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] The present application relates to the field of antibody technology, in particular, to an anti-P24 antibody and application thereof. BACKGROUND

[0002] Human immunodeficiency virus (HIV), also known as acquired immune deficiency syndrome virus, is a virus that causes human immune system deficiency. It is a lentivirus that infects human immune system cells and belongs to a type of retrovirus. HIV is the pathogen of AIDS and is mainly transmitted through sexual contact, blood and mother-to-child transmission. In recent years, the number of HIV-infected patients has been on the rise. At present, the determination of serum HIV antibody is a routine experimental method for diagnosing HIV infection, but there are limitations in determining HIV antibody: more than 70% of HIV-infected persons can be detected for antibodies after 6 months of infection, and this number is more than 80% in the homosexual population, and the detection of antibodies increases the risk of HIV "window period" transmission; in addition, newborns need to be born for one year to produce antibodies, and HIV antibodies from the mother will cause false positives; because HIV antibodies persist during the course of the disease, they disappear only in the late stages of AIDS, and cannot be used as a stable indicator for treatment monitoring.

[0003] P24 is the main structural protein of HIV viral particles and is the product of the structural gene GAG, which plays an important role in the packaging and maturation of the virus. The amino acid sequence of P24 protein is highly conserved among HIV strains, and the deletion of P24 will result in the failure of virus assembly. P24 protein is highly specific and has no cross-reaction with most other retroviruses. When HIV infects the human body, the first viral marker appearing in the blood of the infected person is the viral P24 protein, and there is a long window period between virus infection and detection of HIV antibodies, so detecting HIV-P24 antigen has played an important role in early diagnosis of HIV infection, prognosis judgment of patients, screening and evaluation of anti-HIV drugs, and discovery of mother-to-child transmission, etc.

[0004] The detection of P24 antigen uses serological diagnosis methods, mainly including double antibody sandwich ELISA method, immune complex lysis detection method, super-sensitive EIA method, enzyme-linked immunofluorescence method, etc. At present, the double antibody sandwich method is commonly used to detect human immunodeficiency virus P24 antigen, and the antibody against P24 is the key to realize the detection of the double antibody sandwich method. Therefore, there is a strong demand in the art for an antibody that effectively binds to P24 and detects it. SUMMARY

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

[0006] To achieve the above-mentioned purpose, according to one aspect of the present application, an anti-P24 antibody or antigen-binding fragment thereof is provided, which has three complementarity determining regions of a heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 17 and three complementarity determining regions of a light chain variable region shown in the amino acid sequence of SEQ ID NO: 19.

[0007] To achieve the above-mentioned purpose, according to a second aspect of the present application, an anti-P24 antibody or antigen-binding fragment thereof is provided, which comprises the following complementarity determining regions:

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

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

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

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

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

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

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

[0015] To achieve the above-mentioned purpose, according to a fourth aspect of the present application, an anti-P24 antibody or antigen-binding fragment thereof is provided, which comprises a heavy chain and / or a light chain, 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.

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

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

[0018] To achieve the above object, according to a seventh aspect of the present application, there is provided a method of detecting P24 or HIV, comprising: a) contacting the anti-P24 antibody or antigen-binding fragment thereof, the antibody conjugate, or the reagent or kit described above with P24 in a sample to be tested under conditions sufficient for an antibody / antigen binding reaction to occur to form an immunocomplex; and b) detecting the presence of the immunocomplex, the presence of the complex indicating the presence of the P24 or HIV in the test sample.

[0019] To achieve the above object, according to an eighth aspect of the present application, there is provided a nucleic acid encoding the anti-P24 antibody or antigen-binding fragment thereof described above.

[0020] To achieve the above object, according to a ninth aspect of the present application, there is provided a vector comprising the nucleic acid described above.

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

[0022] To achieve the above object, according to an eleventh aspect of the present application, there is provided a method of producing the anti-P24 antibody or antigen-binding fragment thereof described above, comprising culturing the cell described above.

[0023] To achieve the above object, according to a twelfth aspect of the present application, there is provided use of the anti-P24 antibody or antigen-binding fragment thereof, the antibody conjugate, or the reagent or kit described above in the manufacture of a product for detecting P24 or HIV. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the embodiments will be briefly introduced as follows. It should be understood that the following drawings only show some of the embodiments of the present application, and therefore should not be considered as a limitation on the scope, and for those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0025] Figure 1Results of reducing SDS-PAGE of Anti-P24 6F6 Rmb1. DETAILED DESCRIPTION

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

[0027] In the present application, the term "antibody" is used in the broadest sense, and can include full-length monoclonal antibodies, bispecific or multispecific antibodies, and chimeric antibodies, as long as they exhibit the desired biological activity.

[0028] In the present application, the term "complementarity determining region", "CDR" or "CDRs" refers to the highly variable regions of the heavy and light chains of immunoglobulins, which refer to the regions containing one or more, or even all, of the amino acid residues that play a major role in the binding of the antibody or antigen binding fragment to its recognized antigen or epitope.

[0029] In the present application, the heavy chain complementarity determining region is denoted as HCDR, which includes HCDR1, HCDR2 and HCDR3; the light chain complementarity determining region is denoted as LCDR, which includes LCDR1, LCDR2 and LCDR3.

[0030] The definition method of CDR is well known in the art, and the CDR definition method includes: Kabat definition, Chothia definition, IMGT definition, Contact definition and AbM definition. As described herein, the "Kabat definition" refers to the definition system described in Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). The "Chothia definition" is described in Chothia et al., J Mol Biol 196:901-917 (1987). There can be other CDR definition methods that do not strictly follow one of the above schemes, but still overlap at least part of the CDR regions defined by Kabat, although they can be shortened or lengthened according to predictions or experimental results of specific residues or groups of residues. Exemplary defined CDRs are listed in Table 1 below, and the definitions in different literatures are slightly different. In the case of the variable region amino acid sequence of a given antibody, a person skilled in the art can routinely determine which residues contain a particular CDR. It should be noted that the CDRs defined by other methods in Table 1 are also within the protection scope of the present disclosure.

[0031] Table 1: CDR definitions 1

[0032] CDR Kabat AbM2 IMGT Chothia HCDR1 [H31-H35 3 ]] [H26-H35 3 ]] [H26~H33..5 5 ]] H26-H32..34 4 ]] 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

[0033] 1 The numbering of all CDR definitions in Table 1 is according to the Kabat numbering system (see below), with "H+ numbers" for amino acid positions on the heavy chain and "L+ numbers" for amino acid positions on the light chain. One of ordinary skill in the art can unambiguously correlate this Kabat numbering system to any variable region sequence without reliance on any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0034] 2 "AbM" as used in Table 1 with a lower case "b" refers to CDRs defined by the "AbM" antibody modeling software of Oxford Molecular.

[0035] 3 If neither H35A nor H35B is present, then CDR-H1 ends at position 35; if only H35A is present, then CDR-H1 ends at position 35A; if both H35A and H35B are present, then CDR-H1 ends at position 35B.

[0036] 4 If neither H35A nor H35B is present, then CDR-H1 ends at position 32; if only H35A is present, then CDR-H1 ends at position 33; if both H35A and H35B are present, then CDR-H1 ends at position 34.

[0037] 5 If neither H35A nor H35B is present, then CDR-H1 ends at position 33; if only H35A is present, then CDR-H1 ends at position 34; if both H35A and H35B are present, then CDR-H1 ends at position 35.

[0038] According to embodiments of the application, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3 is defined by any one of the Kabat, Chothia, IMGT, AbM, or Contact systems, or a combination of multiple systems.

[0039] In some embodiments of the application, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Chothia system.

[0040] In some embodiments of the application, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Chothia system.

[0041] In some embodiments of the application, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the IMGT system.

[0042] In some embodiments of the application, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the AbM system.

[0043] In some embodiments of the application, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Contact system.

[0044] In some embodiments of the application, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by a combination of the Kabat, Chothia, IMGT, AbM or Contact system.

[0045] According to embodiments of the application, the Kabat, Chothia, AbM or IMGT system defined HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 amino acid sequence corresponds to the Kabat numbering position as follows:

[0046] 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

[0047] In some embodiments of the application, the anti-P24 antibody or antigen binding fragment thereof comprises the following complementarity determining regions:

[0048] HCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 1;

[0049] HCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 2;

[0050] HCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 3;

[0051] LCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 4;

[0052] LCDR2, which comprises or consists of the amino acid sequence set forth in SEQ ID NO: 5; and

[0053] LCDR3, which comprises or consists of the amino acid sequence set forth in SEQ ID NO: 6.

[0054] In a second aspect, embodiments of the present application provide an anti-P24 antibody or antigen binding fragment thereof, comprising the following complementarity determining regions:

[0055] HCDR1, which comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1;

[0056] HCDR2, which comprises or consists of the amino acid sequence set forth in SEQ ID NO: 2;

[0057] HCDR3, which comprises or consists of the amino acid sequence set forth in SEQ ID NO: 3;

[0058] LCDR1, which comprises or consists of the amino acid sequence set forth in SEQ ID NO: 4;

[0059] LCDR2, which comprises or consists of the amino acid sequence set forth in SEQ ID NO: 5; and

[0060] LCDR3, which comprises or consists of the amino acid sequence set forth in SEQ ID NO: 6.

[0061] According to embodiments of the present application, the HCDRs and LCDRs are defined by the Kabat system.

[0062] In the present application, the "framework region" or "FR" region, which includes the heavy chain framework region and the light chain framework region, refers to the region of the antibody heavy chain variable region and the light chain variable region other than the CDR; wherein the heavy chain framework region can be further subdivided into adjacent regions separated by CDRs, comprising HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework region can be further subdivided into adjacent regions separated by CDRs, comprising LFR1, LFR2, LFR3 and LFR4 framework regions.

[0063] In the present application, the heavy chain variable region is obtained by arranging and connecting the following numbered CDRs and FRs in the following combination: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; and the light chain variable region is obtained by arranging and connecting the following numbered CDRs and FRs in the following combination: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0064] In optional embodiments, the anti-P24 antibody or antigen-binding fragment thereof of the first aspect or the second aspect comprises a heavy chain variable region comprising HFR1, HFR2, HFR3, HFR4, and a light chain variable region comprising LFR1, LFR2, LFR3, and LFR4.

[0065] In optional embodiments, the HFR1 comprises / is an amino acid sequence of SEQ ID NO: 7 or at least 80% identical thereto;

[0066] the HFR2 comprises / is an amino acid sequence of SEQ ID NO: 8 or at least 80% identical thereto;

[0067] the HFR3 comprises / is an amino acid sequence of SEQ ID NO: 9 or at least 80% identical thereto;

[0068] the HFR4 comprises / is an amino acid sequence of SEQ ID NO: 10 or at least 80% identical thereto;

[0069] the LFR1 comprises / is an amino acid sequence of SEQ ID NO: 11 or at least 80% identical thereto;

[0070] the LFR2 comprises / is an amino acid sequence of SEQ ID NO: 12 or at least 80% identical thereto;

[0071] the LFR3 comprises / is an amino acid sequence of SEQ ID NO: 13 or at least 80% identical thereto; and

[0072] the LFR4 comprises / is an amino acid sequence of SEQ ID NO: 14 or at least 80% identical thereto.

[0073] It is noted that in other embodiments, each of the framework regions of the anti-P24 antibody or antigen-binding fragment thereof provided herein can 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 to the corresponding framework region (SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, or 14).

[0074] In optional embodiments, the anti-P24 antibody or antigen-binding fragment thereof binds to P24 with a KD<8.44 x 10 -7 M.

[0075] 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.

[0076] In an optional embodiment, the anti-P24 antibody or its antigen-binding fragment has a KD ≤ 1.75 × 10⁻⁶. -8 M binds to P24 with affinity.

[0077] 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).

[0078] 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.

[0079] 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.

[0080] 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.

[0081] In an optional implementation, the constant region includes a heavy chain constant region and / or a light chain constant region.

[0082] 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.

[0083] 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.

[0084] In an alternative embodiment, the IgG is selected from IgGl, IgG2, IgG3 or IgG4.

[0085] In an alternative embodiment, the light chain constant region is selected from a kappa or lambda light chain constant region.

[0086] In an alternative embodiment, the species origin of the constant region is bovine, equine, bovine, porcine, ovine, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose, turkey, cock or human.

[0087] In an alternative embodiment, the species origin of the constant region is rabbit.

[0088] In an alternative embodiment, the heavy chain constant region sequence (CH) is as set forth in SEQ ID NO: 15 and the light chain constant region (CL) sequence is as set forth in SEQ ID NO: 16.

[0089] It is noted that in other embodiments, the constant region sequence can 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 to the constant region set forth in SEQ ID NO: 15 or 16.

[0090] In an alternative embodiment, the antigen binding fragment is selected from any one of F(ab)2, F(ab')2, Fab', Fab, Fv and scFv of the antibody.

[0091] The antigen binding fragment of the antibody described above generally has the same binding specificity as the antibody from which it is derived. It is readily understood by one skilled in the art that the antigen binding fragment of the antibody described above can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by methods of chemical reduction to split disulfide bonds, based on the structure of the complete antibody disclosed in the present application. The antigen binding fragment described above can be readily obtained by one skilled in the art based on the structure of the complete antibody disclosed in the present application.

[0092] The antigen binding fragment of the antibody described above can also be obtained by recombinant genetic techniques also known to one skilled in the art or by synthesis, for example, using an automatic peptide synthesizer, such as an automatic peptide synthesizer sold by Applied BioSystems and the like.

[0093] In an alternative embodiment, the anti-P24 antibody or antigen-binding fragment thereof of the first, second or third aspect described above comprises a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 18 and / or a light chain having an amino acid sequence as set forth in SEQ ID NO: 20.

[0094] In a fourth aspect, the present application provides an anti-P24 antibody or antigen-binding fragment thereof comprising a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 18 and / or a light chain having an amino acid sequence as set forth in SEQ ID NO: 20.

[0095] In a fifth aspect, the present application provides an antibody conjugate comprising the anti-P24 antibody or antigen-binding fragment thereof described above.

[0096] In an alternative embodiment, the antibody conjugate described above further comprises biotin or a biotin derivative conjugated to the anti-P24 antibody or antigen-binding fragment thereof.

[0097] In an alternative embodiment, the antibody conjugate described above further comprises a label conjugated to the anti-P24 antibody or antigen-binding fragment thereof.

[0098] In an alternative embodiment, the label described above refers to a type of substance having a property such as luminescence, color development, radioactivity, etc. that can be directly observed by the naked eye or detected or probed by an instrument, and through which qualitative or quantitative detection of the corresponding target substance can be achieved.

[0099] In an alternative embodiment, the label described above includes, but is not limited to, a fluorescent dye, an enzyme, a radioisotope, a chemiluminescent reagent, and a nanoparticle-based label.

[0100] In actual use, a person skilled in the art can select a suitable label according to the detection conditions or actual needs, and regardless of the label used, it falls within the protection scope of the present application.

[0101] In optional embodiments, the fluorescent dye includes, but is not limited to, fluorescein-based dyes and their derivatives (such as, but not limited to, fluorescein isothiocyanate (FITC), hydroxycoumarin (FAM), tetra-chloro-fluorescein (TET), and the like or analogs thereof), rhodamine-based dyes and their derivatives (such as, but not limited to, red rhodamine (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), and the like or analogs thereof), Cy series dyes and their derivatives (such as, but not limited to, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy3, and the like or analogs thereof), Alexa series dyes and their derivatives (such as, but not limited to, Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, and the like or analogs thereof), and protein-based dyes and their derivatives (such as, but not limited to, phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (preCP), and the like).

[0102] In optional embodiments, the enzyme includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphogluconate dehydrogenase.

[0103] In optional embodiments, the radioisotope includes, but is 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.

[0104] In optional embodiments, the chemiluminescent reagent includes, but is not limited to, luminol and its derivatives, lucigenin, crustacyanin, ruthenium bispyridyl and its derivatives, acridan ester and its derivatives, dioxetane and its derivatives, luminol and its derivatives, and peroxyoxalate and its derivatives.

[0105] In optional embodiments, the nanoparticle-based label includes, but is not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0106] In optional embodiments, the colloid includes, but is not limited to, colloidal metals, dispersed dyes, dye-labeled microspheres, and latex.

[0107] In optional embodiments, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver and colloidal selenium.

[0108] In optional embodiments, the colloidal metal is colloidal gold.

[0109] In optional embodiments, the antibody conjugate further comprises a solid support coupled to the anti-P24 antibody or antigen-binding fragment thereof.

[0110] In optional embodiments, the solid support is selected from the group consisting of microspheres, plates and membranes.

[0111] In optional embodiments, the solid support includes, but is not limited to, magnetic microspheres, plastic microspheres, plastic microparticles, microwell plates, glass, capillary tubes, nylon and nitrocellulose membranes.

[0112] In a sixth aspect, the present application provides a reagent or kit comprising the anti-P24 antibody or antigen-binding fragment thereof or the antibody conjugate as described above.

[0113] As described above, the anti-P24 antibody or antigen-binding fragment thereof in some embodiments or examples of the present application can effectively bind to P24, and thus the reagent or kit comprising the P24 anti-P24 antibody or antigen-binding fragment thereof can effectively qualitatively or quantitatively detect P24. The reagent or kit provided by the present application can be used in, for example, immunoblotting, immunoprecipitation and other detection methods involving the specific binding of P24 and its antibody. As described above, the anti-P24 antibody or antigen-binding fragment thereof in some embodiments or examples of the present application has higher binding activity or affinity to P24, and thus the reagent or kit comprising the anti-P24 antibody or antigen-binding fragment thereof has higher detection sensitivity or specificity.

[0114] In a seventh aspect, the present application provides a method for detecting P24 or HIV, comprising: a) contacting the anti-P24 antibody or antigen-binding fragment thereof, the antibody conjugate, the reagent or the kit as described above with P24 in a sample to be tested under conditions sufficient to allow an antibody / antigen binding reaction to occur to form an immunocomplex; and b) detecting the presence of the immunocomplex, wherein the presence of the complex indicates the presence of the P24 or HIV in the test sample.

[0115] In optional embodiments, the immunocomplex further comprises a second antibody that binds to the anti-P24 antibody or antigen-binding fragment thereof.

[0116] In optional embodiments, the immunocomplex further comprises a second antibody that binds to P24.

[0117] In an eighth aspect, the present application provides a nucleic acid molecule encoding the anti-P24 antibody or antigen-binding fragment thereof.

[0118] In a ninth aspect, the present application provides a vector comprising the nucleic acid molecule.

[0119] In a tenth aspect, the present application provides a cell comprising the vector.

[0120] In an eleventh aspect, the present application provides a method for preparing the anti-P24 antibody or antigen-binding fragment thereof, comprising culturing the cell as described above.

[0121] In a twelfth aspect, the present application provides use of the anti-P24 antibody or antigen-binding fragment thereof, antibody conjugate, or reagent or kit as described above in the manufacture of a product for detecting P24 or HIV.

[0122] In a thirteenth aspect, the present application provides use of the anti-P24 antibody or antigen-binding fragment thereof, antibody conjugate, or reagent or kit as described above in the detection of P24 or HIV-related diseases.

[0123] Based on the amino acid sequences of the anti-P24 antibody or antigen-binding fragment thereof disclosed in the present application, those skilled in the art can easily conceive of using genetic engineering technology or other technologies (chemical synthesis, recombinant expression) to prepare the anti-P24 antibody or antigen-binding fragment thereof, for example, by isolating and purifying the anti-P24 antibody or antigen-binding fragment thereof from the culture product of recombinant cells capable of recombinantly expressing the anti-P24 antibody or antigen-binding fragment thereof as described in any one of the above, which is easily achieved by those skilled in the art. Therefore, regardless of the technology used to prepare the anti-P24 antibody or antigen-binding fragment thereof of the present application, it falls within the scope of the present application.

[0124] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below. Unless otherwise specified, the specific conditions in the embodiments are carried out according to conventional conditions or manufacturer's recommended conditions. Unless otherwise specified, the reagents or instruments used are conventional products that can be obtained by market purchase.

[0125] 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 belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the preparations or unit doses herein, some methods and materials are now described. The techniques employed or contemplated herein are standard methodologies unless otherwise indicated. Materials, methods, and examples are illustrative only and not limiting.

[0126] Unless otherwise indicated, the practice of the present application will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, Second Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Animal Cell Culture (R. I. Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); and Current Protocols in Immunology (J. E. Coligan et al., eds., 1991), each of which is incorporated herein by reference in its entirety.

[0127] The features and advantages of the present application will be further described in the following detailed description of the application with reference to the accompanying drawings.

[0128] Example 1 Preparation of Anti-P24 6F6 Monoclonal Antibody

[0129] Restriction enzymes and Prime Star DNA polymerase used in this example were purchased from Takara. MagExtractor-RNA extraction kit was purchased from TOYOBO. BD SMART TMThe RACE cDNA Amplification Kit was purchased from Takara Company. The plasmid extraction kit was purchased from Tiangen Company. The primer synthesis and gene sequencing were completed by Invitrogen Company. The rabbit monoclonal antibody against P24 was obtained by hybridoma technology, phage display technology or single B cell cloning technology. The specific implementation method of the phage display technology is as follows:

[0130] 1. Gene acquisition

[0131] (1) Immunization of rabbits

[0132] An emulsified preparation of P24 prepared with incomplete Freund's adjuvant was used to stimulate the immune response of 4-6 week old New Zealand white rabbits by subcutaneous injection, and the pre-immune and post-immune sera were collected on days 0, 14, 28, 42 and 69, respectively. The rabbit spleen was also surgically removed to prepare a spleen cell suspension.

[0133] (2) Construction of phage library

[0134] RNA was extracted from rabbit spleen cells and reverse transcribed into cDNA. The rabbit antibody gene amplification primers were specifically designed, and the VHand VLgene fragments were amplified using the cDNA as a template. Then, the VHand VLgene fragments were sequentially inserted into the phage vector V02 by enzyme digestion and ligation. Finally, the ligated phage plasmid was electroporated into TG1 competent cells, and single clone colonies were selected for PCR identification the next day. Antibody gene sequencing was performed to evaluate the quality of the phage library, and the qualified phage library was screened.

[0135] (3) Screening of phage library

[0136] The phage library TG1 bacterial liquid was inoculated into a flask and cultured to an appropriate bacterial liquid concentration (OD600 of 0.8-1.0). Then, helper phage was added for 1 hour of infection, and then the culture was continued overnight. The next day, the bacterial liquid was centrifuged, and the supernatant was collected. The displayed phage library was purified by salt precipitation.

[0137] The phage library was subjected to 3-4 rounds of magnetic bead panning. Then, single clone phage infection colonies were selected for antibody supernatant expression. Subsequently, ELISA screening was used to screen and identify the monoclonal phage antibody expression supernatant, and the monoclonal phage against P24 was obtained.

[0138] Phage antibody gene sequencing: The monoclonal phage against P24 was subjected to antibody gene sequencing, and through sequence analysis, repeated and invalid sequences were removed to obtain unique rabbit monoclonal antibody sequences.

[0139] 2. Construction of expression plasmid

[0140] 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.

[0141] 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.

[0142] 3. Sample preparation of recombinant antibodies

[0143] 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).

[0144] The resulting antibody was named Anti-P24 6F6Rmb1, and its heavy chain (H) and light chain (L) sequences are shown in SEQ ID NO:18 and SEQ ID NO:20, respectively.

[0145] 1. Affinity Analysis

[0146] Using AMC sensor, purified antibody was diluted to 10 ug / ml with PBST, P24 antigen (obtained from FIP Biotech) was gradient diluted with PBST.

[0147] Running procedure: equilibration in buffer 1 (PBST) for 60 s, antibody immobilization in antibody solution for 300 s, incubation in buffer 2 (PBST) for 180 s, binding in antigen solution for 420 s, dissociation in buffer 2 for 1200 s, sensor regeneration with 10 mM pH 1.69 GLY solution and buffer 3 (PBST), and output data. (KD represents the equilibrium dissociation constant, i.e. affinity; kon represents the association rate; kdis represents the dissociation rate. PBST mainly consists of Na2HPO4+NaCl+TW-20).

[0148] Table 2 Affinity data

[0149] Sample name KD(M) kon(1 / Ms) kdis(1 / s) Control 8.44E-07 1.08E+04 9.12E-03 Anti-P24 6F6 Mb1 1.75E-08 5.92E+04 1.04E-03

[0150] 2. Activity identification

[0151] Coating solution (mainly consisting of NaHCO3) was used to dilute P24 recombinant antigen to 3 ug / ml, 100 uL per well, and incubated at 4°C overnight; the next day, washing solution (mainly consisting of Na2HPO4+NaCl) was used to wash twice, and then dried by tapping; blocking solution (20% BSA+80% PBS) was added, 120 uL per well, and incubated at 37°C for 1 h, and then dried by tapping; diluted purified antibody and control antibody were added, 100 uL per well, and incubated at 37°C for 30 min; washing solution was used to wash 5 times, and then dried by tapping; goat anti-rabbit IgG-HRP was added, 100 uL per well, and incubated at 37°C for 30 min; washing solution was used to wash 5 times, and then dried by tapping; color developing solution A (50 uL per well) was added, color developing solution B (50 uL per well) was added, and incubated for 10 min; stop solution was added, 50 uL per well; and OD value was read on a microplate reader at 450 nm (reference 630 nm).

[0152] Note: Solution A (mainly consisting of citric acid+NaAc+acetanilide+urea peroxide); solution B (mainly consisting of citric acid+EDTA·2Na+TMB+concentrated HCL); stop solution (EDTA·2Na+concentrated H2SO4).

[0153] Table 3 Activity data

[0154]

[0155]

[0156] 3. Stability evaluation

[0157] The above antibody was placed at 4°C (refrigerator), -80°C (refrigerator), 37°C (incubator) for 21 days, and samples taken at 7 days, 14 days, and 21 days were observed for state, and the 21-day sample was detected for activity. The results showed that under the three test conditions, the antibody was not observed to have obvious protein state changes after 21 days, and the activity did not show a downward trend with the increase of the test temperature, indicating that the above antibody is stable. The following table is the enzyme immunoactivity detection OD result of the antibody Anti-P24 6F6Rmb1 after 21 days of testing.

[0158] Table 4 Stability data

[0159] Sample concentration (ng / ml) 31.250 15.625 0.000 4℃, 21 days sample 1.433 0.823 0.002 -80℃, 21 days sample 1.419 0.831 0.030 37℃, 21 days sample 4℃, 21 days sample -80℃, 21 days sample 37℃, 21 days sample 1.438 0.829 0.001

[0160] The preferred embodiments of the present application have been described above with reference to the drawings, but the present application is not limited to the above examples, and various modifications and changes can be made by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

[0161] Part of the amino acid sequences involved in the present application are shown in Table 5:

[0162]

[0163]

Claims

1. An anti-P24 antibody or its antigen-binding fragment, characterized in that, The anti-P24 antibody or its antigen-binding fragment contains three complementary determinant regions of the heavy chain variable region as shown in SEQ ID NO:17 and three complementary determinant regions of the light chain variable region as shown in SEQ ID NO:

19. The complementary determination region of the variable region is defined by any one of the systems Kabat, Chothia, IMGT, or AbM.

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 antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that, The antibody or its antigen-binding fragment 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.

4. The 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 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 < 8.44 × 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; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

19.

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

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

13. The 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 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 antibody or antigen-binding fragment thereof 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 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.

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 conjugated to the anti-P24 antibody or its antigen-binding fragment.

18. 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 a label or solid-phase carrier conjugated to the anti-P24 antibody or its antigen-binding fragment.

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

20. 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 any one of claims 17 to 19.

21. Use of the anti-P24 antibody or antigen-binding fragment thereof according to any one of claims 1 to 16, the antibody conjugate according to any one of claims 17 to 19, or the reagent or kit according to claim 20 in the preparation of products for detecting P24 or HIV.

22. The use according to claim 21, characterized in that it comprises: 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 to 16, the antibody conjugate according to any one of claims 17 to 19, or the reagent or kit according to claim 20 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 sample to be tested.

23. The use according to claim 21, characterized in that the immune complex further comprises a second antibody, the second antibody being bound to the anti-P24 antibody or its antigen-binding fragment.

24. The use according to claim 21, characterized in that the immune complex further comprises a second antibody, the second antibody being bound to P24.

25. A nucleic acid, characterized in that, It encodes the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 16.

26. A carrier, characterized in that, It contains the nucleic acid as described in claim 25.

27. A cell characterized in that, It contains the nucleic acid as described in claim 25 or the vector as described in claim 26.

28. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 16, characterized in that, It includes: Culture the cells as described in claim 27.

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