Conjugate and application thereof

Abstract

The invention relates to the field of biotechnology and molecular detection, and particularly provides a conjugate and application thereof, the conjugate comprises a solid phase carrier; the substance is coupled with the solid phase carrier and specifically binds to the p24 protein. The conjugate provided by the invention can accurately evaluate the p24 protein content of lentivirus particles without being influenced by the content of free p24 protein in a solution, so that an accurate lentivirus titer result is obtained, and the process stability and accuracy in the lentivirus production process are improved.

Description

A conjugate and its application

[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] The present invention relates to the fields of biotechnology and molecular detection, and in particular to a conjugate and applications thereof. Background Art

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

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

[0006] However, during the lentiviral production process, in addition to producing p24 protein on lentiviral particles, 293 cells during transfection also release free p24 protein into the supernatant. This part of p24 protein can account for a large part of the total p24 protein in the viral supernatant. The traditional p24 ELISA method cannot distinguish between p24 protein on lentiviral particles and free p24 protein. Therefore, when testing lentiviral titers, the ELISA method usually overestimates the amount of lentivirus. In addition, since the ratio of free p24 protein to p24 protein on lentiviral particles varies greatly in different batches, different processes, and different samples of the lentiviral vector production system, it can also cause misjudgment of lentiviral titers. The "Technical Guidelines for Pharmaceutical Research and Evaluation of In Vitro Gene Modification Systems (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 lentiviral 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 the p24 protein on lentiviral particles.

[0007] Currently, the primary method for removing free p24 protein is to precipitate the lentivirus by adding a lentiviral precipitation reagent. However, the free p24 protein does not precipitate and remains in the supernatant. After centrifugation to obtain the lentivirus, the lentivirus is re-dissolved to obtain a high-purity lentivirus. Subsequently, an ELISA kit can be used to detect only the p24 protein present on the lentiviral particles. However, this method is time-consuming and complex. Furthermore, due to the wide variety of reagents and widely varying conditions involved, other impurities are often introduced into the sample, interfering with the p24 protein detection results and further affecting the accuracy of lentiviral titer determinations.

[0008] Summary of the Invention

[0009] In order to solve the above problems and deficiencies in the prior art, the first object of the present invention is to provide a conjugate, which comprises: a solid phase carrier; and a substance that specifically binds to the p24 protein and is coupled to the solid phase carrier.

[0010] In some embodiments, the solid support is plastic, microplate, magnetic beads, resin or agarose.

[0011] In some preferred embodiments, the magnetic beads are modified magnetic beads. In some embodiments, 0.2-20 mg of a substance that specifically binds to the p24 protein is coupled to each 1 mL of the modified magnetic beads. In some preferred embodiments, 1-10 mg of a substance that specifically binds to the p24 protein is coupled to each 1 mL of the modified magnetic beads. In some more preferred embodiments, 2-5 mg of a substance that specifically binds to the p24 protein is coupled to each 1 mL of the modified magnetic beads.

[0012] 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.

[0013] In some embodiments, the substance that specifically binds to the p24 protein is selected from an antibody or a nucleic acid aptamer that specifically binds to the p24 protein.

[0014] In some preferred embodiments, the antibody that specifically binds to the p24 protein is one or more of a mouse antibody, a rabbit antibody, a sheep antibody, a chicken antibody, a camelid antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.

[0015] In some preferred embodiments, the antibody that specifically binds to the p24 protein is one or more of a full-length antibody, a single-chain antibody, an antigen-binding fragment, a single-domain antibody, a bispecific antibody, and a multispecific antibody.

[0016] In some embodiments, the modification comprises at least one of chemical modification, activation modification, and biological modification. In some preferred embodiments, the chemical modification comprises at least one of carboxyl modification, amino modification, and hydroxyl modification. In some preferred embodiments, the activation modification comprises at least one of tosyl modification, epoxy modification, chloromethyl modification, N-hydroxysuccinimide modification, and cyanogen bromide modification. In some preferred embodiments, the biological modification comprises at least one of streptavidin, protein A, and protein G modification.

[0017] The second object of the present invention is to provide a method for preparing a conjugate, comprising the following steps: contacting and coupling a solid phase carrier with a substance that specifically binds to the p24 protein, thereby obtaining a conjugate.

[0018] In some embodiments, the solid phase carrier is a plastic product, a microplate, a magnetic bead, a resin or agarose. In some preferred embodiments, the magnetic bead is modified.

[0019] In some embodiments, the contact coupling reaction conditions are room temperature for 2-4 hours or 4°C overnight.

[0020] The present invention also provides use of the conjugate in removing free p24 protein in lentiviral supernatant.

[0021] The present invention also provides use of the conjugate in determining the titer of lentivirus.

[0022] The third object of the present invention is to provide a method for removing free p24 protein from a lentiviral supernatant, comprising contacting the above-mentioned conjugate with a sample to be tested for a certain period of time to remove the free p24 protein from the lentiviral supernatant of the sample to be tested.

[0023] The present invention also provides a method for determining the titer of a lentivirus, comprising the following steps:

[0024] 1) coupling a substance that specifically binds to p24 protein to a solid phase carrier,

[0025] 2) contacting the sample to be tested with the solid phase carrier prepared in step 1) for a certain period of time to remove free p24 protein in the lentiviral supernatant, and

[0026] 3) Determining the titer of the p24 protein in the lentiviral supernatant of the test sample.

[0027] In some embodiments, the substance that specifically binds to the p24 protein is selected from an antibody or a nucleic acid aptamer that specifically binds to the p24 protein.

[0028] In some embodiments, the solid support is plastic, microplate, magnetic beads, resin or agarose.

[0029] In some preferred embodiments, the magnetic beads are modified.

[0030] In some embodiments, the modification comprises at least one of chemical modification, activation modification, and biological modification. In some preferred embodiments, the chemical modification comprises at least one of carboxyl modification, amino modification, and hydroxyl modification. In some preferred embodiments, the activation modification comprises at least one of tosyl modification, epoxy modification, chloromethyl modification, N-hydroxysuccinimide modification, and cyanogen bromide modification. In some preferred embodiments, the biological modification comprises at least one of streptavidin, protein A, and protein G modification.

[0031] In some embodiments, the solid phase carrier is coupled to an antibody or nucleic acid aptamer that specifically binds to the p24 protein.

[0032] In some embodiments, the contact time in step 2) is 10-240 min, preferably 15-30 min; and in step 3), the titer of the lentiviral particle p24 protein in the lentiviral supernatant is determined using an ELISA kit.

[0033] The present invention also provides a kit comprising any one of the conjugates described above and a buffer solution.

[0034] In the technical solution provided by the present invention, by coupling an antibody or nucleic acid aptamer that binds to the p24 protein with high specificity to a solid phase carrier (e.g., plastic products, microplates, magnetic beads, resins, agarose, etc.) and then fully mixing with the sample to be tested and reacting for a certain period of time, a lentiviral supernatant with free p24 protein removed can be obtained. The principle is that the p24 protein on the lentiviral particles is a capsid protein, and the capsid protein is also wrapped by matrix protein and an envelope. Therefore, the p24 protein on the lentiviral vector will not bind to the antibody or nucleic acid aptamer that specifically binds to the p24 protein, while the free p24 protein in the viral supernatant is not blocked by the envelope and can specifically bind to the anti-p24 antibody or nucleic acid aptamer coupled to the solid phase carrier. After removing the free p24 protein in the lentiviral supernatant, the p24 protein detected by the p24 protein detection kit is the p24 protein on the lentiviral particles. Through the above method, the present invention can more accurately and quickly judge the titer of the lentivirus, which is beneficial to the monitoring and release of the lentiviral production process. DETAILED DESCRIPTION

[0035] The present invention will be described more completely below with reference to specific embodiments, in which some but not all embodiments of the present invention are shown. In fact, the present invention can be implemented in many different forms and should not be construed as being limited to the embodiments listed herein. The following definitions and methods will better define the present invention and guide those of ordinary skill in the art to implement the present invention. Those skilled in the art can propose many modifications and other embodiments of the present invention based on the description herein and the teachings of relevant information. Therefore, it should be understood that the present invention is not limited to the disclosed specific embodiments, and that modifications and other embodiments proposed based on the teachings of the present invention are also included within the scope of the appended claims.

[0036] Unless otherwise stated, the terms used herein should be understood according to conventional usage by those of ordinary skill in the art. Generally, the nomenclature used in conjunction with the following and the following techniques are well-known and commonly used in the fields of biochemistry, immunology, enzymology, molecular and cell biology, microbiology and genetics.

[0037] Explanation of terms:

[0038] As used herein, "lentiviral titer" refers to the number of lentiviral particles, i.e., the number of viruses per liter of solution. A p24 protein ELISA is commonly used to quantify viral titer, detecting all p24 proteins. Microplate reader readings can detect at least pg levels of lentiviral p24 protein. Real-time PCR can also be used to determine lentiviral titer.

[0039] As used in this article, "p24 protein" is the product of the lentiviral structural gene Gag. It is the most abundant marker protein in the lentiviral capsid and plays an important role in the packaging and maturation of the virus. The physical titer of the lentivirus is usually determined by evaluating the content of p24 protein in the lentiviral sample.

[0040] The "antibody" mentioned in the present 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.

[0041] The "nucleic acid aptamer" mentioned in the present invention generally refers to an oligonucleotide fragment obtained from a nucleic acid molecule library using in vitro screening technology. The nucleic acid aptamer can bind to the target substance with high specificity and selectivity.

[0042] The "lentiviral particle p24 protein in the lentiviral supernatant" mentioned in the present invention refers to the p24 protein present on the lentiviral particles, which is a capsid protein. The capsid protein is wrapped by a matrix protein and an envelope. Therefore, the p24 protein on the lentiviral particles or lentiviral vectors will not bind to antibodies or nucleic acid aptamers that specifically bind to the p24 protein.

[0043] The term "free p24 protein in the lentiviral supernatant" as used herein refers to the free p24 protein released into the culture supernatant during transfection of cultured cells during the lentiviral production process. Because this free p24 protein in the viral supernatant is not blocked by the viral envelope, it can specifically bind to anti-p24 antibodies or nucleic acid aptamers coupled to a solid support. This free p24 protein typically does not sediment, and its proportion in the total p24 protein in the lentiviral supernatant can vary significantly between different batches, processes, and samples.

[0044] The present invention first provides a conjugate, which comprises: a solid phase carrier; and a substance that specifically binds to p24 protein and is coupled to the solid phase carrier.

[0045] In one embodiment, the solid phase carrier is a plastic product, a microplate, a magnetic bead, a resin or agarose. Using the findings of the present invention, those skilled in the art can arrange the above solid phase carrier in the conjugate, which can achieve the effects of the present invention.

[0046] In one embodiment, the magnetic beads are modified magnetic beads.

[0047] In one embodiment, 0.1-20 mg of a substance that specifically binds to p24 protein is coupled to each 1 mL of modified magnetic beads; preferably, 1-10 mg of a substance that specifically binds to p24 protein is coupled to each 1 mL of modified magnetic beads; more preferably, 2-5 mg of a substance that specifically binds to p24 protein is coupled to each 1 mL of modified magnetic beads.

[0048] For example, in a specific embodiment, each 1 mL of modified magnetic beads can 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 p24 protein.

[0049] In one embodiment, the modified magnetic beads have a particle size of 50 nm to 200 μm, preferably 200 nm to 150 μm, and more preferably 1 μm to 100 μm.

[0050] For example, in a specific embodiment, 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.

[0051] In one embodiment, the substance that specifically binds to the p24 protein of the present invention is selected from an antibody or a nucleic acid aptamer of the p24 protein.

[0052] Preferably, the antibody that specifically binds to the p24 protein is one or more of a mouse antibody, a rabbit antibody, a sheep antibody, a chicken antibody, a camelid antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.

[0053] Preferably, the antibody that specifically binds to p24 protein is one or more of a full-length antibody, a single-chain antibody (scFv), an antigen-binding fragment (Fab, Fab' and F(ab)'2), a single-domain antibody, a bispecific antibody, and a multispecific antibody.

[0054] In one embodiment, the modification comprises at least one of chemical modification, activation modification, and biological modification.

[0055] Preferably, the chemical modification includes at least one of carboxyl modification, amino modification and hydroxyl modification.

[0056] Preferably, the activation modification includes at least one of tosyl modification, epoxy modification, chloromethyl modification, N-hydroxysuccinimide modification and cyanogen bromide modification.

[0057] Preferably, the biological modification comprises at least one of streptavidin, protein A and protein G modification.

[0058] Those skilled in the art can select one or more combinations of the above-mentioned chemical modification, activation modification and biological modification methods based on common sense to enhance the coupling effect between the magnetic beads and the substance that specifically binds to the p24 protein.

[0059] The present invention further provides a method for preparing a conjugate, comprising the following steps: contacting and coupling a solid phase carrier with a substance that specifically binds to the p24 protein, thereby obtaining the conjugate.

[0060] In one embodiment, the solid phase carrier is a plastic product, a microplate, a magnetic bead, a resin or agarose; preferably, the magnetic bead is modified.

[0061] In one embodiment, the modification comprises at least one of chemical modification, activation modification, and biological modification.

[0062] Preferably, the chemical modification includes at least one of carboxyl modification, amino modification and hydroxyl modification.

[0063] Preferably, the activation modification includes at least one of tosyl modification, epoxy modification, chloromethyl modification, N-hydroxysuccinimide modification and cyanogen bromide modification.

[0064] Preferably, the biological modification comprises at least one of streptavidin, protein A and protein G modification.

[0065] In one embodiment, the contact coupling reaction conditions are room temperature for 2-4 hours or 4°C overnight.

[0066] In one embodiment, after the contact coupling is completed, the coupling reaction product can be mixed with a blocking solution to perform blocking, and then the conjugate is obtained.

[0067] The blocking solution mentioned herein can react with the active sites on the magnetic beads to block the redundant sites that are not coupled to the substance specifically binding to the p24 protein. The blocking solution does not bind to the p24 protein and does not affect the performance of the substance specifically binding to the p24 protein coupled to the magnetic beads.

[0068] In one embodiment, the blocking reaction conditions are as follows: blocking at room temperature for 2-4 hours or blocking at 4°C overnight.

[0069] In one embodiment, the preparation steps of the conjugate provided by the present invention include: dialysis to replace the antibody buffer; thoroughly washing the modified magnetic beads; thoroughly mixing 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 a blocking solution at room temperature for 2-4 hours or at 4°C overnight; repeatedly washing the blocked magnetic beads and then adding a preservation solution for storage, to finally obtain the conjugate of the present invention.

[0070] The present invention further provides the use of the conjugate in removing free p24 protein from lentiviral supernatant.

[0071] The present invention further provides use of the conjugate in determining the titer of lentivirus.

[0072] In specific applications, those skilled in the art may choose to directly apply the finished conjugate, or may choose to prepare the conjugate according to the method of the present invention and then apply it subsequently. Accordingly, the present invention provides the following two application methods.

[0073] The present invention further provides a method for removing free p24 protein from a lentiviral supernatant, comprising contacting the conjugate with a sample to be tested for a certain period of time to remove the free p24 protein from the lentiviral supernatant of the sample to be tested.

[0074] The present invention further provides a method for determining the titer of a lentivirus, comprising the following steps:

[0075] 1) coupling a substance that specifically binds to p24 protein to a solid phase carrier,

[0076] 2) contacting the sample to be tested with the solid phase carrier prepared in step 1) for a certain period of time to remove free p24 protein in the lentiviral supernatant, and

[0077] 3) Determining the titer of the p24 protein in the lentiviral supernatant of the test sample.

[0078] Preferably, the substance that specifically binds to the p24 protein is selected from an antibody or a nucleic acid aptamer that specifically binds to the p24 protein.

[0079] In one embodiment, the solid support is plastic, microplate, magnetic beads, resin or agarose.

[0080] In one embodiment, the magnetic beads are modified.

[0081] In one embodiment, the plastic product refers to a plastic substance made of polystyrene or other materials that can adsorb antibodies or nucleic acid aptamers; preferably, the plastic product of the present invention includes a flat plate, a 96-well plate, etc. made of polystyrene or other materials.

[0082] Preferably, the modification includes at least one of chemical modification, activation modification and biological modification.

[0083] More preferably, the chemical modification includes at least one of carboxyl modification, amino modification and hydroxyl modification.

[0084] More preferably, the pre-activation modification includes at least one of tosyl modification, epoxy modification, chloromethyl modification, N-hydroxysuccinimide modification and cyanogen bromide modification.

[0085] More preferably, the biological modification comprises at least one of streptavidin, protein A and protein G modification.

[0086] In one embodiment, in the method for determining the titer of a lentivirus according to the present invention, the contacting time in step 2) is 10-240 min, preferably 15-30 min.

[0087] For example, in one embodiment, the contacting 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.

[0088] In one embodiment, the mixing speed at room temperature in step 2) is 40 to 60 rpm, preferably 50 rpm, to prevent the conjugate from settling.

[0089] In one embodiment, in step 3), an ELISA kit is used to determine the titer of the lentivirus p24 protein in the lentivirus supernatant. In a specific embodiment, the ELISA kit is a Lentivirus Titer p24 ELISA kit.

[0090] A fourth object of the present invention is to provide a kit comprising the conjugate of the present invention and a buffer solution.

[0091] In some embodiments, the kit is used to remove free p24 protein from lentiviral supernatant.

[0092] In some embodiments, the kit is used to determine the titer of a lentivirus.

[0093] In some embodiments, the kit further comprises reagents and buffer for detecting the concentration of p24 protein.

[0094] Those skilled in the art can combine the above-mentioned implementation schemes with common sense to obtain better embodiments of the present invention.

[0095] Taking the conjugate of p24 antibody as an example for illustrative description, in a preferred embodiment, the technical solution of the present invention can be divided into the following three parts:

[0096] 1. Couple the antibody that specifically binds to p24 protein to NHS-activated magnetic beads;

[0097] 2. Remove free p24 protein from lentiviral supernatant;

[0098] 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).

[0099] The specific plan is as follows:

[0100] 1. Conjugate the antibody that specifically binds to p24 protein to NHS-activated magnetic beads to obtain a conjugate.

[0101] (1) The buffers configured in the present invention include: ① Wash buffer: 1 mM HCl; ② Coupling buffer: PBS, pH 7.0; ③ Blocking buffer: 0.1 M Tris-HCl, pH 8.0; ④ Wash buffer A: 0.1 M NaAc, 0.5 M NaCl, pH 4.0; ⑤ Wash buffer B: 0.1 M Tris-HCl, 0.5 M NaCl, pH 8.0; ⑥ Storage buffer: PBS containing 1% BSA, pH 7.4.

[0102] (2) Remove the dialysis bag and dialysis clamp, rinse them with pure water, add the antibody to the dialysis bag, and then place the dialysis bag in a beaker filled with PBS 50-100 times the volume of the antibody. Place the beaker on a magnetic stirrer and dialyze at room temperature of 18-25°C for at least 2 hours. Then remove the antibody from the dialysis bag and detect the concentration after dialysis using Nanodrop.

[0103] (3) Place the NHS-activated magnetic beads on a magnetic rack (the particle size of the magnetic beads after NHS activation is 7 μm), discard the supernatant, and use 10 times the volume of the magnetic beads to wash the NHS-activated magnetic beads three times. Mix them upside down for 1 minute each time to ensure that the NHS-activated magnetic beads are fully washed. Then use 10 times the volume of the magnetic beads to wash the NHS-activated magnetic beads three times. Mix them upside down for 1 minute each time to ensure that the NHS-activated magnetic beads are fully washed. After washing, set aside.

[0104] (4) Add the dialyzed antibody to the washed magnetic beads. After thorough mixing, place the reaction bottle on a plasma mixer and rotate the reaction 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.

[0105] (5) After the reaction of step (4) is completed, the reaction bottle is placed on a magnetic rack. After the supernatant and the reacted magnetic beads are completely separated, the supernatant is discarded, and the reacted magnetic beads are washed three times with a magnetic bead blocking solution of 10 times the volume of the magnetic beads. Then, 2 times the volume of the blocking solution is added, the mixture is thoroughly mixed, and the reaction is placed on a plasma mixer for rotation reaction at a speed of 50 rpm. The reaction time is 2-4 hours at room temperature or overnight at 4°C. During the reaction, ensure that the reacted magnetic beads do not settle.

[0106] (6) After the blocking step (5) is completed, the reaction bottle is placed on a magnetic rack. After the blocking solution and the blocked magnetic beads are completely separated, the supernatant is discarded using a pipette. The blocked magnetic beads are then washed with 5 volumes of pure water, 5 volumes of washing buffer A (pH 4.0), 5 volumes of pure water, and 5 volumes of washing buffer B (pH 8.0). The above process is repeated four times, and the supernatant is discarded.

[0107] (7) Wash the blocked magnetic beads three times with 10 times the volume of magnetic bead storage solution. Mix by inverting for 1 minute each time to ensure that the magnetic beads are fully washed. After washing, add 3 times the volume of magnetic bead storage buffer to store the blocked magnetic beads to obtain a 25% concentration of conjugate.

[0108] 2. Removal of free p24 protein from lentiviral supernatant

[0109] (1) Turn the reagent bottle containing the conjugate (prepared in step 1) upside down to mix the conjugate. Pipette a certain amount of the liquid containing the conjugate into a clean EP tube. Since the conjugate settles quickly, the conjugate should be aspirated immediately after mixing.

[0110] (2) Place the EP tube on a magnetic rack to collect the conjugate, aspirate and discard the supernatant.

[0111] (3) Add 1 mL of PBS to the conjugate and mix thoroughly by inverting the tube several times. After washing thoroughly, place the conjugate on a magnetic rack to collect the conjugate. Aspirate and discard the supernatant. Repeat this step three times.

[0112] (4) Add the sample containing the lentivirus to the conjugate and invert the EP tube several times to mix the conjugate and sample.

[0113] (5) Place the sample-conjugate mixture on a plasma mixer 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.

[0114] (6) After the reaction is completed, the conjugate is placed on a magnetic rack and the supernatant is aspirated to obtain the lentiviral supernatant from which the free p24 protein has been removed.

[0115] 3. ELISA detection of lentiviral particle p24 protein

[0116] (1) Dilute samples and standards in culture medium.

[0117] (2) Add 10 μL of lysate and 100 μL of diluted sample / standard to the corresponding wells and mix on a rotary shaker for 30-60 seconds. Then seal the plate with a sealing film and incubate in a 25°C constant temperature incubator for 1 hour.

[0118] (3) After incubation, carefully remove the sealing film and discard the liquid in the wells. Add at least 260 μL of 1× washing solution to each well and let it soak for 30 seconds. Discard the washing solution and wash the plate four times in succession. Pat dry the remaining liquid for the last time.

[0119] (4) Add 100 μL of Biotin-Anti-p24 Antibody to the corresponding wells, seal the plate with a sealing film, and incubate at 25°C for 15 min.

[0120] (5) After incubation, carefully remove the sealing film and discard the liquid in the wells. Add at least 260 μL of 1× washing solution to each well and let it soak for 30 seconds. Discard the washing solution and wash the plate four times in succession. Pat dry the remaining liquid for the last time.

[0121] (6) Add 100 μL of Streptavidin-HRP to each well, seal the plate with a sealing film, and incubate in a 25°C constant temperature incubator for 10 min.

[0122] (7) After incubation, carefully remove the sealing film and discard the liquid in the wells. Add at least 260 μL of 1× washing solution to each well and let it soak for 30 seconds. Discard the washing solution and wash the plate four times in succession. Pat dry the remaining liquid for the last time.

[0123] (8) Add 100 μL of TMB colorimetric solution to each well of the ELISA plate, seal the plate with a sealing film, and incubate in a 25°C constant temperature incubator in the dark for 15 min.

[0124] (9) Add 50 μL of stop solution to each well, mix gently, and then use a microplate reader to measure the OD value of each well at a single wavelength of 450 nm.

[0125] In order to fully understand the purpose, features and effects of the present invention, the technical solutions of the present invention are described below with reference to examples, but the protection scope of the present invention is not limited to the following embodiments.

[0126] Unless otherwise specified, the instruments and equipment involved in the following examples are all conventional instruments and equipment; the raw materials, reagents or products involved can be purchased commercially or obtained according to conventional preparation methods in the art.

[0127] Example 1: Removal of p24 protein from culture medium

[0128] (1) Turn the reagent bottle containing the conjugate (prepared in step 1 above) upside down to mix the conjugate. Pipette 20 μL of the liquid containing the conjugate, including 5 μL of pure conjugate, and transfer it to a clean EP tube.

[0129] (2) Place the EP tube on a magnetic rack and discard the supernatant.

[0130] (3) Add 1 mL of PBS to the conjugate, mix well by inverting the tube several times, and place the conjugate on a magnetic rack after thorough washing. Aspirate and discard the supernatant, and repeat the washing process three times.

[0131] (4) Add 500 μL of p24 protein of different concentrations diluted with DMEM+10% FBS medium to the conjugate, and rotate the mixture on a plasma mixer at room temperature for 15 minutes at a speed of 50 rpm to ensure that the conjugate does not settle.

[0132] 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 the conjugate was removed was detected by ELISA. The specific results are shown in Table 1 below.

[0133] Table 1

[0134] The results in Table 1 demonstrate that the present invention effectively removes p24 protein from culture medium, achieving a removal efficiency exceeding 90% for various concentrations, such as 13.19 ng / mL to 1747.15 ng / mL. In actual lentiviral production processes, the levels of free p24 protein vary significantly across batches, processes, and samples. However, this product demonstrates effective removal of free p24 at all concentrations, ensuring the broad applicability of the present invention.

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

[0136] (1) Turn the reagent bottle containing the conjugate (prepared in step 1 above) upside down to mix the conjugate. Pipette 40 μL of the liquid containing the conjugate, including 10 μL of pure conjugate, and transfer it to a clean EP tube.

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

[0138] (3) Add 1 mL of PBS to the conjugate, mix well by inverting the tube several times, and place the conjugate on a magnetic rack after thorough washing. Aspirate and discard the supernatant, and repeat the washing process three times.

[0139] (4) Different concentrations of free p24 protein were added to the purified lentiviral sample with a detected p24 protein concentration of 1223.18 ng / mL on the lentiviral particles. Subsequently, 500 μL of lentiviral solution containing different concentrations of free p24 protein was added to the conjugate. The reaction was rotated on a plasma mixer at room temperature for 20 min at a speed of 50 rpm to ensure that the conjugate did not settle.

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

[0141] Table 2

[0142] The above results indicate that when different concentrations of free p24 protein are mixed into a certain concentration of lentiviral sample, Table 2 shows that before using the present invention to remove free p24 protein, the p24 content detected directly using the p24 protein ELISA detection kit is the total p24 content, which includes 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 the lentiviral particles and the free p24 protein concentration. The detected values ​​are very different from the actual p24 content on the lentiviral particles, ranging from 123.90% to 342.43% of the actual content of 1223.18 ng / mL, respectively. The closer the p24 protein detection value / actual p24 protein value is to 100%, the more accurate the quantitative determination of the lentiviral titer is. This shows that traditional p24 protein ELISA detection kits will greatly overestimate the actual lentiviral titer, which is not conducive to the process stability and accuracy of the lentiviral production process. After using the present invention to remove free p24 protein, since the free p24 protein is removed, the total p24 protein content detected by the p24 protein ELISA detection kit is mainly the p24 protein on the lentiviral particles. The total p24 protein content detected by ELISA was 1212.58 ng / mL, 1144.07 ng / mL, and 1336.30 ng / mL, respectively. This detection value is quite close to the actual p24 content of 1223.18 ng / mL on the lentiviral particles, which is 99.13%, 93.53%, and 109.25% of the actual content, respectively, and the accuracy is significantly improved.

[0143] The above results indicate that the invention can accurately assess the content of p24 protein in lentiviral particles and is not 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.

[0144] Example 3: Removal of free p24 protein from lentiviral samples

[0145] (1) Shake the reagent bottle to mix the conjugate (prepared in step 1 above). Pipette 20 μL of the suspension, which contains 5 μL of pure conjugate, and transfer it to a clean EP tube.

[0146] (2) Place the EP tube on a magnetic rack and discard the supernatant.

[0147] (3) Add 1 mL of PBS to the conjugate, mix well by inverting the tube several times, and place the conjugate on a magnetic rack after thorough washing. Aspirate and discard the supernatant, and repeat the washing process three times.

[0148] (4) Add 500 μL of lentiviral culture supernatant to the conjugate and rotate the mixture on a plasma mixer at room temperature for 15 min at 50 rpm to ensure that the conjugate does not settle.

[0149] (5) After the reaction is completed, the conjugate is placed on a magnetic rack, the supernatant is aspirated, and the p24 protein content in the supernatant before and after the conjugate is removed is detected by ELISA. The specific results are shown in Table 3 below.

[0150] Table 3

[0151] The results in Table 3 show that before the present invention was used, the p24 content detected directly using the traditional p24 protein ELISA detection kit was the total p24 content, which included free p24 protein and p24 protein on lentiviral particles. The total p24 protein concentrations detected were 82.65 ng / mL and 234.00 ng / mL, respectively. The proportion of free p24 in different samples was different. The proportion of free p24 protein to total p24 protein in the two samples was 18.46% and 25.56%, respectively, while 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 the present invention, the free p24 protein was removed, and its proportion dropped to 0.97% and 1.43%, respectively. At this time, the total p24 protein detected by the p24 protein ELISA detection kit was mainly lentiviral particle p24 protein. The total p24 protein concentrations detected 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 demonstrate that the present invention can effectively remove free p24 protein and accurately quantify lentiviral titers, facilitating monitoring and release during lentiviral production.

[0152] It can be seen that the present invention has significant beneficial effects:

[0153] 1. The present invention can effectively remove free p24 protein in lentiviral supernatant. In particular, when the levels of free p24 protein vary widely among different batches, different processes, and different samples, the present invention can still maintain a good removal effect of over 90% for free p24 protein of different concentrations, ensuring that the technical solution of the present invention has a wide range of applications.

[0154] 2. The present invention significantly improves the detection accuracy of the p24 protein content of lentiviral particles in actual lentiviral supernatant, and can accurately assess the p24 protein content of lentiviral particles without being affected by the free p24 protein content in the solution, thereby obtaining accurate lentiviral titer results, which is beneficial to improving the process stability and accuracy during the lentiviral production process.

[0155] 3. The measurement results obtained using the technical solution of the present invention are closer to the actual expression level of the p24 protein on the lentiviral particles, and can quickly and accurately quantify the titer of the lentivirus, which is beneficial for the monitoring and release of the lentivirus production process.

[0156] The above-described embodiments merely represent several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art may make various modifications and improvements without departing from the spirit of the present invention, and these modifications and improvements fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be based on the appended claims, and the description may be used to interpret the content of the claims.

Claims

1. A conjugate, characterized in that: The conjugate comprises: a solid support; and A substance that specifically binds to the p24 protein and is coupled to the solid phase carrier.

2. The conjugate according to claim 1, wherein the solid phase carrier is a plastic product, a microplate, a magnetic bead, a resin or agarose.

3. The conjugate according to claim 2, wherein the magnetic beads are modified magnetic beads.

4. The conjugate according to claim 3, wherein 0.2-20 mg of the substance that specifically binds to the p24 protein is coupled to each 1 mL of the modified magnetic beads; preferably, 1-10 mg of the substance that specifically binds to the p24 protein is coupled to each 1 mL of the modified magnetic beads; more preferably, 2-5 mg of the substance that specifically binds to the p24 protein is coupled to each 1 mL of the modified magnetic beads.

5. The conjugate according to claim 3, wherein the particle size of the modified magnetic beads is 50 nm-200 μm, preferably 200 nm-150 μm, and more preferably 1 μm-100 μm. The conjugate according to claim 1 , wherein the substance that specifically binds to the p24 protein is selected from an antibody or a nucleic acid aptamer that specifically binds to the p24 protein.

7. The conjugate according to claim 6, wherein the antibody that specifically binds to the p24 protein is one or more of a mouse antibody, a rabbit antibody, a sheep antibody, a chicken antibody, a camelid antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.

8. The conjugate according to claim 6, wherein the antibody that specifically binds to the p24 protein is one or more of a full-length antibody, a single-chain antibody, an antigen-binding fragment, a single-domain antibody, a bispecific antibody, and a multispecific antibody.

9. The conjugate according to claim 3, wherein the modification comprises at least one of a chemical modification, an activated modification, and a biological modification.

10. The conjugate according to claim 9, wherein the chemical modification comprises at least one of carboxyl modification, amino modification and hydroxyl modification; wherein the activation modification comprises at least one of tosyl modification, epoxy modification, chloromethyl modification, N-hydroxysuccinimide modification and cyanogen bromide modification; and the biological modification comprises at least one of streptavidin, protein A and protein G modification.

11. A method for preparing a conjugate according to any one of claims 1 to 10, characterized in that: The following steps are involved: The solid phase carrier is contacted and coupled with a substance that specifically binds to the p24 protein to obtain a conjugate.

12. The preparation method according to claim 11, wherein the solid phase carrier is a plastic product, a microplate, a magnetic bead, a resin or agarose; preferably, the magnetic bead is modified.

13. The preparation method according to claim 11, wherein the reaction conditions of the contact coupling are room temperature for 2-4 hours or 4°C overnight.

14. Use of the conjugate according to any one of claims 1 to 10 in removing free p24 protein from lentiviral supernatant.

15. Use of the conjugate according to any one of claims 1 to 10 in determining the titer of a lentivirus.

16. A method for removing free p24 protein from lentiviral supernatant, characterized in that: The method comprises contacting the conjugate according to any one of claims 1 to 10 with a sample to be tested for a certain period of time to remove free p24 protein in the lentiviral supernatant of the sample to be tested.

17. A method for determining the titer of a lentivirus, characterized in that: The following steps are involved: 1) coupling a substance that specifically binds to p24 protein to a solid phase carrier, 2) contacting the sample to be tested with the solid phase carrier prepared in step 1) for a certain period of time to remove free p24 protein in the lentiviral supernatant, and 3) Determining the titer of the p24 protein of the lentiviral particles in the lentiviral supernatant of the sample to be tested.

18. The method according to claim 17, wherein the substance that specifically binds to the p24 protein is selected from an antibody or a nucleic acid aptamer that specifically binds to the p24 protein.

19. The method according to claim 17, wherein: The solid phase carrier is a plastic product, a microplate, a magnetic bead, a resin or agarose.

20. The conjugate of claim 19, wherein the magnetic beads are modified.

21. The method according to claim 20, wherein: The modification includes at least one of chemical modification, activation modification and biological modification.

22. The method according to claim 21, wherein: The chemical modification includes at least one of carboxyl modification, amino modification and hydroxyl modification; the activation modification includes at least one of tosyl 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.

23. The method according to claim 17, wherein: The solid phase carrier is coupled with an antibody or a nucleic acid aptamer that specifically binds to the p24 protein.

24. The method of claim 17, wherein: The contact time in step 2) is 10-240 min, preferably 15-30 min; and in step 3), an ELISA kit is used to determine the titer of the p24 protein of the lentiviral particles in the lentiviral supernatant.

25. A kit, characterized in that The invention comprises the conjugate according to any one of claims 1 to 10, and a buffer.