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 anti-P24 antibody and an application thereof. Background Art

[0002] Human Immunodeficiency Virus (HIV), also known as AIDS, is a virus that causes human immune system defects. It is a lentivirus that infects human immune system cells and is 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 antibodies is a routine experimental method for diagnosing HIV infection, but the determination of HIV antibodies has limitations: more than 70% of HIV-infected people can only detect antibodies 6 months after infection. In the homosexual community, this number exceeds 80%. The antibody detection method increases the risk of HIV transmission during the "window period"; in addition, it takes one year for newborns to produce antibodies, and HIV antibodies from the mother will cause false positives; due to the continuous presence of HIV antibodies during the disease process, they will only disappear in the late stage of AIDS and cannot be used as a stable indicator for treatment monitoring.

[0003] P24 is the main structural protein of HIV virus particles and the product of the structural gene GAG. It plays an important role in the packaging and maturation of the virus. The amino acid sequence of the P24 protein is highly conserved among HIV strains. The absence of P24 will cause the virus to be unable to assemble normally. The P24 protein is highly specific and has no cross-reaction with most other retroviruses. When HIV infects the human body, the first viral marker that appears in the blood of the infected person is the viral P24 protein. There is a long window period from viral infection to 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 of patients, the screening and evaluation of anti-HIV drugs, and the detection of mother-to-child transmission.

[0004] The detection of P24 antigen adopts serological diagnosis methods, mainly including double antibody sandwich ELISA method, immune complex lysis detection method, ultra-sensitive EIA method, enzyme-linked immunofluorescence method, etc. At present, the double antibody sandwich method is widely used to detect human immunodeficiency virus P24 antigen, and obtaining anti-P24 antibodies is the key to realize the double antibody sandwich method detection. Therefore, there is a strong demand in the field for antibodies that are effective and bind to P24 and detect it. Summary of the invention

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

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

[0007] In order to achieve the above object, according to a second aspect of the present invention, an anti-P24 antibody or an antigen-binding fragment thereof is provided, wherein the anti-P24 antibody or the antigen-binding fragment thereof comprises the following complementary 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 comprises or consists of the amino acid sequence shown in SEQ ID NO:6.

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

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

[0016] In order to achieve the above object, according to the fifth aspect of the present invention, an antibody conjugate is provided, wherein the antibody conjugate comprises the above anti-P24 antibody or an antigen-binding fragment thereof.

[0017] In order to achieve the above object, according to the sixth aspect of the present invention, a reagent or a kit is provided, wherein the reagent or the kit comprises the above anti-P24 antibody or its antigen-binding fragment or the above antibody conjugate.

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

[0019] In order to achieve the above object, according to the eighth aspect of the present invention, a nucleic acid is provided, which encodes the above anti-P24 antibody or antigen-binding fragment thereof.

[0020] In order to achieve the above object, according to the ninth aspect of the present invention, a vector is provided, wherein the vector comprises the above nucleic acid.

[0021] To achieve the above object, according to the tenth aspect of the present invention, a cell is provided, wherein the cell comprises the above nucleic acid, vector or expresses the above anti-P24 antibody or antigen-binding fragment thereof.

[0022] In order to achieve the above object, according to the eleventh aspect of the present invention, a method for preparing the above anti-P24 antibody or antigen-binding fragment thereof is provided, the method comprising culturing the above cell.

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

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0025] Figure 1The figure shows the result of reducing SDS-PAGE of Anti-P24 16H9Rmb1. DETAILED DESCRIPTION

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

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

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

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

[0030] In the present invention, the term "complementarity determining region", "CDR" or "CDRs" refers to the highly variable regions of the heavy and light chains of immunoglobulins, and refers to the region 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 its recognized antigen or epitope.

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

[0032] The definition methods of CDR are well known in the art, and the CDR definition methods include: Kabat definition, Chothia definition, IMGT definition, Contact definition and AbM definition. As described herein, "Kabat definition" refers to the definition system described by Kabat et al., USDept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). "Chothia definition" refers to 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 schemes, but will still overlap with at least a portion of the CDR region defined by Kabat, although they may be shortened or extended based on predictions or experimental results of specific residues or residue groups. Exemplary defined CDRs are listed in Table 1 below, and the definitions in different documents are slightly different. Given the variable region amino acid sequence of an antibody, a person 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 belong to the scope of protection of the present disclosure.

[0033] Table 1: CDR Definition 1

[0034]

[0035]

[0036] 1 The numbering of all CDR definitions in Table 1 is based on the Kabat numbering system (see below), with the amino acid numbering on the heavy chain represented by "H+number" and the amino acid numbering on the light chain represented by "L+number". One of ordinary skill in the art can unambiguously map the Kabat numbering system to any variable region sequence without relying 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. Patent. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0037] 2 "AbM" as used in Table 1 with a lowercase "b" refers to CDRs defined by Oxford Molecular's "AbM" antibody modeling software.

[0038] 3If both H35A and H35B are absent, CDR-H1 ends at position 35; if only H35A is present, CDR-H1 ends at position 35A; if both H35A and H35B are present, CDR-H1 ends at position 35B.

[0039] 4 If both H35A and H35B are absent, CDR-H1 ends at position 32; if only H35A is present, CDR-H1 ends at position 33; if both H35A and H35B are present, CDR-H1 ends at position 34.

[0040] 5 If both H35A and H35B are absent, CDR-H1 ends at position 33; if only H35A is present, CDR-H1 ends at position 34; if both H35A and H35B are present, CDR-H1 ends at position 35.

[0041] 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 of Kabat, Chothia, IMGT, AbM or Contact.

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

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

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

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

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

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

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

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

[0050] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof comprises the following complementarity determining regions:

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

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

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

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

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

[0056] LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:6.

[0057] In a second aspect, an embodiment of the present invention provides an anti-P24 antibody or an antigen-binding fragment thereof, wherein the anti-P24 antibody or the antigen-binding fragment thereof comprises the following complementary determining regions:

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

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

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

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

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

[0063] LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:6.

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

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

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

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

[0068] In an alternative embodiment, the HFR1 comprises / is SEQ ID NO:7 or an amino acid sequence having at least 80% identity thereto;

[0069] The HFR2 comprises / is SEQ ID NO:8 or an amino acid sequence having at least 80% identity thereto;

[0070] The HFR3 comprises / is SEQ ID NO:9 or an amino acid sequence having at least 80% identity thereto;

[0071] The HFR4 comprises / is SEQ ID NO: 10 or an amino acid sequence having at least 80% identity thereto;

[0072] The LFR1 comprises / is SEQ ID NO:11 or an amino acid sequence having at least 80% identity thereto;

[0073] The LFR2 comprises / is SEQ ID NO: 12 or an amino acid sequence having at least 80% identity thereto;

[0074] The LFR3 comprises / is SEQ ID NO: 13 or an amino acid sequence having at least 80% identity thereto; and

[0075] The LFR4 comprises / is SEQ ID NO: 14 or an amino acid sequence having at least 80% identity thereto.

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

[0077] In an alternative embodiment, the LFR4 comprises / is represented by SEQ ID NO: 21 or an amino acid sequence having at least 80% identity thereto.

[0078] In an optional embodiment, the anti-P24 antibody or antigen-binding fragment thereof has a KD of less than 9.42×10 -7 M binds to P24 with high affinity.

[0079] In an alternative embodiment, the anti-P24 antibody or antigen-binding fragment thereof 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 high affinity.

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

[0081] There are many methods for determining antibody affinity (KD), which can be divided into thermodynamic detection methods, kinetic detection methods and dynamic equilibrium detection methods according to the detection principle. Among them, thermodynamic detection methods are commonly used, such as isothermal titration calorimetry (ITC); kinetic detection methods are commonly used, such as surface plasmon resonance (SPR) and biomembrane interferometry (BLI); dynamic equilibrium detection methods are commonly used, such as enzyme-linked immunosorbent assay (ELISA).

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

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

[0084] In an optional embodiment, the anti-P24 antibody or antigen-binding fragment thereof of the first aspect, the second aspect or the third aspect further comprises a constant region.

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

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

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

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

[0089] In an alternative embodiment, the light chain constant region is selected from a kappa-type or a lambda-type light chain constant region.

[0090] In an alternative embodiment, the constant region is derived from a species of cow, horse, dairy cow, pig, sheep, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose, turkey, fighting cock or human.

[0091] In an alternative embodiment, the species origin of the constant region is rabbit.

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

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

[0094] In an optional 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.

[0095] The antigen-binding fragments of the above antibodies usually have the same binding specificity as the antibodies from which they are derived. It is easy for those skilled in the art to understand based on the contents described in the present invention that the antigen-binding fragments of the above antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by chemical reduction to split disulfide bonds. Based on the structure of the complete antibody disclosed in the present invention, those skilled in the art can easily obtain the above antigen-binding fragments.

[0096] The antigen-binding fragments of the above antibodies can also be synthesized by recombinant genetic techniques also known to those skilled in the art or by, for example, an automatic peptide synthesizer, such as those sold by Applied BioSystems and the like.

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

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

[0099] In a fifth aspect, the present invention provides an antibody conjugate, wherein the antibody conjugate comprises the above-mentioned anti-P24 antibody or an antigen-binding fragment thereof.

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

[0101] In an optional embodiment, the antibody conjugate further comprises a label coupled to the anti-P24 antibody or antigen-binding fragment thereof.

[0102] In an optional embodiment, the above-mentioned marker refers to a class of substances with properties that can be directly observed by the naked eye or detected or detected by an instrument, such as luminescence, color development, radioactivity, etc., through which qualitative or quantitative detection of the corresponding target can be achieved.

[0103] In an alternative embodiment, the label includes but is not limited to fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent agents and nanoparticle labels.

[0104] In actual use, those skilled in the art can select a suitable marker according to the detection conditions or actual needs. No matter which marker is used, it belongs to the protection scope of the present invention.

[0105] In an optional embodiment, the fluorescent dye is not limited to fluorescein dyes and their derivatives (for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (for example, including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy3.7, Cy3.8, Cy3.9, Cy3.10, Cy3.11, Cy3.12, Cy3.13, Cy3.14, Cy3.15, Cy3.16, Cy3.17, Cy3.18, Cy3.19, Cy3.20, Cy3.21, Cy3.22, Cy3.23, Cy3.24, Cy3.25, Cy3.26, Cy3.27, Cy3.28, Cy3.29, Cy3.30, Cy3.31, Cy3.32, Cy3.33, Cy3.34, Cy3.35, Cy3.36, Cy3.37, Cy3.38, Cy3.39, Cy3.40, Cy3.41, Cy3.42, Cy3.43, Cy3.44, Cy3.45, Cy3.46, Cy3.47, Cy3.48, Cy3.49, Cy3.50, Cy3.51, Cy3.52, Cy3.53, Cy3.54, Cy3.55, Cy3.56, Cy3.57, Cy3.58, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.5 y5, Cy5.5, Cy3, etc. or their analogs), Alexa series dyes and their derivatives (for example, including but not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs) and protein dyes and their derivatives (for example, including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), pre-chlorophyll protein (preCP), etc.).

[0106] In alternative embodiments, the enzyme includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphoglucose deoxygenase.

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

[0108] In an optional embodiment, the chemiluminescent reagent includes but is not limited to luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridine ruthenium and its derivatives, acridinium esters and their derivatives, dioxetanes and their derivatives, lophanes and their derivatives, and peroxyoxalates and their derivatives.

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

[0110] In alternative embodiments, the colloid includes, but is not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.

[0111] In an alternative embodiment, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, and colloidal selenium.

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

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

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

[0115] In an optional embodiment, the solid phase carrier includes but is not limited to magnetic microspheres, plastic microspheres, plastic microparticles, microwell plates, glass, capillaries, nylon and nitrocellulose membranes.

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

[0117] As mentioned above, the anti-P24 antibody or its antigen-binding fragment in some embodiments or examples of the present invention can effectively bind to P24, so the reagent or kit containing the anti-P24 antibody or its antigen-binding fragment can effectively detect P24 qualitatively or quantitatively. The reagent or kit provided by the present invention can be used, for example, for detection involving the use of P24 and its antibody specific binding properties such as immunoblotting and immunoprecipitation. As mentioned above, the anti-P24 antibody or its antigen-binding fragment in some embodiments or examples of the present invention has a higher binding activity or affinity with P24, so the reagent or kit containing the anti-P24 antibody or its antigen-binding fragment has a higher detection sensitivity or specificity.

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

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

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

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

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

[0123] In a tenth aspect, the present invention provides a cell containing the above-mentioned vector.

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

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

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

[0127] Based on the amino acid sequence of the anti-P24 antibody or its antigen-binding fragment disclosed in the present invention, those skilled in the art can easily think of using genetic engineering technology or other technologies (chemical synthesis, recombinant expression) to prepare the anti-P24 antibody or its antigen-binding fragment, for example, separating and purifying the anti-P24 antibody or its antigen-binding fragment from the culture product of a recombinant cell that can recombinantly express the anti-P24 antibody or its antigen-binding fragment as described in any of the above items, which is easy to achieve for those skilled in the art. Based on this, no matter what technology is used to prepare the anti-P24 antibody or its antigen-binding fragment of the present invention, it belongs to the protection scope of the present invention.

[0128] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0129] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used for the practice or testing of the preparations or unit doses herein, some methods and materials are now described. Unless otherwise stated, the techniques adopted or considered herein are standard methods. Materials, methods and examples are illustrative and non-restrictive only.

[0130] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. This technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction) (Academic Press, Inc., 1987). Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0131] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

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

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

[0134] 1. Gene acquisition

[0135] (1) Rabbit immunization

[0136] The emulsified preparation of P24 prepared with incomplete Freund's adjuvant was injected subcutaneously to stimulate the immune response of New Zealand white rabbits aged 4 to 6 weeks, and the pre- and post-immunization sera were collected on days 0, 14, 28, 42 and 69, respectively. The spleens of the rabbits were then surgically removed to prepare spleen cell suspensions.

[0137] (2) Construction of phage library

[0138] RNA was extracted from rabbit spleen cells and reverse transcribed into cDNA. Specifically designed rabbit antibody gene amplification primers were used to amplify VH and VL gene fragments using cDNA as templates. Then, the VH and VL gene fragments were inserted into the phage vector V02 in sequence by restriction ligation. Finally, the ligated phage plasmid was electrotransformed into TG1 competent cells. The next day, monoclonal colonies were selected for PCR identification and antibody gene sequencing to evaluate the quality of the phage library. After passing the test, the phage library was screened.

[0139] (3) Screening of phage libraries

[0140] The phage library TG1 bacterial solution was inoculated into a shake flask, and when the bacterial solution reached an appropriate concentration (OD600 of 0.8-1.0), helper phage was added for infection for 1 hour, and then the culture was continued overnight. The next day, the bacterial solution was collected and centrifuged, and the supernatant was taken and purified by salting out precipitation to obtain the displayed phage library.

[0141] The phage library was panned for 3-4 rounds using the magnetic bead panning method, and then the monoclonal phage-infected colonies were selected for antibody supernatant expression. The monoclonal phage antibody expression supernatant was then screened and identified using the ELISA screening method to obtain anti-P24 monoclonal phage.

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

[0143] 2. Expression Plasmid Construction

[0144] pcDNA TM 3.4 vector is a recombinant antibody eukaryotic expression vector constructed, which has been modified to introduce multiple cloning restriction sites and is hereinafter referred to as 3.4A expression vector; based on the variable region genes obtained in the above experiment, VL and VH gene-specific primers were designed, with restriction endonuclease sites and protective bases at both ends, respectively, and 0.72Kb Light Chain gene fragment and 1.40Kb Heavy Chain gene fragment were amplified by PCR amplification method.

[0145] The Heavy Chain and Light Chain gene fragments were double-digested with restriction endonucleases, and the 3.4A vector was double-digested with restriction endonucleases. After the fragments and vectors were purified and recovered, the Heavy Chain gene and the Light Chain gene were respectively connected to the 3.4A expression vector to obtain the recombinant expression plasmids of the Heavy Chain and Light Chain, respectively.

[0146] 3. Recombinant Antibody Sample Preparation

[0147] Resuscitate HEK293 cells in advance and subculture them to 200 ml system to make the cell density reach 3-5×10 6 cells / ml, the cell density reaches the selected antibody concentration and cells, and the cell viability is >95%; the cells are washed by centrifugation and re-dissolved with culture medium, and the cell density is adjusted to 2.9×10 6 cells / ml, wash the cells, and re-dissolve them with culture medium, and use it as a cell diluent. Prepare plasmid DNA and transfection reagent diluents with culture medium respectively. Add the transfection reagent diluent to the plasmid DNA diluent, mix well and let stand at room temperature for 15 minutes; slowly add the mixture to the cell diluent within 1 minute, mix well and take samples to count, record and observe the viability of the cells after transfection, and place them in a 35℃ constant temperature incubator for culture, with a speed of 120rmp and a CO2 content of 8%. Centrifuge and collect samples after 13 days. The centrifuged supernatant was affinity purified using a proteinA affinity chromatography column. Take 6ug layer of purified antibodies for reducing SDS-PAGE, and the electrophoresis is shown in the figure. After reducing SDS-PAGE, two bands are shown, one Mr is 50KD (heavy chain) and the other Mr is 28KD (light chain).

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

[0149] 1. Affinity Analysis

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

[0151] Operation process: 60s of equilibrium in buffer 1 (PBST), 300s of antibody fixation in antibody solution, 180s of incubation in buffer 2 (PBST), 420s of binding in antigen solution, 1200s of dissociation in buffer 2, sensor regeneration with 10mM pH 1.69 GLY solution and buffer 3 (PBST), and data output. (KD represents equilibrium dissociation constant, i.e. affinity; kon represents binding rate; kdis represents dissociation rate. The main components of PBST are Na2HPO4+NaCl+TW-20).

[0152] Table 2 Affinity data

[0153]

[0154]

[0155] 2. Activity identification

[0156] The coating solution (main component NaHCO3) was used to dilute the P24 recombinant antigen to 3ug / ml, 100uL per well, and incubated at 4°C overnight; the next day, the cells were washed twice with the washing solution (main component Na2HPO4+Nacl), and the wells were patted dry; the blocking solution (20% BSA+80% PBS) was added, 120uL per well, incubated at 37°C for 1h, and the wells were patted dry; the diluted purified antibody and control antibody were added, 100uL / well, incubated at 37°C for 30min; the cells were washed 5 times, and the wells were patted dry; the goat anti-rabbit IgG-HRP was added, 100uL per well, incubated at 37°C for 30min; the cells were washed 5 times, and the wells were patted dry; the color developing solution A (50uL / well) and the color developing solution B (50uL / well) were added, and the wells were 10min; the stop solution was added, 50uL / well; the OD value was read at 450nm (reference 630nm) on the microplate reader.

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

[0158] Table 3 Activity data

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

[0160] 3. Stability assessment

[0161] The above antibodies were placed at 4°C (refrigerator), -80°C (refrigerator), and 37°C (constant temperature box) for 21 days, and samples were taken for 7 days, 14 days, and 21 days for status observation, and the activity of the 21-day samples was tested. The results showed that there was no obvious protein state change in the antibodies under the three test conditions for 21 days, and the activity did not show a downward trend with the increase of the test temperature, indicating that the above antibodies are stable. The following table shows the OD results of the enzyme immunoassay activity test for 21 days of antibody test.

[0162] Table 4 Anti-P24 16H9Rmb1 stability data

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

[0164] Table 5 Anti-P24 8D11Mb1 stability data

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

[0166] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

[0167] Some of the amino acid sequences involved in this application are shown in Table 6:

[0168]

Claims

1. An anti-P24 antibody or an antigen-binding fragment thereof, wherein the anti-P24 antibody or the antigen-binding fragment thereof has three complementary determining regions of the heavy chain variable region shown in the amino acid sequence SEQ ID NO: 17 and three complementary determining regions of the light chain variable region shown in the amino acid sequence SEQ ID NO: 19 or SEQ ID NO:

22.

2. The anti-P24 antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The complementarity determining regions of the variable regions are defined by any one of the Kabat, Chothia, IMGT, AbM or Contact systems or a combination of multiple systems.

3. An anti-P24 antibody or an antigen-binding fragment thereof, characterized in that: The anti-P24 antibody or antigen-binding fragment thereof comprises the following complementarity determining regions: HCDR1, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 1; HCDR2, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 2; HCDR3, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 3; LCDR1, which comprises or consists of the amino acid sequence shown in SEQ ID NO:4; LCDR2, which comprises or consists of the amino acid sequence shown in SEQ ID NO:5; and LCDR3, which comprises or consists of the amino acid sequence shown in SEQ ID NO:6; Optionally, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HFR1, HFR2, HFR3, HFR4, and the light chain variable region comprises LFR1, LFR2, LFR3, and LFR4; Optionally, the HFR1 comprises SEQ ID NO:7 or an amino acid sequence having at least 80% identity thereto; The HFR2 comprises SEQ ID NO: 8 or an amino acid sequence having at least 80% identity thereto; The HFR3 comprises SEQ ID NO:9 or an amino acid sequence having at least 80% identity thereto; The HFR4 comprises SEQ ID NO: 10 or an amino acid sequence having at least 80% identity thereto; The LFR1 comprises SEQ ID NO: 11 or an amino acid sequence having at least 80% identity thereto; The LFR2 comprises SEQ ID NO: 12 or an amino acid sequence having at least 80% identity thereto; The LFR3 comprises SEQ ID NO: 13 or an amino acid sequence having at least 80% identity thereto; and The LFR4 comprises SEQ ID NO: 14 or an amino acid sequence having at least 80% identity thereto; Optionally, the anti-P24 antibody or antigen-binding fragment thereof has a KD of less than 9.42×10 -7 M binds to P24 with high affinity.

4. An anti-P24 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and / or 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 or SEQ ID NO: 22; Optionally, the anti-P24 antibody or antigen-binding fragment thereof further comprises a constant region; Optionally, the constant region includes a heavy chain constant region and / or a light chain constant region; Optionally, 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; Optionally, the heavy chain constant region includes CH1 of IgG, hinge region of IgG, CH2 of IgM, CH3 of IgM and / or CH4 of IgM; Optionally, the species of the constant region is cattle, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human; Optionally, the species origin of the constant region is rabbit; Optionally, the heavy chain constant region sequence is as shown in SEQ ID NO: 15 or has at least 80% identity thereto; Optionally, the light chain constant region sequence is as shown in SEQ ID NO: 16 or has at least 80% identity thereto; Optionally, the antigen-binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.

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

23.

6. An antibody conjugate, characterized in that: The antibody conjugate comprises the anti-P24 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5; Optionally, the antibody conjugate further comprises biotin or a biotin derivative conjugated to the anti-P24 antibody or antigen-binding fragment thereof; Optionally, the antibody conjugate further comprises a label coupled to the anti-P24 antibody or antigen-binding fragment thereof; Optionally, the label is selected from fluorescent dyes, enzymes, radioisotopes, chemiluminescent agents and nanoparticle labels; Optionally, the antibody conjugate further comprises a solid phase carrier coupled to the anti-P24 antibody or antigen-binding fragment thereof.

7. A reagent or a kit, characterized in that: The reagent or kit comprises the anti-P24 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 or the antibody conjugate according to claim 6.

8. A method for detecting P24 or HIV, characterized in that: include: a) contacting the anti-P24 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the antibody conjugate according to claim 6, or the reagent or kit according to claim 7 with P24 in a sample to be detected under conditions sufficient for an antibody / antigen binding reaction to form an immune complex; and b) detecting the presence of the immune complex, wherein the presence of the complex indicates the presence of the P24 or HIV in the test sample; Optionally, the immune complex further comprises a second antibody, which binds to the anti-P24 antibody or antigen-binding fragment thereof; Optionally, the immune complex further comprises a second antibody, which binds to P24.

9. A nucleic acid, a vector, a cell or a method for preparing the anti-P24 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, characterized in that: The nucleic acid encodes the anti-P24 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5; the vector contains the above-mentioned nucleic acid; the cell contains the above-mentioned nucleic acid or vector; and the method comprises culturing the above-mentioned cell.

10. Use of the anti-P24 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the antibody conjugate according to claim 6, or the reagent or kit according to claim 7 in the preparation of a product for detecting P24 or HIV.

Images