximize reaction efficiency.

[0104] (5) After the reaction in step (4) is completed, place the reaction bottle on the magnetic rack. After the supernatant is completely separated from the magnetic beads after the reaction, remove the supernatant and wash the magnetic beads after the reaction three times with 10 times the volume of magnetic bead blocking solution. Then add 2 times the volume of blocking solution, mix thoroughly and place on a plasma homogenizer to rotate the reaction at 50 rpm. The reaction time is 2-4 hours at room temperature or overnight at 4°C. During the reaction, ensure that the magnetic beads after the reaction do not settle.

[0105] (6) After the sealing in step (5) is completed, place the reaction bottle on the magnetic rack. After the sealing solution is completely separated from the sealed magnetic beads, use a pipette to remove the supernatant. Then wash the sealed magnetic beads with 5 times the volume of pure water, 5 times the volume of washing buffer A (pH 4.0), 5 times the volume of pure water, and 5 times the volume of washing buffer B (pH 8.0) in sequence. Repeat the above process four times and then remove the supernatant.

[0106] (7) Wash the sealed magnetic beads three times with 10 times the volume of magnetic bead preservation solution. Invert the beads and mix for 1 minute each time to ensure that the magnetic beads are thoroughly washed. After washing, add 3 times the volume of magnetic bead preservation buffer to preserve the sealed magnetic beads, thus obtaining a 25% concentration of the conjugate.

[0107] 2. Removal of free p24 protein from lentivirus supernatant

[0108] (1) Invert the reagent bottle containing the coupling agent (obtained in step 1) to mix the coupling agent thoroughly. Take a certain amount of the liquid containing the coupling agent and transfer it to a clean EP tube. Since the coupling agent settles quickly, it is necessary to remove the coupling agent immediately after mixing in this step.

[0109] (2) Place the EP tube on the magnetic rack to collect the coupling, and aspirate and discard the supernatant.

[0110] (3) Add 1 mL of PBS to the conjugate, invert and mix several times, wash thoroughly, place the conjugate on a magnetic rack to collect the conjugate, aspirate and discard the supernatant, and repeat this step three times.

[0111] (4) Add the sample containing lentivirus to the conjugate and invert the EP tube several times to mix the conjugate and the sample.

[0112] (5) Place the sample-conjugate mixture on a plasma homogenizer and rotate it at room temperature for 15-30 minutes at a speed of 40-70 rpm to ensure that the conjugate does not settle.

[0113] (6) After the reaction is complete, place the conjugate on a magnetic rack and aspirate the supernatant to obtain the lentivirus supernatant from which free p24 protein has been removed.

[0114] 3. ELISA detection of p24 protein in lentiviral particles

[0115] (1) Dilute the samples and standards using culture medium.

[0116] (2) Add 10 μL of lysis buffer and 100 μL of diluted sample / standard to the corresponding wells, mix on a rotary shaker for 30-60 s, then seal the plate with a sealing film and incubate in a 25°C constant temperature incubator for 1 h.

[0117] (3) After incubation, carefully remove the sealing film, discard the liquid in the well, add at least 260 μL of 1× washing solution to each well, let it stand and soak for 30 seconds, discard the washing solution, wash the plate 4 times in a row, and pat dry the remaining liquid on the last time.

[0118] (4) Add 100 μL of Biotin-Anti-p24 Antibody to the corresponding well, seal the plate with a sealing film, and incubate at 25 °C for 15 min.

[0119] (5) After incubation, carefully remove the sealing film, discard the liquid in the wells, add at least 260 μL of 1× washing solution to each well, let it stand and soak for 30 seconds, discard the washing solution, wash the plate 4 times in a row, and pat dry the remaining liquid on the last time.

[0120] (6) Add Streptavidin-HRP at a volume of 100 μL per well, seal the plate with sealing film, and incubate at 25°C for 10 min.

[0121] (7) After incubation, carefully remove the sealing film, discard the liquid in the wells, add at least 260 μL of 1× washing solution to each well, let it stand and soak for 30 seconds, discard the washing solution, wash the plate 4 times in a row, and pat dry the remaining liquid on the last time.

[0122] (8) Add 100 μL of TMB chromogenic solution to each well of the microplate, seal the plate with sealing film, and incubate in a 25°C constant temperature incubator for 15 min in the dark.

[0123] (9) Add 50 μL of stop solution to each well, mix gently, and then use an ELISA reader to detect the OD value of each well at a single wavelength of 450 nm.

[0124] To fully understand the purpose, features and effects of the present invention, the technical solution of the present invention is described below with reference to examples, but the scope of protection of the present invention is not limited to the following embodiments.

[0125] Unless otherwise specified, the instruments and equipment involved in the following embodiments are all conventional instruments and equipment; the raw materials, reagents or products involved are all commercially available or can be obtained according to conventional preparation methods in the art.

[0126] Example 1: Removal of p24 protein from culture medium

[0127] (1) Invert the reagent bottle containing the coupling agent (prepared from step 1 above) to mix the coupling agent, take 20 μL of the liquid containing the coupling agent, including 5 μL of pure coupling agent, and transfer it to a clean EP tube.

[0128] (2) Place the EP tube on the magnetic rack and remove the supernatant.

[0129] (3) Add 1 mL of PBS to the conjugate, invert and mix several times, wash thoroughly, place the conjugate on a magnetic rack, remove the supernatant, and repeat the washing three times.

[0130] (4) Add 500 μL of p24 protein of different concentrations diluted with DMEM + 10% FBS medium to the conjugate, and rotate it at room temperature for 15 min at 50 rpm on a plasma mixer to ensure that the conjugate does not settle.

[0131] After the reaction was completed, the conjugate was placed on a magnetic rack, the supernatant was aspirated, and the p24 protein content in the supernatant before and after conjugate removal was detected by ELISA. The specific results are shown in Table 1 below.

[0132] Table 1

[0133]

[0134] The results in Table 1 show that the present invention can effectively remove p24 protein from the culture medium, with removal efficiencies exceeding 90% for different concentrations, such as 13.19 ng / mL to 1747.15 ng / mL. In actual lentivirus production processes, the levels of free p24 protein vary significantly across different batches, processes, and samples. This product demonstrates good removal efficiency for all concentrations of free p24, thus ensuring the broad applicability of the technical solution of this invention.

[0135] Example 2: Removal of p24 protein after mixing with free p24 protein in lentiviral supernatant at a certain concentration.

[0136] (1) Invert the reagent bottle containing the coupling agent (prepared from step 1 above) to mix the coupling agent, take 40 μL of the liquid containing the coupling agent, including 10 μL of pure coupling agent, and transfer it to a clean EP tube.

[0137] (2) Place the EP tube on the magnetic rack and remove the supernatant.

[0138] (3) Add 1 mL of PBS to the conjugate, invert and mix several times, wash thoroughly, place the conjugate on a magnetic rack, remove the supernatant, and repeat the washing three times.

[0139] (4) Add different concentrations of free p24 protein to the purified lentivirus sample, which was found to have a p24 protein concentration of 1223.18 ng / mL on the lentivirus particles. Then add 500 μL of lentivirus solution containing different concentrations of free p24 protein to the conjugate. Rotate the conjugate at room temperature for 20 min at 50 rpm on a plasma mixer to ensure that the conjugate does not settle.

[0140] (5) After the reaction is complete, place the conjugate on a magnetic rack, aspirate the supernatant, and detect the total content of p24 protein in the supernatant after the conjugate is removed by ELISA. The specific results are shown in Table 2 below.

[0141] Table 2

[0142]

[0143] The above results indicate that when different concentrations of free p24 protein are mixed into a lentiviral sample of a certain concentration, Table 2 shows that before using the p24 protein ELISA kit of the present invention to remove free p24 protein, the p24 content detected directly by the p24 protein ELISA kit is the total p24 content, which includes both free p24 protein and p24 protein on lentiviral particles. The detected total p24 protein concentrations are 1515.50 ng / mL, 2033.94 ng / mL, and 4188.51 ng / mL, respectively. This result is close to the sum of the p24 protein concentration of lentiviral particles and the concentration of free p24 protein. This detected value differs greatly from the actual p24 content on lentiviral particles, ranging from 123.90% to 342.43% of the actual content of 1223.18 ng / mL. The closer the p24 protein detection value / actual p24 protein value is to 100%, the more accurate the lentivirus titer quantification. Therefore, traditional p24 protein ELISA kits significantly overestimate the actual lentivirus titer, which is detrimental to the process stability and accuracy of lentivirus production. However, after removing free p24 protein using this invention, the total p24 protein content detected by the p24 protein ELISA kit is mainly the p24 protein on the lentivirus particles. The total p24 protein content detected by the ELISA was 1212.58 ng / mL, 1144.07 ng / mL, and 1336.30 ng / mL, respectively. These values ​​are very close to the actual p24 content on the lentivirus particles (1223.18 ng / mL), representing 99.13%, 93.53%, and 109.25% of the actual content, respectively, indicating a significant improvement in accuracy.

[0144] The above results indicate that this invention can accurately assess the p24 protein content of lentiviral particles without being affected by the content of free p24 protein in the solution, thereby obtaining accurate lentiviral titer results, which is beneficial for monitoring and release during the lentiviral production process.

[0145] Example 3: Removal of free p24 protein from lentiviral samples

[0146] (1) Shake the reagent bottle to mix the conjugate (prepared from step 1 above), take 20 μL of suspension containing 5 μL of pure conjugate, and transfer it to a clean EP tube.

[0147] (2) Place the EP tube on the magnetic rack and remove the supernatant.

[0148] (3) Add 1 mL of PBS to the conjugate, invert and mix several times, wash thoroughly, place the conjugate on a magnetic rack, remove the supernatant, and repeat the washing three times.

[0149] (4) Add 500 μL of lentivirus culture supernatant to the conjugate and rotate it at room temperature for 15 min at 50 rpm on a plasma mixer to ensure that the conjugate does not settle.

[0150] (5) After the reaction is complete, place the conjugate on a magnetic rack, aspirate the supernatant, and detect the p24 protein content in the supernatant before and after conjugate removal by ELISA. The specific results are shown in Table 3 below.

[0151] Table 3

[0152]

[0153] Table 3 shows that, before adopting this invention, the p24 content detected by the traditional p24 protein ELISA kit was the total p24 content, including both free p24 protein and p24 protein on lentiviral particles. The detected total p24 protein concentrations were 82.65 ng / mL and 234.00 ng / mL, respectively. The proportion of free p24 varied among different samples, with free p24 protein accounting for 18.46% and 25.56% of the total p24 protein in the two samples, respectively. However, the actual p24 protein expression levels on lentiviral particles were 67.39 ng / mL and 174.18 ng / mL, respectively, indicating that this detection method overestimates the actual lentiviral titer. After using this invention, free p24 protein was removed, with its proportion decreasing to 0.97% and 1.43%, respectively. When the p24 protein ELISA kit was used for detection, the total p24 protein detected was mainly p24 protein from lentiviral particles. The total p24 protein concentrations detected at this time were 68.05 ng / mL and 176.70 ng / mL, respectively, which are closer to the actual p24 protein expression levels on lentiviral particles (67.39 ng / mL and 174.18 ng / mL, respectively). These results indicate that the present invention can effectively remove free p24 protein and accurately quantify lentiviral titers, which is beneficial for monitoring and release during lentiviral production.

[0154] Therefore, it can be seen that the present invention has significant beneficial effects:

[0155] 1. This invention can effectively remove free p24 protein from lentivirus supernatant. In particular, when there are large differences in the level of free p24 protein in different batches, different processes, and different samples, this invention can still maintain a good removal effect of more than 90% for different concentrations of free p24 protein, ensuring that the technical solution of this invention has a wide range of applications.

[0156] 2. This invention significantly improves the accuracy of detecting the p24 protein content of lentivirus particles in actual lentivirus supernatant. It can accurately assess the p24 protein content of lentivirus particles without being affected by the content of free p24 protein in the solution, thereby obtaining accurate lentivirus titer results, which is beneficial to improving the process stability and accuracy in lentivirus production.

[0157] 3. The measurement results obtained by using the technical solution of this invention are closer to the actual expression level of p24 protein on lentivirus particles, which can quickly and accurately quantify the titer of lentivirus, which is beneficial for monitoring and release during the lentivirus production process.

[0158] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. A method for determining lentivirus titers, characterized in that, Includes the following steps: 1) A substance that specifically binds to the p24 protein is coupled to a solid support. 2) Contact the sample to be tested with the solid-phase support prepared in step 1) for a certain period of time to remove free p24 protein from the lentivirus supernatant, and 3) Determine the titer of p24 protein in lentivirus particles in the lentivirus supernatant of the sample to be tested.

2. The method according to claim 1, wherein the substance that specifically binds to the p24 protein is selected from antibodies or nucleic acid aptamers that specifically bind to the p24 protein.

3. The method according to claim 2, wherein the antibody that specifically binds to the p24 protein is one or more of the following: mouse antibody, rabbit antibody, sheep antibody, chicken antibody, camel antibody, chimeric antibody, humanized antibody, and fully human antibody.

4. The method according to claim 2, wherein the antibody that specifically binds to the p24 protein is one or more of the following: full-length antibody, single-chain antibody, antigen-binding fragment, single-domain antibody, bispecific antibody, and multispecific antibody.

5. The method according to claim 1, wherein, The solid support is a plastic product, microplate, magnetic beads, resin or agarose.

6. The method of claim 5, wherein the magnetic bead is modified.

7. The method according to claim 6, wherein, Each 1 mL of modified magnetic beads is coupled with 0.2-20 mg of a substance that specifically binds to the p24 protein.

8. The method according to claim 6, wherein, Each 1 mL of modified magnetic beads is coupled with 1-10 mg of a substance that specifically binds to the p24 protein.

9. The method according to claim 6, wherein, Each 1 mL of modified magnetic beads is coupled with 2-5 mg of a substance that specifically binds to the p24 protein.

10. The method according to claim 6, wherein the modified magnetic beads have a particle size of 50 nm to 200 μm.

11. The method according to claim 6, wherein the modified magnetic beads have a particle size of 200 nm-150 μm.

12. The method according to claim 6, wherein the modified magnetic beads have a particle size of 1 μm-100 μm.

13. The method according to claim 6, wherein, The modification includes at least one of chemical modification, activation modification and biological modification.

14. The method according to claim 13, wherein, The chemical modification includes at least one of carboxyl modification, amino modification, and hydroxyl modification; the activation modification includes at least one of toluenesulfonyl modification, epoxy modification, chloromethyl modification, N-hydroxysuccinimide modification, and cyanogen bromide modification; the biological modification includes at least one of streptavidin, protein A, and protein G modification.

15. The method according to claim 1, wherein, Step 1) includes the following steps: contacting and coupling a solid support with a substance that specifically binds to the p24 protein to obtain a conjugate.

16. The method according to claim 15, wherein the reaction conditions for contact coupling are 2-4 hours at room temperature or overnight at 4°C.

17. The method according to claim 1, wherein, The contact time in step 2) is 10-240 min; in step 3), the titer of p24 protein of lentivirus particles in lentivirus supernatant is determined using an ELISA kit.

18. The method according to claim 1, wherein, The contact time in step 2) is 15-30 minutes.

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