Anti-P24 antibody and application thereof

Abstract

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

Description

Technical Field

[0001] The present invention relates to the technical field of antibodies, and in particular, to an antibody against P24 and its application. Background Art

[0002] Human Immunodeficiency Virus (HIV), namely the AIDS virus (AIDS), also known as the acquired immunodeficiency syndrome virus, is a virus that causes defects in the human immune system. In 1981, the human immunodeficiency virus was first discovered in the United States. It is a lentivirus that infects human immune system cells and belongs to a type of retrovirus.

[0003] HIV is the pathogen of AIDS and is mainly transmitted through sexual contact, blood, and mother-to-child transmission. Currently, measuring serum HIV antibodies is a conventional experimental method for diagnosing HIV infection, but there are limitations in measuring HIV antibodies: more than 70% of HIV-infected individuals can only detect antibodies 6 months after infection, and the antibody detection method increases the risk of transmission during the "window period" of HIV; in addition, newborns need 1 year after birth to produce antibodies, and HIV antibodies from the mother can cause false positives; due to the continuous presence of HIV antibodies during the disease process, they only disappear in the late stage of AIDS and cannot be used as a stable indicator for treatment monitoring.

[0004] P24 is the main structural protein of HIV virus particles, is the product of the structural gene GAG, and plays an important role in the packaging and maturation of the virus. The amino acid sequence of the P24 protein is highly conserved among various HIV strains, and the deletion of P24 will cause the virus to be unable to assemble normally. The P24 protein has strong specificity and has no cross-reaction with most other retroviruses. When HIV infects the human body, the first viral marker to appear in the blood of infected individuals is the viral P24 protein. There is a long window period between virus infection and the detection of HIV antibodies. Therefore, the detection of HIV-P24 antigen has played an important role in the early diagnosis of HIV infection, the prognosis judgment of patients, the screening and evaluation of anti-HIV drugs, and the discovery of mother-to-child transmission.

[0005] The detection of HIV-1 P24 antigen uses serological diagnostic methods, mainly including double antibody sandwich ELISA, immunocomplex lysis detection, ultrasensitive EIA, enzyme-linked immunofluorescence method, etc. Currently, the double antibody sandwich method is commonly used to detect the human immunodeficiency virus P24 antigen, and obtaining an antibody against P24 is the key to realizing the double antibody sandwich method for detection.

[0006] Therefore, there is a strong demand in the art for anti-P24 antibodies with good performance. Summary of the Invention

[0007] The present application provides an antibody against P24, which provides an important raw material source for the detection of P24 and has good activity or affinity.

[0008] To achieve the above object, according to one aspect of the present invention, there is provided an antibody against P24, which comprises three complementary determining regions of a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 19 and three complementary determining regions of any one of the light chain variable regions having amino acid sequences SEQ ID NO: 21, 22, 23, 24.

[0009] To achieve the above object, according to a second aspect of the present invention, there is provided an antibody against P24, which comprises the following complementary determining regions:

[0010] HCDR1, which comprises the amino acid sequence shown in SEQ ID NO: 1 or consists of the same;

[0011] HCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 2 or consists of the same;

[0012] HCDR3, which comprises the amino acid sequence shown in SEQ ID NO: 3 or consists of the same;

[0013] LCDR1, which comprises the amino acid sequence shown in SEQ ID NO: 4 or consists of the same;

[0014] LCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 5 or consists of the same;

[0015] LCDR3, which comprises the amino acid sequence shown in SEQ ID NO: 6 or consists of the same.

[0016] To achieve the above object, according to a third aspect of the present invention, there is provided an antibody against P24, which comprises a heavy chain variable region and / or a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 19; and the amino acid sequence of the light chain variable region is any one of SEQ ID NO: 21, 22, 23, 24.

[0017] To achieve the above object, according to a fourth aspect of the present invention, there is provided an antibody against P24, which comprises a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is any one of SEQ ID NO: 20, 30; and the amino acid sequence of the light chain is any one of SEQ ID NO: 25, 26, 27, 28.

[0018] To achieve the above object, according to a fifth aspect of the present invention, there is provided an antibody conjugate, which comprises the above antibody.

[0019] To achieve the above object, according to the sixth aspect of the present invention, there is provided a reagent or a kit, which comprises the above-mentioned antibody or the above-mentioned antibody conjugate.

[0020] To achieve the above object, according to the eighth aspect of the present invention, there is provided a use of the above-mentioned antibody or antibody conjugate in the preparation of a product for detecting P24.

[0021] To achieve the above object, the present invention further provides a nucleic acid, a vector, a cell and a method for preparing the above-mentioned antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] To more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and thus should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other relevant drawings can also be obtained based on these drawings.

[0023] Figure 1 Results of reducing SDS-PAGE for Anti-P24 20C5 Rmb1 to Anti-P24 20C5 Rmb8. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0024] In the first aspect, an embodiment of the present invention provides an antibody against P24, which comprises three complementarity-determining regions of a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 19 and three complementarity-determining regions of a light chain variable region having any one of the amino acid sequences SEQ ID NO: 21, 22, 23, 24.

[0025] It should be noted that HCDR1, HCDR2 and HCDR3 are amino acid sequences identical to HCDR1, HCDR2, HCDR3 of the same heavy chain variable region defined in the antibody described in the first aspect, and LCDR1, LCDR2 and LCDR3 are amino acid sequences identical to LCDR1, LCDR2, LCDR3 of the same light chain variable region defined in the antibody described in the first aspect.

[0026] For example, HCDR1, HCDR2, HCDR3 are amino acid sequences identical to HCDR1, HCDR2, HCDR3 of the heavy chain variable region shown in SEQ ID NO: 19; LCDR1, LCDR2, LCDR3 are amino acid sequences identical to LCDR1, LCDR2, LCDR3 of the light chain variable region shown in SEQ ID NO: 21.

[0027] In the present invention, the term "antibody" is used in the broadest sense and can include full-length monoclonal antibodies, bispecific, multispecific antibodies, chimeric antibodies or antigen-binding fragments, provided that they exhibit the desired biological activity.

[0028] The above-mentioned antigen-binding fragments generally have the same binding specificity as the antibody from which they are derived. It will be readily understood by those skilled in the art from the content described in the present invention that the above-mentioned antigen-binding fragments can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by chemical reduction to cleave disulfide bonds. Based on the disclosure of the structure of the intact antibody in the present invention, those skilled in the art can easily obtain the above-mentioned antigen-binding fragments.

[0029] The above-mentioned antigen-binding fragments can also be obtained by recombinant genetic techniques known to those skilled in the art or by synthesis using, for example, an automated peptide synthesizer, such as those sold by Applied BioSystems and the like.

[0030] In the present invention, the terms "complementary determining region", "CDR" or "CDRs" refer to the highly variable regions of the heavy and light chains of an immunoglobulin, and refer to regions containing one or more or even all of the major amino acid residues that contribute to the binding of an antibody or antigen-binding fragment to the antigen or epitope it recognizes. In the specific embodiments of the present invention, the CDRs refer to the highly variable regions of the heavy and light chains of the antibody.

[0031] In the present invention, the heavy chain complementary determining regions are denoted as HCDR and include HCDR1, HCDR2 and HCDR3; the light chain complementary determining regions are denoted as LCDR and include LCDR1, LCDR2 and LCDR3.

[0032] The 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, the "Kabat definition" refers to the definition system described by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). For the "Chothia definition", see Chothia et al., J Mol Biol 196: 901-917 (1987). There are other CDR definition methods that may not strictly follow one of the above-mentioned schemes but will still overlap at least a part of the CDR region defined by Kabat, although they may be shortened or lengthened according to the prediction 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. Given the amino acid sequence of the variable region of a given 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 Table 1 also fall within the scope of protection of the present disclosure.

[0033] Table 1: CDR Definitions 1

[0034] CDR Kabat AbM2 IMGT Chothia HCDR1 <![CDATA[H31~H35 3 > <![CDATA[H26~H35 3 > <![CDATA[H26~H33..5 5 > <![CDATA[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

[0035] 1 The numbers of all CDR definitions in Table 1 are based on the Kabat numbering system (see below). The amino acid numbers on the heavy chain are represented by "H + number", and the amino acid numbers on the light chain are represented by "L + number". Those of ordinary skill in the art can clearly map this Kabat numbering system to any variable region sequence without relying on any experimental data outside the sequence itself. As described herein, the "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).

[0036] 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.

[0037] 3If 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.

[0038] 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.

[0039] 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.

[0040] According to an embodiment of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by any one system or a combination of multiple systems among Kabat, Chothia, IMGT, AbM or Contact.

[0041] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat system.

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

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

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

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

[0046] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by a combination of the Kabat, Chothia, IMGT, AbM, or Contact systems.

[0047] In a second aspect, an embodiment of the present invention provides an antibody against P24, the antibody comprising the following complementarity-determining regions:

[0048] HCDR1, which comprises the amino acid sequence shown in SEQ ID NO:1, or consists of the same.

[0049] HCDR2, which comprises the amino acid sequence shown in SEQ ID NO:2, or consists of the same.

[0050] HCDR3, which comprises the amino acid sequence shown in SEQ ID NO:3, or consists of the same.

[0051] LCDR1, which comprises the amino acid sequence shown in SEQ ID NO:4, or consists of the same.

[0052] LCDR2, which comprises the amino acid sequence shown in SEQ ID NO:5, or consists of the same.

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

[0054] According to an embodiment of the present invention, the HCDRs and LCDRs are defined by the Kabat system.

[0055] In the present invention, the "framework region" or "FR" region includes the heavy-chain framework region and the light-chain framework region, which refers to the regions other than the CDRs in the heavy-chain variable region and the light-chain variable region of the antibody; wherein, the heavy-chain framework region can be further subdivided into adjacent regions separated by CDRs, including the HFR1, HFR2, HFR3, and HFR4 framework regions; the light-chain framework region can be further subdivided into adjacent regions separated by CDRs, including the LFR1, LFR2, LFR3, and LFR4 framework regions.

[0056] In the present invention, the heavy-chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combined arrangement: 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 combined arrangement: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0057] In an alternative embodiment, the antibody according to the first or second aspect further has HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4.

[0058] In an alternative embodiment, the HFR1 comprises / is as shown in SEQ ID NO:7 or an amino acid sequence having at least 80% identity thereto;

[0059] The HFR2 comprises / is as shown in SEQ ID NO:8 or an amino acid sequence having at least 80% identity thereto;

[0060] The HFR3 comprises / is as shown in SEQ ID NO:9 or an amino acid sequence having at least 80% identity thereto;

[0061] The HFR4 comprises / is as shown in SEQ ID NO:10 or an amino acid sequence having at least 80% identity thereto;

[0062] The LFR1 comprises / is as shown in SEQ ID NO:11 or an amino acid sequence having at least 80% identity thereto;

[0063] The LFR2 comprises / is as shown in SEQ ID NO:12 or an amino acid sequence having at least 80% identity thereto;

[0064] The LFR3 comprises / is as shown in SEQ ID NO:13 or an amino acid sequence having at least 80% identity thereto; and

[0065] The LFR4 comprises / is as shown in SEQ ID NO:14 or an amino acid sequence having at least 80% identity thereto.

[0066] It should be noted that in other embodiments, the amino acid sequences of each framework region of the anti-P24 antibody 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 framework regions (SEQ ID NO:7, 8, 9, 10, 11, 12, 13, or 14).

[0067] In an alternative embodiment, the LFR3 comprises / is as shown in the amino acid sequence of SEQ ID NO:18.

[0068] In an alternative embodiment, the LFR2 comprises / is as shown in the amino acid sequence of SEQ ID NO:17.

[0069] In an alternative embodiment, the antibody has a KD < 7.15×10-8 The affinity of M binds to P24.

[0070] In an alternative embodiment, the antibody has a KD ≤ 10 -8 M, KD ≤ 10 -9 M, KD ≤ 10 -10 M, KD ≤ 10 -11 M or KD ≤ 10 -12 The affinity of M binds to P24.

[0071] In an alternative embodiment, the antibody has a KD ≤ 1.41×10 -9 The affinity of M binds to P24.

[0072] There are many methods for measuring antibody affinity (KD), which can be classified into thermodynamic detection methods, kinetic detection methods, and dynamic equilibrium detection methods according to the detection principle. Among them, common thermodynamic detection methods include isothermal titration calorimetry (ITC); common kinetic detection methods include surface plasmon resonance (SPR) and biolayer interferometry (BLI); common dynamic equilibrium detection methods include enzyme-linked immunosorbent assay (ELISA), etc.

[0073] In an alternative embodiment, the KD is measured using a kinetic detection method; optionally, surface plasmon resonance, for example, by using a biosensor system such as the system's biosensor system.

[0074] In a third aspect, an embodiment of the present invention provides an antibody against P24, 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 as shown in SEQ ID NO: 19, and the amino acid sequence of the light chain variable region is any one of SEQ ID NOs: 21, 22, 23, and 24.

[0075] In an alternative embodiment, the antibodies described in the above first aspect, second aspect, and third aspect further comprise a constant region.

[0076] In an alternative embodiment, the constant region includes a heavy chain constant region and / or a light chain constant region.

[0077] In an alternative embodiment, the heavy chain constant region is selected from the heavy chain constant region of any one of IgG, IgA, IgM, IgE, IgD or a combination of multiple constant region segments.

[0078] In an alternative 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.

[0079] In an alternative embodiment, the IgG is selected from IgG1, IgG2, IgG3, or IgG4.

[0080] In an alternative embodiment, the light chain constant region is selected from a kappa or lambda light chain constant region.

[0081] In an alternative embodiment, the species origin of the constant region is bovine, equine, dairy cow, porcine, ovine, rat, mouse, canine, camel, feline, rabbit, donkey, deer, mink, chicken, duck, goose, turkey, fighting cock or human.

[0082] In an alternative embodiment, the species origin of the constant region is human.

[0083] In the present disclosure, the division of the variable and constant region sequences follows the IMGT division method, see Lefranc, the international ImMunoGeneTics database. Nucl. Acids Res., 29(1):207-209(2001). DOI:10.1093 / nar / 29.1.207. PMID:11125093. and Martinez-Jean C. and Bosc N. or Ehrenmann, Patrice Duroux, Chantal Ginestoux, Gene table: house mouse (Mus musculus) IGHC, IMGT Repertoire. the international ImMunoGenetics information http: / / www.imgt.org . Created: 16 / 03 / 2011. Version: 17 / 01 / 2020. or Ehrenmann, Patrice Duroux, Chantal Ginestoux, Gene table: house mouse (Mus musculus) IGLC, IMGT Repertoire. the international ImMunoGenetics information http: / / www.imgt.org . Created: 16 / 03 / 2011. Version: 17 / 01 / 2020.. There will be some amino acid differences between the variable regions divided by different methods and the C-terminus of the variable region or the N-terminus of the constant region divided by IMGT. The variable or constant regions divided by other methods well-known in the art are also within the scope of protection of the present invention.

[0084] In an alternative embodiment, the heavy chain constant region sequence (CH) is as shown in SEQ ID NO: 15 or 29, and the light chain constant region (CL) sequence is as shown in SEQ ID NO: 16.

[0085] 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 above constant regions (SEQ ID NO: 15 or 16).

[0086] In an alternative embodiment, the antibody includes any one of F(ab)2, F(ab’)2, Fab’, Fab, Fv and scFv.

[0087] In a fourth aspect, the present invention provides an antibody against P24, comprising a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is as shown in any one of SEQ ID NO: 20 and 30, and the amino acid sequence of the light chain is as shown in any one of SEQ ID NO: 25, 26, 27 and 28.

[0088] In an alternative embodiment, the antibody according to the first, second, third or fourth aspect above comprises a heavy chain and a light chain in any of the following combinations:

[0089]

[0090]

[0091] In a fifth aspect, the present invention provides an antibody conjugate, which comprises the above antibody.

[0092] In an alternative embodiment, the above antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody.

[0093] In an alternative embodiment, the antibody conjugate further comprises a label conjugated to the antibody.

[0094] In an alternative embodiment, the above label refers to a class of substances having characteristics such as luminescence, color development, radioactivity, etc. that can be directly observed by the naked eye or detected or detected by an instrument, and qualitative or quantitative detection of the corresponding target can be achieved through this characteristic.

[0095] In an alternative embodiment, the label includes but is not limited to fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents and nanoparticle-based labels.

[0096] In actual use, those skilled in the art can select appropriate markers according to the detection conditions or actual needs. No matter which marker is used, it falls within the protection scope of the present invention.

[0097] In an alternative embodiment, the fluorescent dyes include but are not limited to fluorescein dyes and their derivatives (such as, but not limited to, fluorescein isothiocyanate (FITC), hydroxy fluorescein (FAM), tetrachloro fluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (such as, but not limited to, rhodamine B isothiocyanate (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (such as, but not limited to, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, etc. or their analogs), Alexa series dyes and their derivatives (such as, but not limited to, Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 633, 647, 680, 700, 750, etc. or their analogs), and protein dyes and their derivatives (such as, but not limited to, phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (PerCP), etc.).

[0098] In an alternative embodiment, the enzymes include but are not limited to horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase.

[0099] In an alternative embodiment, the radioisotopes 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.

[0100] In an alternative embodiment, the chemiluminescent reagents include but are not limited to luminol and its derivatives, lucigenin, crustacean luciferin and its derivatives, ruthenium bipyridine and its derivatives, acridinium ester and its derivatives, dioxetane and its derivatives, rofluorescein and its derivatives, and peroxyoxalate and its derivatives.

[0101] In an alternative embodiment, the nanoparticle-based markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0102] In an alternative embodiment, the colloids include, but are not limited to, colloidal metals, colloidal carbon, disperse dyes, dye-labeled microspheres, and latex.

[0103] In an alternative embodiment, the colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0104] In an alternative embodiment, the colloidal metal is colloidal gold.

[0105] In an alternative embodiment, the above antibody conjugate further includes a solid-phase carrier conjugated to the antibody.

[0106] In an alternative embodiment, the solid-phase carrier is selected from microspheres, plates, and membranes.

[0107] In an alternative embodiment, the solid-phase carrier includes, but is not limited to, magnetic microspheres, plastic microspheres, plastic particles, microtiter plates, glass, capillary tubes, nylon, and nitrocellulose membranes.

[0108] In a sixth aspect, the present invention provides a reagent or a kit, which includes the above-mentioned antibody or the above-mentioned antibody conjugate.

[0109] As mentioned above, the antibodies in some embodiments or examples of the present invention can effectively bind to P24. Therefore, the reagent or kit containing the P24 antibody can effectively qualitatively or quantitatively detect P24. Applying the reagent or kit provided by the present invention, for example, can be used for immunoblotting, immunoprecipitation, etc., which involve detecting the specific binding performance of P24 and its antibody. As mentioned above, the antibodies in some embodiments or examples of the present invention have higher binding activity or affinity with P24. Therefore, the reagent or kit containing the antibody has higher detection sensitivity or specificity.

[0110] In a seventh aspect, the present invention provides a method for detecting P24, including: a) contacting the above-mentioned antibody, antibody conjugate, reagent or kit with P24 in a test sample under conditions sufficient for 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 the antigen in the test sample;

[0111] In an alternative embodiment, the immune complex further includes a second antibody that binds to the antibody.

[0112] In an alternative embodiment, the immune complex further comprises a second antibody that binds to P24.

[0113] In an eighth aspect, the present invention provides the use of the above-mentioned anti-P24 antibody and antibody conjugate in the preparation of a product for detecting P24.

[0114] It should be noted that the products of the present invention include but are not limited to reagents, reagent kits, test strips or detection plates.

[0115] In a ninth aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned antibody.

[0116] In a tenth aspect, the present invention provides a vector containing the above-mentioned nucleic acid molecule.

[0117] In an eleventh aspect, the present invention provides a cell containing the above-mentioned vector.

[0118] In a twelfth aspect, the present invention provides a method for preparing an anti-P24 antibody, which comprises: culturing the cells as described above.

[0119] Based on the disclosure of the amino acid sequence of the anti-P24 antibody in the present invention, those skilled in the art can easily conceive of preparing the anti-P24 antibody by using genetic engineering techniques or other techniques (chemical synthesis, recombinant expression), for example, isolating and purifying the antibody from the culture product of recombinant cells capable of recombinantly expressing the antibody described in any one of the above, which is easily achievable for those skilled in the art. Based on this, no matter what technique is used to prepare the anti-P24 antibody of the present invention, it falls within the protection scope of the present invention.

[0120] 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. Those not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. Those reagents or instruments not specified by the manufacturer are all conventional products that can be obtained through commercial purchase.

[0121] 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 formulations or unit doses herein, some methods and materials are now described. Unless otherwise noted, the techniques employed or contemplated herein are standard methods. The materials, methods and examples are illustrative only and not limiting.

[0122] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. Such techniques are fully explained in the literature, such as "Molecular Cloning: A Laboratory Manual", Second Edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (ed. M.J. Gait, 1984); "Animal Cell Culture" (ed. R.I. Freshney, 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (eds. D.M. Weir and C.C. Blackwell); "Gene Transfer Vectors for Mammalian Cells" (eds. J.M. Miller and M.P. Calos, 1987); "Current Protocols in Molecular Biology" (eds. F.M. Ausubel et al., 1987); "PCR: The Polymerase Chain Reaction" (eds. Mullis et al., 1994); and "Current Protocols in Immunology" (eds. J.E. Coligan et al., 1991), each of which is hereby expressly incorporated by reference.

[0123] The features and properties of the present invention will be further described in detail below in conjunction with examples.

[0124] Example 1 Preparation of Anti-P24 20C5 Monoclonal Antibody

[0125] In this example, the restriction endonuclease 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 pMD-18T vector was purchased from Takara. The plasmid extraction kit was purchased from Tiangen. Primer synthesis and gene sequencing were completed by Invitrogen. The hybridoma cell line secreting the mAnti-P24 20C5 monoclonal antibody was the hybridoma cell line prepared in this laboratory and was revived for standby.

[0126] (1) Preparation of antibody genes

[0127] mRNA was extracted from the hybridoma cell line secreting the mAnti-P24 20C5 monoclonal antibody, and DNA products were obtained by RT-PCR. After adding A to the products with rTaq DNA polymerase, they were inserted into the pMD-18T vector and transformed into DH5α competent cells. After colonies grew, the Heavy Chain and Light Chain gene clones were taken respectively, and 4 clones each were sent to a gene sequencing company for sequencing.

[0128] (2) Sequence analysis of the variable region genes of the Anti-P24 20C5 antibody

[0129] The gene sequences obtained from the above sequencing were analyzed in the kabat antibody database, and the VNTI11.5 software was used for analysis to determine that the genes amplified by the heavy chain and light chain primer pairs were correct. Among the gene fragments amplified by the Light Chain, the VL gene sequence was 330 bp, and there was a 57-bp leader peptide sequence in front of it; among the gene fragments amplified by the Heavy Chain primer pair, the VH gene sequence was 378 bp, belonging to the VH1 gene family, and there was a 57-bp leader peptide sequence in front of it. Then, the obtained murine antibody was humanized through humanization technology to obtain the humanized antibody and its sequence.

[0130] (3) Construction of recombinant antibody expression plasmids

[0131] pcDNA TM 3.4 The pcDNA3.4 vector was the recombinant antibody eukaryotic expression vector constructed. Multiple cloning enzyme digestion sites such as HindIII, BamHI, and EcoRI had been introduced into this expression vector, and it was named the pcDNA3.4A expression vector, hereinafter simply referred to as the 3.4A expression vector; according to the sequence of the above humanized antibody, specific primers for the VL and VH genes of this antibody were designed, with HindIII and EcoRI enzyme digestion sites and protection bases at both ends, and a 0.71-kb Light Chain gene fragment and a 1.42-kb Heavy Chain gene fragment were amplified by PCR amplification.

[0132] The Heavy Chain and Light Chain gene fragments were digested with HindIII / EcoRI double enzymes respectively, and the 3.4A vector was digested with HindIII / EcoRI double enzymes. After purifying and recovering the fragments and the vector, the Heavy Chain gene and the Light Chain gene were respectively ligated into the 3.4A expression vector to obtain the recombinant expression plasmids of Heavy Chain and Light Chain respectively.

[0133] 2. Recombinant antibody production

[0134] Resuscitate HEK293 cells in advance, subculture them to a 200 ml system to make the cell density reach 3 - 5×10 6 cells / ml. Select antibodies with the appropriate concentration and cells with a cell viability > 95%; centrifuge and wash the cells, resuspend them with the medium, and at the same time adjust the cell density to 2.9×10 6 cells / ml. Wash the cells, resuspend them with the medium, and at the same time, use it as the cell dilution solution. Prepare the plasmid DNA and transfection reagent dilution solutions with the medium respectively. Add the transfection reagent dilution solution to the plasmid DNA dilution solution, mix well and let it stand at room temperature for 15 min; slowly add this mixture to the cell dilution solution within 1 min, mix well, sample and count, record and observe the viability of the cells after transfection, and place them in an incubator at 35°C for culture, with a rotation speed of 120 rmp and a CO2 content of 8%. After 13 days, collect the samples by centrifugation. Affinity purify the centrifuged supernatant with a protein A affinity chromatography column. Take 6 μg of the purified antibody for reducing SDS-PAGE, and the electrophoresis pattern is shown in the figure. After reducing SDS-PAGE, two bands are shown, one with Mr of 50 KD (heavy chain) and the other with Mr of 28 KD (light chain).

[0135] The obtained antibody was named Anti-P24 20C5Rmb1, and the Anti-P24 20C5Rmb1 was mutated to obtain a mutant antibody. The sequences of the heavy chain (H) and light chain (L) of the above antibody are shown in the following table:

[0136] Table 2 Antibody sequences

[0137] Antibody Name Heavy Chain Light Chain Anti - P24 20C5Rmb1 SEQ ID NO:20 SEQ ID NO:25 Anti - P24 20C5Rmb2 SEQ ID NO:20 SEQ ID NO:27 Anti - P24 20C5Rmb3 SEQ ID NO:20 SEQ ID NO:26 Anti - P24 20C5Rmb4 SEQ ID NO:20 SEQ ID NO:28 Anti - P24 20C5Rmb5 SEQ ID NO:30 SEQ ID NO:25 Anti - P24 20C5Rmb6 SEQ ID NO:30 SEQ ID NO:27 Anti - P24 20C5Rmb7 SEQ ID NO:30 SEQ ID NO:26 Anti - P24 20C5Rmb8 SEQ ID NO:30 SEQ ID NO:28

[0138] Example 2 Performance detection of the antibody

[0139] 1. Affinity analysis

[0140] Dilute the purified antibody in advance, and simultaneously perform gradient dilution on the P24 recombinant antigen (from Fapon Biotech); use the CM5 chip that has been pre-coupled with Protein A to test the binding and dissociation curves of the antigen and antibody on the Biacore 8K+ device, and the instrument automatically fits to obtain the affinity constant, binding rate, and dissociation rate. (KD represents the equilibrium dissociation constant, i.e., the affinity constant; ka represents the binding rate; kd represents the dissociation rate)

[0141] Table 3 Affinity Data

[0142] Sample Name KD ka kd Control 7.15E-08 5.76E+03 4.12E-04 Anti - P24 20C5Rmb1 1.88E-10 3.18E+05 5.98E-05 Anti - P24 20C5Rmb2 4.45E-10 2.04E+05 9.08E-05 Anti - P24 20C5Rmb3 3.31E-10 2.66E+05 8.80E-05 Anti - P24 20C5Rmb4 3.01E-10 3.13E+05 9.42E-05 Anti - P24 20C5Rmb5 1.95E-10 1.80E+05 3.51E-05 Anti - P24 20C5Rmb6 4.49E-10 2.35E+05 1.06E-04 Anti - P24 20C5Rmb7 1.07E-11 1.85E+05 1.98E-06 Anti - P24 20C5Rmb8 1.41E-09 6.97E+04 9.83E-05

[0143] 2. Activity Identification

[0144] Dilute the P24 recombinant antigen (from Fapon Biotech) with the coating solution (main component NaHCO3) to 3 μg / ml, 100 μL per well, and incubate overnight at 4°C; the next day, wash 2 times with the washing solution (main components Na2HPO4 + NaCl), and pat dry; add the blocking solution (20% BSA + 80% PBS), 120 μL per well, at 37°C for 1 h, and pat dry; add the diluted purified antibody and control antibody, 100 μL / well, at 37°C for 30 min; wash 5 times with the washing solution, and pat dry; add goat anti-human IgG-HRP, 100 μL per well, at 37°C for 30 min; wash 5 times with the washing solution, and pat dry; add chromogenic solution A (50 μL / well), add chromogenic solution B (50 μL / well), for 10 min; add the stop solution, 50 μL / well; read the OD value at 450 nm (reference 630 nm) on the microplate reader.

[0145] Note: 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)

[0146] Table 4 Activity Data

[0147] Concentration (ng / ml) 125.00 62.50 31.250 15.63 7.81 0.00 Control 1.157 0.802 0.313 0.118 0.023 0.006 Anti - P24 20C5Rmb1 1.799 1.263 0.635 0.382 0.213 0.025 Anti - P24 20C5Rmb2 1.661 1.372 0.727 0.485 0.204 0.024 Anti - P24 20C5Rmb3 1.740 1.232 0.715 0.407 0.164 0.034 Anti - P24 20C5Rmb4 1.525 1.142 0.401 0.295 0.170 0.020 Anti - P24 20C5Rmb5 1.737 1.469 0.842 0.426 0.259 0.023 Anti - P24 20C5Rmb6 1.725 1.294 0.678 0.322 0.206 0.012 Anti - P24 20C5Rmb7 1.870 1.416 0.827 0.475 0.261 0.023 Anti - P24 20C5Rmb8 1.877 1.384 0.791 0.375 0.169 0.036

[0148] 3. Stability Assessment

[0149] Place the above antibody at 4°C (refrigerator), -80°C (refrigerator), and 37°C (incubator) for 21 days. Take samples at 7 days, 14 days, and 21 days for status observation, and perform activity detection on the 21-day samples. The results show that there is no obvious change in the protein status of the antibody after being placed for 21 days under the three assessment conditions, and the activity does not show a downward trend with the increase of the assessment temperature, indicating that the above antibody is stable. The following Table 5 shows the OD results of the enzyme immunoassay activity detection of the antibody Anti-P24 20C5Rmb5 after 21 days of assessment.

[0150] Table 5 Stability Data

[0151] Sample Concentration (ng / ml) 62.50 31.25 0.00 Sample at 4°C for 21 days 1.463 0.845 0.037 Sample at - 80°C for 21 days 1.452 0.827 0.031 Sample at 37°C for 21 days 1.439 0.835 0.028

[0152] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

[0153] Some of the amino acid sequences involved in this application are shown in Table 6 as follows:

[0154]

[0155]

Claims

1. An anti-P24 antibody, wherein the antibody comprises three complementary determining regions of a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 19 and three complementary determining regions of a light chain variable region having any one of the amino acid sequences SEQ ID NO: 21, 22, 23, 24.

2. The antibody according to claim 1, wherein, the complementary determining regions of the variable regions are defined by any one system or a combination of multiple systems among Kabat, Chothia, IMGT, AbM or Contact.

3. An anti-P24 antibody, wherein, the antibody comprises the following complementary determining regions: HCDR1, which comprises the amino acid sequence shown in SEQ ID NO: 1, or consists of the same; HCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 2, or consists of the same; HCDR3, which comprises the amino acid sequence shown in SEQ ID NO: 3, or consists of the same; LCDR1, which comprises the amino acid sequence shown in SEQ ID NO: 4, or consists of the same; LCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 5, or consists of the same; LCDR3, which comprises the amino acid sequence shown in SEQ ID NO: 6, or consists of the same; Optionally, the HFR1 comprises SEQ ID NO: 7 or an amino acid sequence having at least 80% identity therewith; the HFR2 comprises SEQ ID NO: 8 or an amino acid sequence having at least 80% identity therewith; the HFR3 comprises SEQ ID NO: 9 or an amino acid sequence having at least 80% identity therewith; the HFR4 comprises SEQ ID NO: 10 or an amino acid sequence having at least 80% identity therewith; the LFR1 comprises SEQ ID NO: 11 or an amino acid sequence having at least 80% identity therewith; the LFR2 comprises SEQ ID NO: 12 or an amino acid sequence having at least 80% identity therewith; the LFR3 comprises SEQ ID NO: 13 or an amino acid sequence having at least 80% identity therewith; and the LFR4 comprises SEQ ID NO: 14 or an amino acid sequence having at least 80% identity therewith; Optionally, the antibody binds to P24 with an affinity of KD < 7.15×10 -8 M.

4. An anti-P24 antibody 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 as shown in SEQ ID NO: 19; the amino acid sequence of the light chain variable region is as shown in any one of SEQ ID NO: 21, 22, 23, 24; Optionally, the antibody further comprises a constant region; Optionally, the constant region comprises a heavy chain constant region and / or a light chain constant region; Optionally, the heavy chain constant region is selected from the heavy chain constant regions of any one of IgG, IgA, IgM, IgE, IgD or a combination of multiple constant region segments; Optionally, the heavy chain constant region comprises CH1 of IgG, the hinge region of IgG, CH2 of IgM, CH3 of IgM and / or CH4 of IgM; Optionally, the species origin of the constant region is bovine, equine, porcine, ovine, caprine, rat, mouse, dog, camel, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human; Optionally, the species origin of the constant region is human; Optionally, the heavy chain constant region sequence is as shown in SEQ ID NO: 15 or 29 or has at least 80% identity therewith; Optionally, the light chain constant region sequence is as shown in SEQ ID NO: 16 or has at least 80% identity therewith; Optionally, the antibody comprises any one of F(ab’)2, Fab’, Fab, Fv and scFv.

5. An anti-P24 antibody, comprising a heavy chain and / or a light chain, characterized in that the amino acid sequence of the heavy chain is as shown in any one of SEQ ID NO: 20 and 30; the amino acid sequence of the light chain is as shown in any one of SEQ ID NO: 25, 26, 27 and 28.

6. An antibody conjugate, characterized in that the antibody conjugate comprises the antibody according to any one of claims 1 to 5; Optionally, the antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody; Optionally, the antibody conjugate further comprises a label conjugated to the antibody; Optionally, the label is selected from fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents and nanoparticle-based labels; Optionally, the antibody conjugate further comprises a solid-phase carrier conjugated to the antibody.

7. A reagent or kit, characterized in that the reagent or kit comprises the antibody according to any one of claims 1 to 5 or the antibody conjugate according to claim 6.

8. Use of the antibody according to any one of claims 1-5, the antibody conjugate according to claim 6 in the preparation of a product for detecting P24; Optionally, the use comprises: a) contacting the antibody according to any one of claims 1-5, the antibody conjugate according to claim 6, or the reagent or kit according to claim 7 with P24 in a test sample under conditions sufficient to effect 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 the antigen in the test sample; Optionally, the immune complex further comprises a second antibody that binds to the antibody; Optionally, the immune complex further comprises a second antibody that binds to P24.

9. A nucleic acid, vector, cell or method for preparing the antibody according to any one of claims 1-5, the nucleic acid encoding the antibody according to any one of claims 1 to 5; the vector contains the nucleic acid encoding the antibody according to any one of claims 1 to 5; the cell contains the above nucleic acid or vector; the method comprises the above cell.

Images