Anti-p24 antibodies and uses thereof

Abstract

The application discloses an anti-P24 antibody and application thereof, and relates to the field of antibodies. The anti-P24 antibody disclosed by the application comprises a heavy chain complementarity determining region and a light chain complementarity determining region, the antibody provides an important raw material source for detection of P24, and has good affinity or activity.

Description

Technical Field

[0001] This invention relates to the field of antibody technology, and more specifically, to an anti-P24 antibody and its application. Background Technology

[0002] Human Immunodeficiency Virus (HIV), also known as the HIV virus causing AIDS, is a virus that causes defects in the human immune system. It is a lentivirus that infects cells of the human immune system and belongs to the retrovirus family. HIV is the pathogen of AIDS and is mainly transmitted through sexual contact, blood, and from mother to child. In recent years, the number of people infected with HIV has been on the rise. Currently, measuring serum HIV antibodies is the routine laboratory method for diagnosing HIV infection. However, measuring HIV antibodies has limitations: more than 70% of HIV-infected individuals cannot detect antibodies until 6 months after infection, and in the homosexual community, this number exceeds 80%. Antibody detection increases the risk of transmission during the HIV "window period"; furthermore, newborns do not develop antibodies until one year after birth, and HIV antibodies from the mother can cause false positives; because HIV antibodies persist throughout the disease process and only disappear in the late stages of AIDS, they cannot serve as a stable indicator for treatment monitoring.

[0003] P24 is a major structural protein of the HIV viral particle, a product of the structural gene GAG, and plays a crucial role in viral packaging and maturation. The amino acid sequence of the P24 protein is highly conserved across different HIV strains; the absence of P24 prevents normal viral assembly. The P24 protein is highly specific and shows no cross-reactivity with most other retroviruses. In HIV infection, the first viral marker to appear in the blood of infected individuals is the viral P24 protein. A relatively long window period exists between viral infection and the detection of HIV antibodies; therefore, the detection of HIV-P24 antigen has played a vital role in the early diagnosis of HIV infection, patient prognosis, screening and evaluation of anti-HIV drugs, and the detection of mother-to-child transmission.

[0004] The detection of P24 antigen employs serological diagnostic methods, primarily including double-antibody sandwich ELISA, immune complex lysis detection, ultrasensitive EIA, and enzyme-linked immunofluorescence assays. Currently, the double-antibody sandwich method is widely used for detecting the human immunodeficiency virus (HIV) P24 antigen, and obtaining anti-P24 antibodies is crucial for achieving this detection method. Therefore, there is a strong demand in this field for antibodies that effectively bind to and detect P24. Summary of the Invention

[0005] This application provides an anti-P24 antibody or its antigen-binding fragment, which provides an important source of raw materials for the detection of P24 and has good activity or affinity.

[0006] To achieve the above objectives, according to one aspect of the present invention, an anti-P24 antibody or an antigen-binding fragment thereof is provided, the anti-P24 antibody or the antigen-binding fragment thereof having three complementary determinant regions of the heavy chain variable region shown in amino acid sequence SEQ ID NO:17 and three complementary determinant regions of the light chain variable region shown in amino acid sequence SEQ ID NO:19 or SEQ ID NO:22.

[0007] To achieve the above objective, according to a second aspect of the present invention, an anti-P24 antibody or an antigen-binding fragment thereof is provided, said anti-P24 antibody or antigen-binding fragment comprising the following complementarity-determining region:

[0008] HCDR1, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:1;

[0009] HCDR2, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:2;

[0010] HCDR3, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:3;

[0011] LCDR1, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:4;

[0012] LCDR2, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:5; and

[0013] LCDR3, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:6.

[0014] To achieve the above objectives, according to a third aspect of the present invention, an anti-P24 antibody or an antigen-binding fragment thereof is provided, comprising a heavy chain variable region and / or a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:17; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:19 or SEQ ID NO:22.

[0015] To achieve the above objectives, according to a fourth aspect of the present invention, an anti-P24 antibody or an antigen-binding fragment thereof is provided, comprising a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO:18; and the amino acid sequence of the light chain is shown in SEQ ID NO:20 or SEQ ID NO:23.

[0016] To achieve the above objectives, according to a fifth aspect of the present invention, an antibody conjugate is provided, the antibody conjugate comprising the above-described anti-P24 antibody or its antigen-binding fragment.

[0017] To achieve the above objectives, according to a sixth aspect of the present invention, a reagent or kit is provided, the reagent or kit comprising the above-described anti-P24 antibody or its antigen-binding fragment or the above-described antibody conjugate.

[0018] To achieve the above objectives, according to a seventh aspect of the present invention, a method for detecting P24 or HIV is provided, comprising: a) contacting the aforementioned anti-P24 antibody or its antigen-binding fragment, antibody-drug conjugate, or reagent or kit with P24 in a sample to be tested under conditions sufficient to induce an antibody / antigen binding reaction to form an immune complex; and b) detecting the presence of the immune complex, the presence of which indicates the presence of P24 or HIV in the test sample.

[0019] To achieve the above objectives, according to an eighth aspect of the present invention, a nucleic acid is provided that encodes the aforementioned anti-P24 antibody or its antigen-binding fragment.

[0020] To achieve the above objectives, according to a ninth aspect of the present invention, a vector is provided, the vector comprising the above-described nucleic acid.

[0021] To achieve the above objectives, according to a tenth aspect of the present invention, a cell is provided, said cell comprising the above-described nucleic acid, vector, or expressing the above-described anti-P24 antibody or its antigen-binding fragment.

[0022] To achieve the above objectives, according to the eleventh aspect of the present invention, a method for preparing the above-described anti-P24 antibody or its antigen-binding fragment is provided, the method comprising culturing the above-described cells.

[0023] To achieve the above objectives, according to the twelfth aspect of the present invention, the use of the above-described anti-P24 antibody or its antigen-binding fragment, antibody conjugate, reagent or kit in the preparation of products for detecting P24 or HIV is provided. Attached Figure Description

[0024] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1The results of SDS-PAGE for the reduction of Anti-P24 16H9Rmb1. Detailed Implementation

[0026] In a first aspect, embodiments of the present invention provide an anti-P24 antibody or an antigen-binding fragment thereof, wherein the anti-P24 antibody or the antigen-binding fragment thereof has three complementary determinant regions of the heavy chain variable region shown in amino acid sequence SEQ ID NO:17 and three complementary determinant regions of the light chain variable region shown in amino acid sequence SEQ ID NO:19 or SEQ ID NO:22.

[0027] It should be noted that HCDR1, HCDR2, and HCDR3 are amino acid sequences identical to those of HCDR1, HCDR2, and HCDR3 in the same heavy chain variable region defined in the anti-P24 antibody or its antigen-binding fragment as described in the first aspect, and LCDR1, LCDR2, and LCDR3 are amino acid sequences identical to those of LCDR1, LCDR2, and LCDR3 in the same light chain variable region defined in the anti-P24 antibody or its antigen-binding fragment as described in the first aspect.

[0028] For example, HCDR1, HCDR2, and HCDR3 are amino acid sequences consistent with HCDR1, HCDR2, and HCDR3 of the heavy chain variable region shown in SEQ ID NO:17; LCDR1, LCDR2, and LCDR3 are amino acid sequences consistent with LCDR1, LCDR2, and LCDR3 of the light chain variable region shown in SEQ ID NO:19.

[0029] In this invention, the term "antibody" is used in the broadest sense, and may include full-length monoclonal antibodies, bispecific or multispecific antibodies, and chimeric antibodies, as long as they exhibit the desired biological activity.

[0030] In this invention, the terms "complementarity-determining region," "CDR," or "CDRs" refer to highly variable regions of the heavy and light chains of immunoglobulins, specifically regions containing one or more, or even all, of the major amino acid residues that act on the binding of an antibody or antigen-binding fragment to the antigen or epitope it recognizes.

[0031] In this invention, the heavy chain complementarity determination region is represented by HCDR, which includes HCDR1, HCDR2 and HCDR3; the light chain complementarity determination region is represented by LCDR, which includes LCDR1, LCDR2 and LCDR3.

[0032] Methods for defining CDRs are well-known in the art and include: Kabat definition, Chothia definition, IMGT definition, Contact definition, and AbM definition. As described herein, "Kabat definition" refers to the definition system described in Kabat et al., USDept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). "Chothia definition" is found in Chothia et al., J Mol Biol 196:901-917 (1987). Other CDR definition methods may not strictly follow one of the above schemes but will still overlap with at least a portion of the CDR region defined by Kabat, although they may be shortened or lengthened based on predictions or experimental results of specific residues or residue groups. Exemplary defined CDRs are listed in Table 1 below, although definitions vary slightly in different literature. Given the amino acid sequence of the variable region of an antibody, those skilled in the art can routinely determine which residues contain a specific CDR. It should be noted that CDRs defined by other methods, not limited to those in Table 1, are also within the scope of this disclosure.

[0033] Table 1: CDR Definition 1

[0034]

[0035]

[0036] 1 The CDRs defined in Table 1 are numbered according to the Kabat numbering system (see below), with amino acid numbers on the heavy chain represented by "H + number" and amino acid numbers on the light chain represented by "L + number". Those skilled in the art can readily map this Kabat numbering system to any variable region sequence without relying on any experimental data outside the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described by Kabat et al., USD ept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0037] 2 As used in Table 1, “AbM” with a lowercase “b” refers to the CDR defined by the “AbM” antibody modeling software of Oxford Molecular.

[0038] 3If neither H35A nor H35B exists, then CDR-H1 ends at bit 35; if only H35A exists, then CDR-H1 ends at bit 35A; if both H35A and H35B exist, then CDR-H1 ends at bit 35B.

[0039] 4 If neither H35A nor H35B exists, then CDR-H1 ends at bit 32; if only H35A exists, then CDR-H1 ends at bit 33; if both H35A and H35B exist, then CDR-H1 ends at bit 34.

[0040] 5 If neither H35A nor H35B exists, then CDR-H1 ends at bit 33; if only H35A exists, then CDR-H1 ends at bit 34; if both H35A and H35B exist, then CDR-H1 ends at bit 35.

[0041] According to embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by any one or a combination of systems such as Kabat, Chothia, IMGT, AbM or Contact.

[0042] In some optional embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Kabat system.

[0043] In some optional embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Chothia system.

[0044] In some alternative embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the IMGT system.

[0045] In some optional embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the AbM system.

[0046] In some alternative embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Contact system.

[0047] In some alternative embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by a combination of Kabat, Chothia, IMGT, AbM, or Contact systems.

[0048] According to embodiments of the present invention, the Kabat numbering positions corresponding to the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3 defined by the Kabat, Chothia, AbM, or IMGT systems are as follows:

[0049] CDR Kabat AbM IMGT Chothia HCDR1 H31~H35 H26~H35 H26~H33 H26~H32 HCDR2 H50~H65 H50~H58 H51~H57 H52~H56 HCDR3 H95~H102 H95~H102 H93~H102 H95~H102 LCDR1 L24~L34 L24~L34 L27~L32 L24~L34 LCDR2 L50~L56 L50~L56 L50~L51 L50~L56 LCDR3 L89~L97 L89~L97 L89~L97 L89~L97

[0050] According to embodiments of the present invention, the antibody or its antigen-binding fragment includes the following complementarity-determining region:

[0051] HCDR1, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:1;

[0052] HCDR2, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:2;

[0053] HCDR3, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:3;

[0054] LCDR1, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:4;

[0055] LCDR2, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:5; and

[0056] LCDR3, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:6.

[0057] Secondly, embodiments of the present invention provide an anti-P24 antibody or its antigen-binding fragment, wherein the anti-P24 antibody or its antigen-binding fragment includes the following complementarity-determining region:

[0058] HCDR1, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:1;

[0059] HCDR2, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:2;

[0060] HCDR3, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:3;

[0061] LCDR1, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:4;

[0062] LCDR2, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:5; and

[0063] LCDR3, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:6.

[0064] According to an embodiment of the present invention, the HCDRs and LCDRs are defined by the Kabat system.

[0065] In this invention, the "frame region" or "FR" region includes the heavy chain frame region and the light chain frame region, referring to the regions in the antibody heavy chain variable region and light chain variable region other than the CDR; wherein, the heavy chain frame region can be further subdivided into adjacent regions separated by the CDR, including the HFR1, HFR2, HFR3 and HFR4 frame regions; the light chain frame region can be further subdivided into adjacent regions separated by the CDR, including the LFR1, LFR2, LFR3 and LFR4 frame regions.

[0066] In this invention, the heavy chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combination: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combination: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0067] In an optional embodiment, the anti-P24 antibody or its antigen-binding fragment described in the first or second aspect includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes HFR1, HFR2, HFR3, and HFR4, and the light chain variable region includes LFR1, LFR2, LFR3, and LFR4.

[0068] In an optional embodiment, the HFR1 includes / such as SEQ ID NO:7 or an amino acid sequence having at least 80% identity with it;

[0069] The HFR2 includes / is such as SEQ ID NO:8 or an amino acid sequence having at least 80% identity with it;

[0070] The HFR3 includes / is, for example, SEQ ID NO:9 or an amino acid sequence having at least 80% identity with it;

[0071] The HFR4 includes / is, for example, SEQ ID NO:10 or an amino acid sequence having at least 80% identity with it;

[0072] The LFR1 includes / such as SEQ ID NO:11 or an amino acid sequence having at least 80% identity with it;

[0073] The LFR2 includes / is, for example, SEQ ID NO:12 or an amino acid sequence having at least 80% identity with it;

[0074] The LFR3 includes / is, for example, SEQ ID NO:13 or an amino acid sequence having at least 80% identity with it; and

[0075] The LFR4 includes / such as SEQ ID NO:14 or an amino acid sequence having at least 80% identity with it.

[0076] It should be noted that, in other embodiments, the amino acid sequences of each frame region of the anti-P24 antibody or its antigen-binding fragment provided by the present invention may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the corresponding frame regions (SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, or 14) mentioned above.

[0077] In an optional embodiment, the LFR4 includes / such as SEQ ID NO:21 or an amino acid sequence having at least 80% identity with it.

[0078] In an optional embodiment, the anti-P24 antibody or its antigen-binding fragment has a KD < 9.42 × 10⁻⁶. -7 M binds to P24 with affinity.

[0079] In an optional embodiment, the anti-P24 antibody or its antigen-binding fragment has a KD ≤ 10. -7 M, KD≤10 -8 M, KD≤10 -9 M, KD≤10 -10 M, KD≤10 -11 M, KD≤10 -12 M, KD≤10 -13 M binds to P24 with affinity.

[0080] In an optional embodiment, the anti-P24 antibody or its antigen-binding fragment has a KD ≤ 3.25 × 10⁻⁶. -9 M binds to P24 with affinity.

[0081] There are many methods for determining antibody affinity (KD), which can be categorized into thermodynamic detection methods, kinetic detection methods, and dynamic equilibrium detection methods based on their detection principles. Common thermodynamic detection methods include isothermal titration calorimetry (ITC); common kinetic detection methods include surface plasmon resonance (SPR) and biomembrane optical interferometry (BLI); and common dynamic equilibrium detection methods include enzyme-linked immunosorbent assay (ELISA).

[0082] In an optional embodiment, the anti-P24 antibody or its antigen-binding fragment described in the first or second aspect above includes a heavy chain variable region and / or a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:17, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:19 or SEQ ID NO:22.

[0083] Thirdly, embodiments of the present invention provide an anti-P24 antibody or its antigen-binding fragment, comprising a heavy chain variable region and / or a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:17, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:19 or SEQ ID NO:22.

[0084] In an optional embodiment, the anti-P24 antibody or its antigen-binding fragment described in the first, second, or third aspect above further includes a constant region.

[0085] In an optional implementation, the constant region includes a heavy chain constant region and / or a light chain constant region.

[0086] In an optional implementation, the heavy chain constant region is selected from any one of the heavy chain constant regions of IgG, IgA, IgM, IgE, and IgD, or a combination of multiple constant region segments.

[0087] In an optional embodiment, the heavy chain constant region includes CH1 of IgG, the hinge region of IgG, CH2 of IgM, CH3 of IgM, and / or CH4 of IgM.

[0088] In an optional implementation, the IgG is selected from IgG1, IgG2, IgG3 or IgG4.

[0089] In an optional implementation, the light chain constant region is selected from the κ-type or λ-type light chain constant region.

[0090] In an optional implementation, the species source of the constant region is cattle, horses, dairy cows, pigs, sheep, rats, mice, dogs, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, turkeys, fighting cocks, or humans.

[0091] In an optional implementation, the species source of the constant region is rabbit.

[0092] In an optional embodiment, the heavy chain constant region sequence (CH) is as shown in SEQ ID NO:15, and the light chain constant region sequence (CL) is as shown in SEQ ID NO:16.

[0093] It should be noted that, in other embodiments, the constant region sequence may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the aforementioned constant region (SEQ ID NO: 15 or 16).

[0094] In an optional embodiment, the antigen-binding fragment is selected from any one of the antibody's F(ab)2, F(ab')2, Fab', Fab, Fv, and scFv.

[0095] The antigen-binding fragments of the aforementioned antibodies typically possess the same binding specificity as the source antibody. Those skilled in the art will readily understand, based on the description of this invention, that the antigen-binding fragments of the aforementioned antibodies can be obtained, for example, by enzymatic digestion (including pepsin or papain) and / or by chemical reduction of disulfide bonds. Based on the complete antibody structure disclosed in this invention, those skilled in the art can readily obtain the aforementioned antigen-binding fragments.

[0096] The antigen-binding fragments of the aforementioned antibodies can also be obtained by recombinant genetic techniques known to those skilled in the art or by synthesizing, for example, automated peptide synthesizers sold by Applied BioSystems.

[0097] In an optional embodiment, the anti-P24 antibody or its antigen-binding fragment described in the first, second or third aspects above includes a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO:18 and the amino acid sequence of the light chain is shown in SEQ ID NO:20 or SEQ ID NO:23.

[0098] Fourthly, the present invention provides an anti-P24 antibody or an antigen-binding fragment thereof, comprising a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO:18, and the amino acid sequence of the light chain is shown in SEQ ID NO:20 or SEQ ID NO:23.

[0099] Fifthly, the present invention provides an antibody conjugate comprising the above-described anti-P24 antibody or its antigen-binding fragment.

[0100] In an optional embodiment, the antibody conjugate further includes biotin or a biotin derivative conjugated to the anti-P24 antibody or its antigen-binding fragment.

[0101] In an optional embodiment, the antibody conjugate further includes a marker conjugated to the anti-P24 antibody or its antigen-binding fragment.

[0102] In an optional implementation, the aforementioned marker refers to a type of substance that has properties such as luminescence, color development, and radioactivity that can be directly observed by the naked eye or detected or probed by instruments. Through these properties, qualitative or quantitative detection of the corresponding target can be achieved.

[0103] In optional embodiments, the markers include, but are not limited to, fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle markers.

[0104] In practical use, those skilled in the art can select appropriate markers according to the detection conditions or actual needs. Regardless of the marker used, it falls within the protection scope of this invention.

[0105] In optional embodiments, the fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (e.g., including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc., or their analogues), rhodamine dyes and their derivatives (e.g., including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc., or their analogues), and Cy series dyes and their derivatives (e.g., including but not limited to Cy2, Cy3, Cy3B, Cy3.5, C...). y5, Cy5.5, Cy3 and other similar substances), Alexa series dyes and their derivatives (including but not limited to Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750 and other similar substances) and protein dyes and their derivatives (including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydiophytoxanthin-chlorophyll protein (preCP) and other similar substances).

[0106] In optional embodiments, the enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase.

[0107] In optional embodiments, the radioactive isotopes include, but are not limited to, 212Bi, 131I, 111In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi, 32P, 94mTc, 99mTc, 203Pb, 67Ga, 68Ga, 43Sc, 47Sc, 110mIn, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 121Sn, 161Tb, 166Ho, 105Rh, 177Lu, 172Lu, and 18F.

[0108] In optional embodiments, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, luciferin, fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine ester and its derivatives, dioxane and its derivatives, rofenine and its derivatives, and peroxazone and its derivatives.

[0109] In optional embodiments, the nanoparticle-based markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0110] In optional embodiments, the colloid includes, but is not limited to, colloidal metals, dispersed dyes, dye-labeled microspheres, and latexes.

[0111] In optional embodiments, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0112] In an optional embodiment, the colloidal metal is colloidal gold.

[0113] In an optional embodiment, the antibody conjugate further includes a solid-phase carrier conjugated to the anti-P24 antibody or its antigen-binding fragment.

[0114] In an optional embodiment, the solid support is selected from microspheres, plates, and membranes.

[0115] In optional embodiments, the solid support includes, but is not limited to, magnetic microspheres, plastic microspheres, plastic microparticles, microporous plates, glass, capillaries, nylon, and nitrocellulose membranes.

[0116] In a sixth aspect, the present invention provides a reagent or kit comprising the above-described anti-P24 antibody or its antigen-binding fragment or the above-described antibody conjugate.

[0117] As previously stated, the anti-P24 antibody or its antigen-binding fragment in some embodiments or examples of the present invention can effectively bind to P24. Therefore, reagents or kits containing the anti-P24 antibody or its antigen-binding fragment can effectively perform qualitative or quantitative detection of P24. The reagents or kits provided by the present invention can be used, for example, for detections involving the specific binding properties of P24 and its antibody, such as immunoblotting and immunoprecipitation. As previously stated, the anti-P24 antibody or its antigen-binding fragment in some embodiments or examples of the present invention has higher binding activity or affinity for P24. Therefore, reagents or kits containing the anti-P24 antibody or its antigen-binding fragment have higher detection sensitivity or specificity.

[0118] In a seventh aspect, the present invention provides a method for detecting P24 or HIV, comprising: a) contacting the above-described anti-P24 antibody or its antigen-binding fragment, antibody conjugate, reagent or kit with P24 in a sample to be tested under conditions sufficient to cause an antibody / antigen binding reaction to form an immune complex; and b) detecting the presence of the immune complex, the presence of the complex indicating the presence of P24 or HIV in the test sample.

[0119] In an optional embodiment, the immune complex further includes a second antibody that binds to the anti-P24 antibody or its antigen-binding fragment.

[0120] In an optional embodiment, the immune complex further includes a second antibody that binds to P24.

[0121] Eighthly, the present invention provides a nucleic acid molecule encoding the above-mentioned anti-P24 antibody or its antigen-binding fragment.

[0122] In a ninth aspect, the present invention provides a carrier containing the above-mentioned nucleic acid molecules.

[0123] In a tenth aspect, the present invention provides cells containing the above-described carrier.

[0124] In one aspect, the present invention provides a method for preparing an anti-P24 antibody or an antigen-binding fragment thereof, comprising: culturing cells as described above.

[0125] In a twelfth aspect, the present invention provides the use of the above-described anti-P24 antibody or its antigen-binding fragment, antibody conjugate, or the above-described reagent or kit in the preparation of products for detecting P24 or HIV.

[0126] In a thirteenth aspect, the present invention provides the use of the above-described antibody or its antigen-binding fragment, antibody conjugate, or the above-described reagent or kit in the detection of P24 or HIV-related diseases.

[0127] Based on the amino acid sequence of the anti-P24 antibody or its antigen-binding fragment disclosed in this invention, those skilled in the art will readily conceive of preparing the anti-P24 antibody or its antigen-binding fragment using genetic engineering or other techniques (chemical synthesis, recombinant expression). For example, the anti-P24 antibody or its antigen-binding fragment can be isolated and purified from the culture product of recombinant cells capable of recombinantly expressing the anti-P24 antibody or its antigen-binding fragment as described in any of the preceding claims. This is easily achievable by those skilled in the art. Therefore, regardless of the technique used to prepare the anti-P24 antibody or its antigen-binding fragment of this invention, it falls within the protection scope of this invention.

[0128] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0129] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of formulations or unit doses herein, some methods and materials are described hereby. Unless otherwise stated, the techniques employed or considered herein are standard methods. Materials, methods, and examples are illustrative and not limiting in nature.

[0130] Unless otherwise specified, the practice of this invention will employ conventional techniques of cell biology, molecular biology (including recombinant technologies), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. This technique is well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.P. Calos, 1987); *Current Protocols in Molecular Biology* (edited by F.M. Mausubel et al., 1987); and *PCR: The Polymerase Chain Reaction*. The references cited in the references are: "Reaction" (Mullis et al., ed., 1994); and "Current Protocols in Immunology" (JEColigan et al., ed., 1991), each of which is explicitly incorporated herein by reference.

[0131] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0132] Example 1: Preparation of Anti-P24 16H9 Monoclonal Antibody

[0133] In this embodiment, restriction endonucleases and Prime Star DNA polymerase were purchased from Takara. The MagExtractor RNA extraction kit was purchased from TOYOBO. BD SMART TMThe RACE cDNA Amplification Kit was purchased from Takara. The plasmid extraction kit was purchased from Tiangen Pharmaceuticals. Primer synthesis and gene sequencing were performed by Invitrogen. The rabbit monoclonal antibody against P24 could be obtained using hybridoma technology, phage display technology, or single B cell cloning technology. The specific implementation method for phage display technology is as follows:

[0134] 1. Gene Acquisition

[0135] (1) Rabbit's immunity

[0136] An emulsion of P24 prepared with incomplete Freund's adjuvant was injected subcutaneously to stimulate an immune response in 4-6 week old New Zealand white rabbits. Serum was collected before and after immunization on days 0, 14, 28, 42 and 69. The rabbit spleen was then surgically removed to prepare a spleen cell suspension.

[0137] (2) Construction of phage libraries

[0138] RNA was extracted from rabbit spleen cells and reverse transcribed into cDNA. Using specifically designed rabbit antibody gene amplification primers and cDNA as a template, VH and VL gene fragments were amplified separately. Then, the VH and VL gene fragments were sequentially inserted into the phage vector VO2 using enzyme digestion and ligation. Finally, the ligated phage plasmids were electroporated into TG1 competent cells. The next day, single colonies were selected for PCR identification and antibody gene sequencing to assess the quality of the phage library. Once qualified, the phage library was screened.

[0139] (3) Screening of phage libraries

[0140] The TG1 phage library was inoculated into a shake flask and cultured until a suitable bacterial concentration (OD600 of 0.8-1.0) was reached. Helper phages were then added for infection for 1 hour, followed by overnight incubation. The bacterial culture was collected the next day, centrifuged, and the supernatant was purified by salting out to obtain the displayed phage library.

[0141] The phage library was panned 3-4 times using magnetic bead panning. Then, monoclonal phage-infected colonies were selected for antibody expression in the supernatant. Subsequently, the monoclonal phage antibody expression supernatant was screened and identified using ELISA to obtain anti-P24 monoclonal phages.

[0142] Phage antibody gene sequencing: Anti-P24 monoclonal phage antibody gene sequencing was performed. Through sequence analysis, duplicate and invalid sequences were removed to obtain a unique rabbit monoclonal antibody sequence.

[0143] 2. Expression plasmid construction

[0144] pcDNA TM 3.4 The vector is a recombinant antibody eukaryotic expression vector that has been modified to include polyclonal restriction enzyme sites, and will be referred to as the 3.4A expression vector. Based on the variable region gene obtained in the above experiments, VL and VH gene-specific primers were designed, with restriction endonuclease sites and protective bases at both ends, respectively. The 0.72Kb Light Chain gene fragment and the 1.40Kb Heavy Chain gene fragment were amplified by PCR.

[0145] The Heavy Chain and Light Chain gene fragments were double-digested with restriction endonucleases, and the 3.4A vector was also double-digested with restriction endonucleases. After purification and recovery of the fragments and vector, the Heavy Chain gene and Light Chain gene were ligated into the 3.4A expression vector to obtain recombinant expression plasmids of Heavy Chain and Light Chain, respectively.

[0146] 3. Sample preparation of recombinant antibodies

[0147] HEK293 cells were revived early and passaged to a 200ml volume to achieve a cell density of 3–5 × 10⁻⁶ cells / mL. 6 Cell density reached the required antibody concentration and cell viability >95%; cells were washed by centrifugation, reconstituted with culture medium, and the cell density was adjusted to 2.9 × 10⁻⁶ cells / ml. 6 Cells were washed at a concentration of cells / ml and reconstituted with culture medium, which served as a cell dilution buffer. Plasmid DNA and transfection reagent dilution buffers were prepared separately using culture medium. The transfection reagent dilution buffer was added to the plasmid DNA dilution buffer, mixed well, and incubated at room temperature for 15 min. This mixture was then slowly added to the cell dilution buffer over 1 min, mixed well, and samples were taken for cell counting. Cell viability after transfection was recorded and observed. The cells were then incubated at 35°C with a rotation speed of 120 rpm and a CO2 concentration of 8%. After 13 days, the samples were centrifuged and collected. The supernatant was purified using a protein A affinity chromatography column. 6 μg of the purified antibody was subjected to reducing SDS-PAGE, as shown in the figure. The reducing SDS-PAGE showed two bands: one with a Mr of 50 kDa (heavy chain) and the other with a Mr of 28 kDa (light chain).

[0148] The resulting antibody was named Anti-P24 16H9Rmb1, and its heavy chain (H) and light chain (L) sequences are shown in SEQ ID NO:18 and SEQ ID NO:20, respectively. Anti-P24 16H9Rmb1 was mutated to obtain Anti-P24 8D11Rmb1, and its heavy chain (H) and light chain (L) sequences are shown in SEQ ID NO:18 and SEQ ID NO:23, respectively.

[0149] 1. Affinity Analysis

[0150] Using the AMC sensor, the purified antibody was diluted to 10ug / ml with PBST, and the P24 antigen (obtained from Phytobio) was serially diluted with PBST.

[0151] Operating procedure: Equilibrate in PBST for 60 s, immobilize antibody in antibody solution for 300 s, incubate in PBST for 180 s, bind in antigen solution for 420 s, dissociate in PBST for 1200 s, regenerate the sensor using 10 mM pH 1.69 GLY solution and PBST, and output data. (KD represents the equilibrium dissociation constant, i.e., affinity; kon represents the binding rate; kdis represents the dissociation rate. The main components of PBST are Na2HPO4 + NaCl + TW-2O).

[0152] Table 2 Affinity Data

[0153]

[0154]

[0155] 2. Activity identification

[0156] Dilute the P24 recombinant antigen to 3 μg / ml with coating buffer (mainly NaHCO3), add 100 μL per well, and incubate overnight at 4°C. The next day, wash twice with washing buffer (mainly Na2HPO4 + NaCl) and blot dry. Add blocking buffer (20% BSA + 80% PBS), add 120 μL per well, incubate at 37°C for 1 h, and blot dry. Add diluted purified antibody and control antibody, 100 μL / well, incubate at 37°C for 30 min. Wash 5 times with washing buffer and blot dry. Add goat anti-rabbit IgG-HRP, 100 μL per well, incubate at 37°C for 30 min. Wash 5 times with washing buffer and blot dry. Add chromogenic solution A (50 μL / well) and chromogenic solution B (50 μL / well), incubate for 10 min. Add stop solution, 50 μL / well. Read the OD value at 450 nm (630 nm for reference) on the microplate reader.

[0157] Notes: Solution A (main components: citric acid + sodium acetate + acetanilide + urea peroxide); Solution B (main components: citric acid + EDTA·2Na + TMB + concentrated HCl); Stop solution (EDTA·2Na + concentrated H2SO4).

[0158] Table 3 Activity Data

[0159] Concentration (ng / ml) 15.625 7.813 3.906 1.953 0.977 0 Comparison 1.021 0.601 0.346 0.189 0.099 0.012 Anti-P24 16H9Rmb1 1.515 0.831 0.415 0.252 0.138 0.034 Anti-P24 8D11Mb1 1.556 1.05 0.606 0.348 0.197 0.024

[0160] 3. Stability assessment

[0161] The antibodies were placed at 4℃ (refrigerator), -80℃ (refrigerator), and 37℃ (incubator) for 21 days. Samples were taken at 7, 14, and 21 days for observation of their state, and the activity of the 21-day sample was tested. The results showed that no significant changes in protein state were observed under the three testing conditions after 21 days, and the activity did not decrease with increasing testing temperature, indicating that the antibodies were stable. The table below shows the OD results of the enzyme immunoassay for antibody activity after 21 days of testing.

[0162] Table 4. Stability data for Anti-P24 16H9Rmb1

[0163] Sample concentration (ng / ml) 15.625 7.813 0 4℃, 21-day sample 1.531 0.825 0.004 -80℃, 21-day sample 1.527 0.843 0.035 37℃, 21-day sample 1.519 0.84 0.021

[0164] Table 5. Stability data for Anti-P24 8D11Mb1

[0165] Sample concentration (ng / ml) 15.625 7.813 0 4℃, 21-day sample 1.536 1.059 0.032 -80℃, 21-day sample 1.555 1.166 0.017 37℃, 21-day sample 1.539 1.057 0.014

[0166] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0167] The partial amino acid sequences involved in this application are shown in Table 6:

[0168]

Claims

1. An anti-P24 antibody or its antigen-binding fragment, characterized in that, The complementarity-determining regions of the anti-P24 antibody or its antigen-binding fragment are: three complementarity-determining regions of the heavy chain variable region shown in amino acid sequence SEQ ID NO:17 and three complementarity-determining regions of the light chain variable region shown in amino acid sequence SEQ ID NO:19 or SEQ ID NO:22, wherein the complementarity-determining regions of the variable region are defined by any one of the Kabat, Chothia, IMGT, AbM or Contact systems.

2. An anti-P24 antibody or its antigen-binding fragment, characterized in that, The anti-P24 antibody or its antigen-binding fragment includes the following complementarity-determining region: HCDR1, whose amino acid sequence is shown in SEQ ID NO:1; HCDR2, the amino acid sequence of which is shown in SEQ ID NO:2; HCDR3, the amino acid sequence of which is shown in SEQ ID NO:3; LCDR1, whose amino acid sequence is shown in SEQ ID NO:4; LCDR2, whose amino acid sequence is shown in SEQ ID NO:5; and LCDR3, whose amino acid sequence is shown in SEQ ID NO:

6.

3. The anti-P24 antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that, The anti-P24 antibody or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region. The heavy chain variable region includes HFR1, HFR2, HFR3, and HFR4, and the light chain variable region includes LFR1, LFR2, LFR3, and LFR4.

4. The anti-P24 antibody or its antigen-binding fragment according to claim 3, characterized in that, The HFR1 includes SEQ ID NO:7 or an amino acid sequence that has at least 80% identity with it; The HFR2 comprises SEQ ID NO:8 or an amino acid sequence having at least 80% identity with it; The HFR3 includes SEQ ID NO:9 or an amino acid sequence that has at least 80% identity with it; The HFR4 includes SEQ ID NO:10 or an amino acid sequence that is at least 80% identical to it; The LFR1 includes SEQ ID NO:11 or an amino acid sequence that has at least 80% identity with it; The LFR2 includes SEQ ID NO:12 or an amino acid sequence that has at least 80% identity with it; The LFR3 comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:13; and The LFR4 includes SEQ ID NO:14 or an amino acid sequence that is at least 80% identical to it.

5. The anti-P24 antibody or its antigen-binding fragment according to any one of claims 1, 2, and 4, characterized in that, The anti-P24 antibody or its antigen-binding fragment has a KD < 9.42 × 10⁻⁶. -7 M binds to P24 with affinity.

6. An anti-P24 antibody or its antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region, characterized in that, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:17, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:19 or SEQ ID NO:

22.

7. The anti-P24 antibody or its antigen-binding fragment according to any one of claims 1, 2, 4, and 6, characterized in that, The anti-P24 antibody or its antigen-binding fragment also includes a constant region.

8. The anti-P24 antibody or its antigen-binding fragment according to claim 7, characterized in that, The constant region includes the heavy chain constant region and the light chain constant region.

9. The anti-P24 antibody or its antigen-binding fragment according to claim 8, characterized in that, The heavy chain constant region is selected from any one of the heavy chain constant regions of IgG, IgA, IgM, IgE, and IgD, or a combination of multiple constant region segments.

10. The anti-P24 antibody or its antigen-binding fragment according to claim 8, characterized in that, The heavy chain constant region includes CH1 of IgG, the hinge region of IgG, CH2 of IgM, CH3 of IgM, and / or CH4 of IgM.

11. The anti-P24 antibody or its antigen-binding fragment according to claim 7, characterized in that, The species source of the constant region is cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, or humans.

12. The anti-P24 antibody or its antigen-binding fragment according to claim 7, characterized in that, The species origin of the constant region is rabbit.

13. The anti-P24 antibody or its antigen-binding fragment according to claim 8, characterized in that, The heavy chain constant region sequence is as shown in SEQ ID NO:15 or has at least 80% identity with it.

14. The anti-P24 antibody or its antigen-binding fragment according to claim 8, characterized in that, The light chain constant region sequence is as shown in SEQ ID NO:16 or has at least 80% identity with it.

15. The anti-P24 antibody or its antigen-binding fragment according to any one of claims 1, 2, 4, and 6, characterized in that, The antigen-binding fragment is selected from any one of the antibody's F(ab')2, Fab', Fab, Fv, and scFv.

16. An anti-P24 antibody or an antigen-binding fragment thereof, said anti-P24 antibody comprising a heavy chain and a light chain, characterized in that, The amino acid sequence of the heavy chain is shown in SEQ ID NO:18; the amino acid sequence of the light chain is shown in SEQ ID NO:20 or SEQ ID NO:

23.

17. An antibody conjugate, characterized in that, The antibody-drug conjugate comprises the anti-P24 antibody or its antigen-binding fragment as described in any one of claims 1 to 16, and biotin, a label, or a solid-phase carrier conjugated to the anti-P24 antibody or its antigen-binding fragment.

18. The antibody conjugate according to claim 17, characterized in that, The markers are selected from fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent reagents, and nanoparticle markers.

19. A reagent or kit, characterized in that, The reagent or kit comprises the anti-P24 antibody or its antigen-binding fragment as described in any one of claims 1 to 16, or the antibody conjugate as described in claim 17 or 18.

20. A nucleic acid, characterized in that, It encodes the antibody as described in any one of claims 1 to 16.

21. A carrier, characterized in that, It contains the nucleic acid as described in claim 20.

22. A cell, characterized in that, It contains the nucleic acid as described in claim 20 or the vector as described in claim 21.

23. A method for preparing the anti-P24 antibody according to any one of claims 1 to 16, characterized in that, It includes: Culture the cells as described in claim 22.

24. Use of the anti-P24 antibody or antigen-binding fragment thereof according to any one of claims 1-16, the antibody conjugate according to claim 17 or 18, or the reagent or kit according to claim 19 in the preparation of products for detecting P24 or HIV.

25. The use according to claim 24, characterized in that, include: a) Under conditions sufficient to induce an antibody / antigen binding reaction, the anti-P24 antibody or its antigen-binding fragment according to any one of claims 1-16, the antibody conjugate according to claim 17 or 18, or the reagent or kit according to claim 19 is contacted with P24 in the sample to be tested to form an immune complex; and b) Detect the presence of the immune complex, the presence of which indicates the presence of P24 or HIV in the test sample.

26. The use according to claim 25, characterized in that, The immune complex further includes a second antibody, which binds to the anti-P24 antibody or its antigen-binding fragment.

27. The use according to claim 25, characterized in that, The immune complex also includes a second antibody that binds to P24.

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