Antibodies or antigen-binding fragments that specifically bind to HIV p24 protein, detection kits and their applications

Abstract

This invention belongs to the technical field of molecular detection, and discloses an antibody or antigen-binding fragment thereof that specifically binds to the HIV p24 protein, a detection kit, and their applications. The antibody or antigen-binding fragment provided by this invention includes a heavy chain variable region and a light chain variable region; the amino acid sequences of HCDR1~HCDR3 in the heavy chain variable region are shown in SEQ ID NO:1~3, and the amino acid sequences of LCDR1~LCDR3 in the light chain variable region are shown in SEQ ID NO:5~7. This antibody or antigen-binding fragment exhibits excellent specificity in recognizing and binding to different genotypes of p24 protein, and possesses excellent broad-spectrum detection, specificity, and sensitivity, showing great promise for applications in HIV-1 prevention, diagnosis, basic research, and drug and vaccine development.

Description

Technical Field

[0001] This invention belongs to the technical field of molecular detection, and particularly relates to an antibody or its antigen-binding fragment that specifically binds to HIV p24 protein, a detection kit, and their applications. Background Technology

[0002] AIDS, also known as Acquired Immunodeficiency Syndrome, is a disease caused by infection with the Human Immunodeficiency Virus (HIV). HIV is a spherical, enveloped virus, approximately 120 nm in diameter. The outer membrane of HIV is a lipid envelope derived from the host cell and contains the viral proteins gp120 and gp41. gp41 is a transmembrane protein, while gp120 is located on the surface and binds to gp41 through non-covalent interactions. Inwardly, there is a spherical matrix formed by the p17 protein and a semi-conical capsid formed by the p24 protein. The capsid exhibits high electron density under an electron microscope. The capsid contains the viral RNA genome, reverse transcriptase, integrase, proteases, and other enzyme molecules, as well as components from the host cell (such as tRNAlys3, which serves as a primer for reverse transcription). HIV is an RNA retrovirus with two serotypes—type I and type II—that are very similar in genetic characteristics and exhibit extensive cross-antigens. Type I is prevalent worldwide. Although type II has been discovered for more than ten years, it is still mainly confined to West Africa as a local epidemic, and there is no obvious trend of spreading to other places.

[0003] After HIV-1 invades the human body, the level of p24 protein develops along with the viral RNA level and can appear as early as the acute infection phase. It is generally considered an indirect marker of viral replication and is closely related to disease progression. Therefore, the detection of p24 protein in blood and other bodily fluid samples can shorten the window period, which is helpful for the early diagnosis, prognosis, and evaluation of the effectiveness of antiviral therapy for HIV-1, and has good practical application value. However, HIV-1 has multiple subtypes, including type A, type B, and type C, as well as recombinant types, and is highly mutable. Existing antibodies used to detect p24 protein cannot effectively identify different subtypes, different mutant strains, and different forms of p24 protein in the acute and chronic infection phases, which may lead to missed subtypes, missed mutations, and missed stages, thereby delaying patient treatment and increasing the risk of transmission.

[0004] Therefore, obtaining an anti-p24 protein antibody with excellent broad-spectrum activity, sensitivity, and specificity is of great significance for HIV-1 prevention, diagnosis, basic research, and drug and vaccine development. Summary of the Invention

[0005] The primary objective of this invention is to address the shortcomings of existing anti-p24 antibodies in terms of their less-than-ideal detection breadth, sensitivity, and specificity, and to provide an antibody or its antigen-binding fragment thereof.

[0006] A second objective of this invention is to provide a reagent kit.

[0007] A third objective of this invention is to provide the application of the above-mentioned antibody or its antigen-binding fragment in HIV detection for non-diagnostic purposes.

[0008] The fourth objective of this invention is to provide an HIV testing kit.

[0009] Specifically, the antibody or its antigen-binding fragment provided by the present invention includes a heavy chain variable region and a light chain variable region; wherein, the amino acid sequence of HCDR1 in the heavy chain variable region is shown in SEQ ID NO:1, the amino acid sequence of HCDR2 is shown in SEQ ID NO:2, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:3; the amino acid sequence of LCDR1 in the light chain variable region is shown in SEQ ID NO:5, the amino acid sequence of LCDR2 is shown in SEQ ID NO:6, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:7.

[0010] Furthermore, the heavy chain variable region includes an amino acid fragment with the sequence shown in SEQ ID NO:4.

[0011] Furthermore, the light chain variable region includes an amino acid fragment with the sequence shown in SEQ ID NO:8.

[0012] Furthermore, the antibody or its antigen-binding fragment is selected from one or more of monoclonal antibodies, dsFv fragments, scFv fragments, Fd fragments, Fab fragments, (Fab')2 fragments, CDR fragments, and nanobodies.

[0013] Furthermore, the antibody or its antigen-binding fragment is provided with a detectable label, which is selected from one or more of fluorescent substances, chemiluminescent substances, radioactive isotopes, enzymes, biotin, colloidal gold, and magnetic particles.

[0014] The kit provided by this invention includes a nucleic acid fragment encoding the above-mentioned antibody or its antigen-binding fragment.

[0015] This invention provides the application of the above-mentioned antibody or its antigen-binding fragment in HIV detection for non-diagnostic purposes.

[0016] Furthermore, the antibody or its antigen-binding fragment is used to detect the presence or level of p24 protein in a sample.

[0017] The HIV test kit provided by this invention includes the above-mentioned antibody or its antigen-binding fragment.

[0018] Furthermore, the test kit includes a magnetic bead-coated anti-p24 protein antibody and an acridinium ester-labeled antibody or its antigen-binding fragment, and the anti-p24 protein antibody and the antibody or its antigen-binding fragment do not compete for epitopes.

[0019] Beneficial effects:

[0020] The antibodies or antigen-binding antibodies provided by this invention, with sequences such as HCDR1~HCDR3 shown in SEQ ID NO:1~3 and LCDR1~LCDR3 shown in SEQ ID NO:5~7, together form a non-linear three-dimensional binding interface with suitable conformational flexibility. This interface can effectively recognize the conformation of the NTD domain in the p24 protein, thereby endowing the antibody or its antigen-binding fragment with the ability to specifically recognize and bind to different genotypes of p24 protein. It possesses excellent broad-spectrum detection, specificity, and sensitivity, and has very promising application prospects in the prevention, diagnosis, basic research, and drug and vaccine development of HIV-1. Detailed Implementation

[0021] Firstly, the antibody or antigen-binding fragment provided by this invention belongs to the category of anti-HIV p24 protein antibodies, specifically recognizing the conformation of the NTD domain in the p24 protein. More specifically, the antibody or antigen-binding fragment includes a heavy chain variable region and a light chain variable region. The HCDR1~HCDR3 of the heavy chain variable region and the LCDR1~LCDR3 of the light chain variable region together form a three-dimensional binding interface with excellent recognition and binding ability for the NTD domains of different p24 protein genotypes, endowing the obtained antibody or antigen-binding fragment with excellent detection broad-spectrum, specificity, and sensitivity.

[0022] In this invention, the amino acid sequence of HCDR1 in the heavy chain variable region of the antibody or its antigen-binding fragment is specifically shown as SEQ ID NO:1, the amino acid sequence of HCDR2 is specifically shown as SEQ ID NO:2, and the amino acid sequence of HCDR3 is specifically shown as SEQ ID NO:3.

[0023] In this invention, the heavy chain variable region of the antibody or its antigen-binding fragment specifically includes a heavy chain framework region. Therefore, the heavy chain framework region forms the folded framework of the heavy chain variable region. In immunoglobulins of mammals commonly used for immunization to prepare antibodies, such as humans, mice, rabbits, and sheep, the heavy chain framework region has high conservation, especially the key residues that maintain the folding of the variable region. It can be one of various existing options. Those skilled in the art can make adaptive designs based on the existing disclosed framework region structures according to actual needs. This invention does not particularly limit it. Specific examples include, but are not limited to, one or more of the following: heavy chain framework regions derived from mouse immunoglobulins, heavy chain framework regions derived from rabbit immunoglobulins, heavy chain framework regions derived from sheep immunoglobulins, and heavy chain framework regions derived from human immunoglobulins.

[0024] In some specific embodiments, the heavy chain variable region preferably includes an amino acid fragment with a sequence as shown in SEQ ID NO:4.

[0025] In this invention, the amino acid sequence of LCDR1 in the light chain variable region of the antibody or its antigen-binding fragment is specifically shown in SEQ ID NO:5, the amino acid sequence of LCDR2 is specifically shown in SEQ ID NO:6, and the amino acid sequence of LCDR3 is specifically shown in SEQ ID NO:7.

[0026] In this invention, the light chain variable region of the antibody or its antigen-binding fragment specifically includes a light chain framework region. Therefore, the light chain framework region forms the folded framework of the light chain variable region. In immunoglobulins of mammals commonly used for immunization to prepare antibodies, such as humans, mice, rabbits, and sheep, the light chain framework region has high conservation, especially the key residues that maintain the folding of the variable region. It can be various existing options. Those skilled in the art can make adaptive designs based on the existing disclosed framework region structures according to actual needs. This invention does not particularly limit it. Specific examples include, but are not limited to, one or more of the following: light chain framework regions derived from mouse immunoglobulins, light chain framework regions derived from rabbit immunoglobulins, light chain framework regions derived from sheep immunoglobulins, and light chain framework regions derived from human immunoglobulins.

[0027] In some specific embodiments, the light chain variable region preferably includes an amino acid fragment with a sequence as shown in SEQ ID NO:8.

[0028] In this invention, the antibody or its antigen-binding fragment can be a monoclonal antibody with a complete antibody structure or a functional fragment that retains the ability to specifically bind to antigens. These are various existing options, and those skilled in the art can make adaptive choices according to actual needs. This invention does not impose any particular limitation on it. Specific examples include, but are not limited to, one or more of the following: monoclonal antibodies, dsFv fragments, scFv fragments, Fd fragments, Fab fragments, (Fab')2 fragments, CDR fragments, and nanobodies.

[0029] In some specific embodiments, when the antibody or its antigen-binding fragment is a monoclonal antibody, it specifically includes a heavy chain constant region and a light chain constant region, which can be various existing options. Those skilled in the art can make adaptive designs based on actual needs and the existing disclosed constant region structures. This invention does not impose any particular limitations on it.

[0030] In this invention, the antibody or its antigen-binding fragment is preferably provided with a detectable label. The detectable label refers to a substance that can be detected and quantified by optical, chemical, photochemical, biochemical, immunological, and electrical means. Various existing options are available, and those skilled in the art can make adaptive designs based on actual needs. This invention does not particularly limit its use. Specific examples include, but are not limited to, one or more of the following: fluorescent substances, chemiluminescent substances, radioactive isotopes, enzymes, biotin, colloidal gold, and magnetic particles. Specific examples of the fluorescent substances include, but are not limited to, one or more of the following: fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate, green fluorescent protein, Cy3, Cy5, Cy5.5, and Cy7. Specific examples of the chemiluminescent substances include, but are not limited to, one or more of the following: luminol, isoluminol, phenanthrene, and acridine ester. Specific examples of the radioactive isotopes include, but are not limited to: 3 H, 125 I, 35 S, 14 C and 32 One or more of P. Specific examples of the enzyme include, but are not limited to, one or more of glucose oxidase, horseradish peroxidase, alkaline phosphatase, and β-galactosidase. Specific examples of the biotin include, but are not limited to, avidin and / or streptavidin.

[0031] Secondly, the kit provided by the present invention is used to prepare the above-mentioned antibody or its antigen-binding fragment. Specifically, the kit includes a nucleic acid fragment encoding the above-mentioned antibody or its antigen-binding fragment.

[0032] In this invention, the nucleic acid fragment is a conventional technical means used in the existing antibody production field, and there are various existing options. Those skilled in the art can make adaptive designs according to actual needs, and this invention does not impose any special limitations on it.

[0033] Thirdly, the present invention provides the application of the above-mentioned antibody or its antigen-binding fragment in HIV detection for non-diagnostic purposes.

[0034] In this invention, specific examples of HIV testing scenarios for non-diagnostic purposes include, but are not limited to, one or more of the following: efficacy evaluation scenarios in drug development, immune effect assessment scenarios in vaccine development, quality control scenarios in biobanks, calibration and quality control scenarios of laboratory testing methods, and virus transmission dynamics research scenarios.

[0035] In this invention, the antibody or its antigen-binding fragment has good specific recognition and binding activity for different subtypes of p24 protein, and can be used to achieve high specificity and sensitivity detection of the presence or level of p24 protein in a sample.

[0036] Fourthly, the HIV detection kit provided by the present invention specifically includes the aforementioned antibody or its antigen-binding fragment.

[0037] In this invention, the key to the HIV detection kit's ability to detect HIV lies in introducing the aforementioned antibody or its antigen-binding fragment to capture p24 protein of different genotypes. Various existing methods can be used to detect the complex formed by the binding of the antibody or its antigen-binding fragment to the p24 protein. Those skilled in the art can make adaptive selections from existing molecular detection technologies according to actual needs. This invention does not impose any particular limitations on these methods. Specific examples include, but are not limited to, one or more of enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, immunochromatography, and colloidal gold assay.

[0038] In this invention, the detection kit preferably comprises a magnetic bead-coated anti-p24 protein antibody and an acridil ester-labeled antibody or its antigen-binding fragment, wherein the anti-p24 protein antibody and the antibody or its antigen-binding fragment do not compete for epitopes. The anti-p24 protein antibody is a type of anti-p24 protein in the prior art whose recognition epitopes do not overlap with those of the antibody or its antigen-binding fragment; various existing options are acceptable, and this invention does not impose any particular limitation on it.

[0039] The embodiments of the present invention are described in detail below. These embodiments are intended to explain the present invention and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0040] Preparation Example 1

[0041] This preparation example illustrates the preparation of p24 proteins of different genotypes. Based on the amino acid sequences of different subtypes of p24 proteins shown in Table 1, the company synthesized recombinant plasmids carrying the corresponding coding genes and expressed and purified p24 proteins to obtain p24 proteins of each genotype.

[0042] Table 1.

[0043]

[0044] Preparation Example 2

[0045] This preparation example illustrates the preparation of truncated peptides derived from the p24 protein. The truncated peptides were designed using the known HIV-1 p24 protein as a reference. The amino acid sequences of each truncated peptide are shown in Table 2. The biotechnology company synthesized recombinant plasmids carrying the corresponding coding genes and expressed and purified the truncated peptides to obtain each truncated peptide.

[0046] Table 2.

[0047]

[0048] Example 1

[0049] This embodiment illustrates the preparation and screening of monoclonal antibodies that specifically bind to the p24 protein. The monoclonal antibodies are prepared by using the p24 protein P3P24 provided in Preparation Example 1 as an immunogen, and performing immune cell fusion, hybridoma and subclonal screening, as well as antibody identification and preparation. Specifically, the steps include:

[0050] 1. Preparation of immunogens

[0051] The p24 protein P3P24 was diluted to 0.4 mg / mL with ultrapure water and then mixed with equal volumes of Freund's complete adjuvant (Sigma, catalog number F5881) and Freund's incomplete adjuvant (Sigma, catalog number F5506) to obtain the immunogen reagent.

[0052] 2. Basic immunization in mice

[0053] Primary immunization was performed by subcutaneous injection of immunogen reagent (French complete adjuvant) at multiple sites in 6-8 week old BALB / c female mice at an injection dose of 500 μL / mouse. Secondary booster immunizations were then performed every 2 weeks using immunogen reagent (French incomplete adjuvant), with 200 μL of ocular venous blood collected before each immunization and serum titer measured by indirect ELISA. Immunization was stopped once the mouse serum titer reached a plateau, and fusion was initiated within 2 weeks. Three days before the start of fusion, a booster immunization was performed using p24 protein (P3P24) at a concentration of 1.0 mg / mL at an injection dose of 100 μL / mouse.

[0054] 3. Preparation and screening of fusion hybridomas

[0055] (1) Preparation of feeder cells: A BALB / c mouse of about 6 weeks old was euthanized by cervical induction. After soaking in 75% ethanol solution for 5 min, the peritoneal cavity of the mouse was opened in a clean bench to obtain peritoneal cells. The cell concentration was adjusted to 2×10⁻⁶ cells using HT culture medium (purchased from Xiamen Imbio Biotechnology Co., Ltd.) 5 The cells / mL were used to obtain the feeder cell culture medium for later use.

[0056] (2) Preparation of myeloma cells: Mouse myeloma cell line Sp2 / 0-Ag14 (Sp2 / 0) was revived and then seeded into RPMI-1640 medium (Shanghai Yuanpei Biotechnology Co., Ltd., catalog number L220KJ) and cultured until the logarithmic growth phase. The cell concentration was adjusted to 2×10⁻⁶ cells / year using RPMI-1640 medium. 7 The cell / mL ratio was used to obtain myeloma cell culture medium for later use.

[0057] (3) Preparation of immune spleen cells: (i) BALB / C mice that have undergone basic immunization were euthanized by removing their eyeballs and bleeding out. The blood of the mice was collected to prepare antiserum, which was used as a positive control for antibody detection.

[0058] (ii) Rinse the mice with tap water, soak them in 75% ethanol solution for 5 minutes, open the abdominal cavity of the mice in a clean bench to obtain the spleen, cut the spleen into small pieces with scissors and place them on a 200-mesh cell sieve, then grind them with a grinding rod (inner core of a syringe), while adding RPMI-1640 culture medium dropwise with a blowpipe to obtain the broken spleen tissue.

[0059] (iii) Add an appropriate amount of RPMI-1640 culture medium to the lysed spleen tissue, let stand for 5 min, transfer the upper 2 / 3 of the suspension to a 50 mL plastic centrifuge tube, repeat the above operation 3 times, and collect the suspension obtained in each operation; centrifuge the collected suspension at 1000 rpm for 10 min, discard the supernatant, wash the cells with RPMI-1640 culture medium, and adjust the cell concentration to 2 × 10⁻⁶. 7 The cells / mL yielded the immunosplenic spleen cell culture medium.

[0060] (4) Cell fusion: (i) Take 1 mL of PEG-1450 (sigma, catalog number P7181), 50 mL of RPMI-1640 culture medium and 200 mL of RPMI-1640 culture medium containing 20% ​​fetal bovine serum (Rongye, catalog number RYS-GF12-05), and preheat to 37°C;

[0061] (ii) Mix myeloma cell culture medium and immune spleen cell culture medium in a 50 mL centrifuge tube (the number of myeloma cells is 1×10⁻⁶). 7 cells, the number of immune spleen cells is 1×10 8 Centrifuge the cells at 1500 rpm for 5 min, remove the supernatant, and gently tap the bottom of the tube to loosen the cells into a paste;

[0062] (iii) Add 0.8 mL of PEG-1450 to a centrifuge tube while gently stirring. The PEG-1450 should be added within 60 seconds. Then add 10 mL of RPMI-1640 culture medium containing 20% ​​fetal bovine serum. After gently stirring and mixing, add more RPMI-1640 culture medium to a final volume of 40 mL to obtain a mixed cell culture medium.

[0063] (iv) Centrifuge the mixed cell culture medium at 1000 rpm for 5 min, remove the supernatant, take a small amount of HT culture medium, carefully disperse the cells and transfer them into 300 mL of HT culture medium; add 0.1 mL / well to a 96-well cell culture plate and incubate at 37℃ and 5% CO2 for 12 h; then add HAT complete medium (Sigma, catalog number H0262) at 0.1 mL / well and continue culturing for 5 days; take HT culture medium to replace 100% of the cell supernatant in the wells, and continue culturing for 3 days to obtain hybridoma cell solution.

[0064] (5) Screening of hybridoma cells: (i) Coating, washing and blocking the enzyme-labeled plate with p24 protein P3P24 (concentration of 1 μg / mL) at a coating amount of 0.1 mL / well to obtain an enzyme-labeled plate coated with antigen;

[0065] (ii) Add hybridoma cell solution to the ELISA plate at a rate of 50 μL / well, incubate at 37°C for 1 h, and wash the plate 5 times. Then add goat anti-mouse IgG-HRP (antibody purchased from Beijing Wanyu Meilan Technology Co., Ltd., HRP labeled by NaIO4 oxidation method, the same below), incubate at 37°C for 30 min, and wash the plate 5 times. Add TMB chromogenic reagent (purchased from Beijing Wantai Biological Pharmaceutical Co., Ltd.) to the ELISA plate at a rate of 50 μL / well, react in the dark for 15 min, and add stop solution (purchased from Beijing Wantai Biological Pharmaceutical Co., Ltd.) to the ELISA plate at a rate of 50 μL / well. Use an ELISA reader to detect the 450 / 620 nm values ​​of each well and screen for positive clones.

[0066] (6) Cloning of hybridoma cells: The cells in the positive clone wells were serially diluted to a certain concentration using the limiting dilution method, and then seeded into 96-well cell culture plates, with the goal of having only one cell growing in each well. The cloning was repeated 2-3 times until 100% positive, and stable clones such as Ab071, Ab073, Ab074, Ab076, Ab077, and Ab078 were obtained.

[0067] 4. Production of monoclonal antibody ascites

[0068] (1) Preparation of injection fluid: Stable clones were centrifuged at 1500 rpm for 5 min, and the cell density was adjusted to 5 × 10⁶ cells / mL using RPMI-1640 medium. 6 The number of cells / mL was used to obtain the injected cell solution.

[0069] (2) Take 3 BALB / c mice and inject 0.5 mL of liquid paraffin oil into the peritoneum. After feeding them with a standard diet for 1 week, inject the injected cell fluid into the mice with a dosage of 0.5 mL / mouse. Feed them with a standard diet for 7-10 days until the mice's abdomen is obviously distended. Then, euthanize the mice by cervical inclination, soak them in 75% ethanol solution for 1 min, open the peritoneum of the mice in a clean bench to aspirate all the ascites in the peritoneum, centrifuge at 12000 rpm for 10 min, collect the supernatant, and obtain the monoclonal antibody ascites.

[0070] (3) The monoclonal antibody ascites was purified by ammonium sulfate precipitation and Protein A affinity chromatography (purchased from GE, USA) to obtain high-purity monoclonal antibodies Ab071, Ab073, Ab074, Ab076, Ab077 and Ab078.

[0071] Example 2

[0072] This embodiment illustrates the reactivity of the monoclonal antibody provided in Example 1 to different p24 protein subtypes, specifically including:

[0073] 1. The concentrations of p24 protein P3P24 and different genotypes of p24 protein provided in Preparation Example 1 were diluted to 1 μg / mL using CB buffer (50 mM, pH=9.6), and the concentration of goat anti-mouse IgG was diluted to 5 μg / mL using PB buffer (10 mM, pH=7.4). The diluted solutions were used to coat, wash, and block the enzyme-labeled plates at a coating volume of 100 μL / well to obtain enzyme-labeled plates coated with antigens.

[0074] 2. The protein concentration of the monoclonal antibody provided in Example 1 was diluted to 1 μg / mL, 100 ng / mL, 10 ng / mL and 1 ng / mL using PBS buffer (10 mM, pH=7.4) to obtain the antibody solution to be tested;

[0075] 3. Add 100 μL of each antibody solution to the microplate and incubate at 37°C for 30 min, then wash the plate 5 times. Next, add 100 μL of goat anti-mouse IgG-HRP (1:5000 dilution) or p24 protein P3P24-HRP (NaIO4 oxidation method labeled HRP, 1:1000 dilution) to the microplate and incubate at 37°C for 30 min, then wash the plate 5 times. Add 50 μL of TMB chromogenic reagent to the microplate and react in the dark for 10 min. Add 50 μL of stop solution to the microplate and use a microplate reader to detect the 450 / 620 nm values ​​of each well. The results are shown in Table 3.

[0076] Table 3.

[0077]

[0078] As shown in Table 3, the monoclonal antibodies Ab071, Ab073, Ab074, Ab076, Ab077 and Ab078 provided in Example 1 all exhibit excellent reactivity to p24 proteins of different genotypes such as P3P24, B94 and NL43.

[0079] Example 3

[0080] This embodiment illustrates the epitope identification of the monoclonal antibody provided in Example 1, specifically including:

[0081] (1) The truncated antigen provided in Preparation Example 2 was diluted to 1 μg / mL using CB buffer (50 mM, pH=9.6); the diluted solution was used to coat, wash and block the enzyme-labeled plate according to the coating amount of 100 μL / well to obtain the enzyme-labeled plate coated with antigen.

[0082] (2) The protein concentration of the monoclonal antibody provided in Example 1 was diluted to 10 μg / mL using PBS buffer (10 mM, pH=7.4) to obtain the antibody solution to be tested.

[0083] (3) Add 100 μL of the antibody solution to each well into the enzyme-labeled plate coated with the antigen, incubate at 37°C for 30 min and wash the plate 5 times. Then add 100 μL of goat anti-mouse IgG-HRP (NaIO4 oxidation method HRP) diluted 1:5000 into each well, incubate at 37°C for 30 min and wash the plate 5 times. Add 100 μL of TMB chromogenic agent into the enzyme-labeled plate, react in the dark for 10 min, and add 50 μL of stop solution into the enzyme-labeled plate. Use an enzyme-labeled plate reader to detect the 450 / 620 nm values ​​of each well, and use PBS buffer (10 mM, pH=7.4) as a blank control. The results are shown in Table 4.

[0084] Table 4.

[0085]

[0086] As shown in Table 4, the monoclonal antibodies Ab071, Ab073, Ab074, Ab076, Ab077, and Ab078 provided in Example 1 all reacted to the NTD truncated antigen but not to the CTD truncated antigen. Among them, monoclonal antibodies Ab074, Ab076, Ab077, and Ab078 can be further identified as epitope monoclonal antibodies recognized by the N2 truncated antigen, while Ab071 and Ab073 are more likely to be conformational monoclonal antibodies against the NTD truncated antigen.

[0087] Example 4

[0088] This embodiment illustrates the preparation of a chemiluminescence detection kit and the screening of monoclonal antibodies coated with magnetic beads therein, specifically including:

[0089] 1. Preparation of Chemiluminescence Detection Kit

[0090] (1) Preparation of M reagent: (i) Take Dynabeads™ M-270 Carboxylic acid (Thermo Fisher Scientific, catalog number 34310D, particle size 1.5μm~3μm) and rotate it in a rotator for 30 min to prepare EDC solution;

[0091] Add 10 mg / mL of Dynabeads™ M-270 Carboxylic acid to MES solution (50 mM) after equilibration. After mixing well, use a magnet to adsorb the mixture for 1 min. Fix the centrifuge tube, invert it, and discard the supernatant. Add EDC solution to the precipitate according to the addition ratio of Dynabeads™ M-270 Carboxylic acid:EDC = 10 mg:30 mL. After mixing well, place the mixture in a gyroscope and activate it at room temperature for 30 min. Discard the supernatant to obtain activated magnetic microparticles.

[0092] (ii) Dilute the anti-p24 protein monoclonal antibody-I (purchased from Xiamen Imbio Biotechnology Co., Ltd., catalog number M11020) with an appropriate amount of MES solution (50mM) to obtain an antibody solution; according to the addition ratio of monoclonal antibody: activated magnetic microparticles = 20μg: 1mg, mix the antibody solution and activated magnetic microparticles, and react in a gyroscope at 4℃ for 24h; wash the reacted magnetic microparticles three times with PBS buffer (10mM, pH=7.4), and add an equal volume of magnetic bead preservation solution (purchased from Xiamen Imbio Biotechnology Co., Ltd.) to obtain reagent M.

[0093] (2) Preparation of R reagent: (i) Take 50 μg of the monoclonal antibody provided in Example 1 and mix it with an appropriate amount of PBS buffer (10 mM, pH=7.4); add acridine ester solution (10 μg / mL) according to the addition ratio of antibody: acridine ester = 10 μg: 1 μg, and adjust the volume to 300 μL with PBS buffer (10 mM, pH=7.4), and react at room temperature in the dark for 30 min; add 200 μL of acridine ester termination buffer (purchased from Xiamen Yingbomai Biotechnology Co., Ltd.), and react at room temperature in the dark for 30 min; transfer the product to a dialysis bag, use PBS buffer (10 mM, pH=7.4) as the dialysate, and dialyze at 4℃ and 300 rpm in the dark, changing the PBS buffer every 2 h for a total of 4 times to remove unlabeled acridine ester and obtain protein solution.

[0094] (ii) After removing the protein solution, add 500 μL of glycerol and 10 μL of 10% (W / V) bovine serum albumin, and filter through a 0.22 μm filter membrane in the dark to obtain reagent R.

[0095] 2. Screening of acridine ester-labeled monoclonal antibodies

[0096] (1) Preparation of test solution: The p24 proteins of the three genotypes P3P24, B94 and NL43 provided in Preparation Example 1 were diluted to 5 ng / mL and 0.5 g / mL with 20% NBS buffer (purchased from Xiamen Imbio Biotechnology Co., Ltd.) to obtain the test solution.

[0097] (2) Preparation of detection reagent: (i) The concentration of reagent M was diluted to 0.4 mg / mL using magnetic bead preservation solution to obtain detection reagent M.

[0098] (ii) Dilute reagent R with acridinium ester diluent (purchased from Xiamen Imbio Biotechnology Co., Ltd.) at a dilution ratio of 1:500 to obtain test reagent R.

[0099] (3) Detection of the test solution: Take 50 μL of the test solution and 50 μL of the detection reagent M, mix them evenly, and incubate at 37℃ for 10 min; wash with PBS buffer (10 mM, pH=7.4) containing 0.05% (W / V) Tween-20, then add 50 μL of the detection reagent R, shake to mix, and incubate at 37℃ for 10 min; wash with PBS buffer (10 mM, pH=7.4) containing 0.05% (W / V) Tween-20, add 100 μL of 1% (w / v) hydrogen peroxide solution, pre-activate at 37℃, add 100 μL of sodium hydroxide solution (1 mol / L) for activation, and then detect. 20% NBS buffer is used as a control. The results are shown in Table 5.

[0100] Table 5.

[0101]

[0102] As shown in Table 5, the monoclonal antibody Ab073 exhibits superior reactivity against the three different genotypes of p24 protein provided in Preparation Example 1: P3P24, B94, and NL43.

[0103] 3. Structural identification of monoclonal antibody Ab073

[0104] The selected monoclonal antibody Ab073 was sent for sequencing to obtain its specific amino acid sequence information, as shown in Table 6.

[0105] Table 6.

[0106]

[0107] Example 5

[0108] This embodiment illustrates the detection performance of the chemiluminescence detection kit provided in Example 4. This kit uses monoclonal antibody Ab073 as an acridinium ester-labeled monoclonal antibody, and the specific tests include:

[0109] 1. Detection capability for different genotypes of P24 protein

[0110] Referring to the method provided in Example 4, the different genotypes of p24 protein provided in Preparation Example 1 were detected using a chemiluminescence detection kit. A commercially available chemiluminescence detection kit (purchased from Roche, USA) and reagent 1 (using anti-p24 protein monoclonal antibody-I as the magnetic bead-coated monoclonal antibody and anti-p24 protein monoclonal antibody-II [purchased from Xiamen Imbio Biotechnology Co., Ltd., catalog number M11015] as the acrid ester-labeled monoclonal antibody, and other raw materials and preparation methods were the same as in Example 4) were used as controls. The results are shown in Table 7.

[0111] Table 7.

[0112]

[0113] As shown in Table 7, the chemiluminescent detection kit provided in Example 4 of this invention, which uses monoclonal antibody Ab073 as an acridine ester-labeled monoclonal antibody, has excellent detection capabilities for different genotypes of p24 protein.

[0114] 2. Detection capability for national reference materials of human immunodeficiency virus p24 antigen

[0115] (1) Preparation of test samples: The national reference material of human immunodeficiency virus p24 antigen (China Institute of Food and Drug Control, batch number: 220015-202307, the same below) was used as the test sample and stored frozen at -20℃. Before testing, it was placed in a refrigerator at 4℃ to thaw and mixed before use.

[0116] (2) Detection of the sample to be tested: The national reference material of human immunodeficiency virus p24 antigen was detected by chemiluminescence detection kit according to the method provided in Example 4, and reagent 1 was used as a control. The detection results are shown in Table 8.

[0117] As shown in Table 8, the chemiluminescent detection kit provided in Example 4 of this invention, which uses acridine ester-labeled monoclonal antibody Ab073 as the basis for detection, exhibits excellent detection sensitivity and specificity for HIV-1 p24 protein.

[0118] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

[0119] Table 8.

[0120]

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to HIV p24 protein, characterized in that, The antibody or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region; The amino acid sequence of HCDR1 in the heavy chain variable region is shown in SEQ ID NO:1, the amino acid sequence of HCDR2 is shown in SEQ ID NO:2, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:

3. The amino acid sequence of LCDR1 in the light chain variable region is shown in SEQ ID NO:5, the amino acid sequence of LCDR2 is YVS, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:

7.

2. The antibody or antigen-binding fragment thereof that specifically binds to HIV p24 protein according to claim 1, characterized in that, The heavy chain variable region includes an amino acid fragment with the sequence shown in SEQ ID NO:

4.

3. The antibody or antigen-binding fragment thereof that specifically binds to HIV p24 protein according to claim 1, characterized in that, The light chain variable region includes an amino acid fragment with the sequence shown in SEQ ID NO:

8.

4. The antibody or antigen-binding fragment thereof that specifically binds to HIV p24 protein according to claim 1, characterized in that, The antibody or its antigen-binding fragment is selected from one of the following: monoclonal antibody, dsFv fragment, scFv fragment, Fab fragment, and (Fab')2 fragment.

5. The antibody or antigen-binding fragment thereof that specifically binds to HIV p24 protein according to claim 1, characterized in that, The antibody or its antigen-binding fragment is provided with a detectable marker, which is selected from one or more of fluorescent substances, chemiluminescent substances, radioactive isotopes, enzymes, biotin, colloidal gold, and magnetic particles.

6. A reagent kit, characterized in that, The kit comprises a nucleic acid fragment encoding an antibody or antigen-binding fragment thereof that specifically binds to the HIV p24 protein as described in any one of claims 1 to 5.

7. The use of the antibody or antigen-binding fragment thereof that specifically binds to HIV p24 protein as described in any one of claims 1 to 5 in HIV detection for non-diagnostic purposes.

8. The application of the antibody or antigen-binding fragment thereof that specifically binds to HIV p24 protein according to claim 7 in HIV detection for non-diagnostic purposes, characterized in that, The antibody or its antigen-binding fragment that specifically binds to the HIV p24 protein is used to detect the presence or level of the p24 protein in a sample.

9. An HIV testing kit, characterized in that, The test kit includes an antibody or antigen-binding fragment thereof that specifically binds to the HIV p24 protein as described in any one of claims 1 to 5.

10. The HIV test kit according to claim 9, characterized in that, The test kit includes a magnetic bead-coated anti-p24 protein antibody and an acridinium ester-labeled antibody or its antigen-binding fragment thereof, and the anti-p24 protein antibody and the antibody or its antigen-binding fragment thereof do not compete for epitopes.

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