Anti-P24 antibodies, reagents and kits for detecting P24

Abstract

The present invention discloses an anti-P24 antibody, a reagent for detecting P24, and a kit, relating to the field of antibody technology. The anti-P24 antibody disclosed in the present invention comprises a heavy chain complementary determining region and a light chain complementary determining region. The antibody provides an important raw material source for the detection of P24 and has improved affinity or activity.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This disclosure claims priority to Chinese patent application number 202310077910.9, filed with the Chinese Patent Office on February 3, 2023, entitled “Anti-P24 Antibodies, Reagents and Kits for Detecting P24,” the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to the technical field of antibodies, and in particular to an anti-P24 antibody, a reagent and a kit for detecting P24. Background Art

[0004] Human immunodeficiency virus (HIV), also known as AIDS, is a virus that causes immune system defects in humans. First discovered in the United States in 1981, HIV is a type of retrovirus that infects cells of the human immune system.

[0005] P24 is the primary structural protein of HIV virus particles and a product of the structural gene GAG. It plays a crucial role in viral packaging and maturation. The amino acid sequence of the P24 protein is highly conserved across HIV strains, and the absence of P24 prevents normal viral assembly. The P24 protein is highly specific and does not cross-react with most other retroviruses. When HIV infects the human body, the viral P24 protein is the first viral marker to appear in the blood. There is a long window period between viral infection and the detection of HIV antibodies. Therefore, the detection of HIV-P24 antigen has played a vital role in the early diagnosis of HIV infection, patient prognosis, screening and evaluation of anti-HIV drugs, and detecting mother-to-child transmission.

[0006] HIV-1 p24 antigen detection utilizes serological diagnostic methods, primarily including double-antibody sandwich ELISA, immune complex lysis assay, ultrasensitive EIA, and enzyme-linked immunofluorescence assay. Currently, the double-antibody sandwich assay is widely used to detect the human immunodeficiency virus p24 antigen. Producing anti-p24 antibodies is crucial for successful double-antibody sandwich detection. Therefore, there is a strong demand in the field for antibodies that effectively bind to and detect p24. Summary of the Invention

[0007] The present application provides an anti-P24 antibody, which provides an important source of raw materials for the detection of P24.

[0008] To achieve the above objectives, according to a first aspect of the present invention, an anti-P24 antibody is provided, which comprises HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, wherein the HCDR1, HCDR2, HCDR3 comprise / are amino acid sequences consistent with the HCDR1, HCDR2, HCDR3 of the heavy chain variable region shown in SEQ ID NO: 17; and the LCDR1, LCDR2, LCDR3 comprise / are amino acid sequences consistent with the LCDR1, LCDR2, LCDR3 of the light chain variable region shown in SEQ ID NO: 18.

[0009] In order to achieve the above object, according to a second aspect of the present invention, an anti-P24 antibody is provided, wherein the antibody comprises the following complementary determining regions:

[0010] HCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1;

[0011] HCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 2;

[0012] HCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 3;

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

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

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

[0016] To achieve the above object, according to the third aspect of the present invention, an anti-P24 antibody 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: 18.

[0017] To achieve the above object, according to the fourth aspect of the present invention, an anti-P24 antibody 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: 19; the amino acid sequence of the light chain is shown in SEQ ID NO: 20.

[0018] In order to achieve the above object, according to a fifth aspect of the present invention, an antibody conjugate is provided, wherein the antibody conjugate comprises the above antibody.

[0019] In order to achieve the above object, according to the sixth aspect of the present invention, a reagent or kit is provided, wherein the reagent or kit comprises the above antibody or the above antibody conjugate.

[0020] To achieve the above-mentioned object, according to the seventh aspect of the present invention, a method for detecting P24 is provided, comprising: a) contacting the above-mentioned antibody, antibody conjugate, or reagent or kit with the P24 antigen 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 antigen in the test sample.

[0021] 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 antibody.

[0022] 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.

[0023] 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 antibody.

[0024] In order to achieve the above object, according to the eleventh aspect of the present invention, a method for preparing the above antibody is provided, which comprises culturing the above cell.

[0025] In order to achieve the above-mentioned object, according to the twelfth aspect of the present invention, there is provided a use of the above-mentioned antibody, antibody conjugate, reagent or kit in detecting or preparing a P24 detection product. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0027] Figure 1 The results of reducing SDS-PAGE of Anti-P24 17D2mut are shown. DETAILED DESCRIPTION

[0028] In a first aspect, an embodiment of the present invention provides an anti-P24 antibody, comprising HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise / are amino acid sequences consistent with the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region shown in SEQ ID NO: 17; and the LCDR1, LCDR2, and LCDR3 comprise / are amino acid sequences consistent with the LCDR1, LCDR2, and LCDR3 of the light chain variable region shown in SEQ ID NO: 18.

[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, chimeric antibodies and antigen-binding fragments, so long as they exhibit the desired antigen-binding activity.

[0030] Antigen-binding fragments typically have the same binding specificity as the antibody from which they are derived. Those skilled in the art will readily appreciate, based on the disclosure herein, that such 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 structure of the intact antibody disclosed herein, those skilled in the art can readily obtain such antigen-binding fragments.

[0031] Antigen-binding fragments can also be synthesized by recombinant genetic techniques also known to those skilled in the art or by, for example, an automated peptide synthesizer, such as those sold by Applied BioSystems and the like.

[0032] As used herein, the terms "complementarity determining region," "CDR," or "CDRs" refer to the hypervariable regions of the heavy and light chains of immunoglobulins, and include one or more, or even all, of the amino acid residues that contribute substantially to the binding of an antibody or antigen-binding fragment to its recognized antigen or epitope. In specific embodiments of the present invention, CDRs refer to the hypervariable regions of the heavy and light chains of the antibody.

[0033] 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.

[0034] Methods for defining CDRs are well known in the art, including the Kabat definition, the Chothia definition, the IMGT definition, the Contact definition, and the AbM definition. As used herein, the "Kabat definition" refers to the definition system described by Kabat et al., U.S. Patent No. 200200201154, "Sequence of Proteins of Immunological Interest" (1983). The "Chothia definition" refers to the definition system described by Chothia et al., J Mol Biol 196: 901-917 (1987). Other CDR definition methods may not strictly follow one of the above schemes, but will still overlap with at least a portion of the CDR region defined by Kabat, although they may be shortened or lengthened based on predictions or experimental results of specific residues or residue groups. Exemplary defined CDRs are listed in Table 1 below, and the definitions in different literature vary slightly. Given the variable region amino acid sequence of an antibody, a person skilled in the art can routinely determine which residues comprise a specific CDR. It should be noted that CDRs defined by other methods other than those in Table 1 also fall within the scope of protection of the present disclosure.

[0035] Table 1: CDR Definition 1

[0036]

[0037]

[0038] 1 The numbering of all CDR definitions in Table 1 is based on the Kabat numbering system (see below), with amino acid numbers on the heavy chain represented by "H+number" and amino acid numbers on the light chain represented by "L+number." One of ordinary skill in the art can unambiguously assign this 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 in Kabat et al., U.S. Pat. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0039] 2 "AbM" as used in Table 1 with a lowercase "b" refers to CDRs defined by Oxford Molecular's "AbM" antibody modeling software.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] According to an embodiment of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by any one of the Kabat, Chothia, IMGT, AbM or Contact systems or a combination of multiple systems.

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

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

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

[0047] In some optional embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the AbM system.

[0048] In some optional embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by a Contact system.

[0049] 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.

[0050] In a second aspect, an embodiment of the present invention provides an anti-P24 antibody, wherein the antibody comprises the following complementary determining regions:

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

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

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

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

[0055] LCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 5.

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

[0057] According to an embodiment of the present invention, the HCDRs and LCDRs are defined by the Kabat system.

[0058] 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 of the antibody heavy chain variable region and the light chain variable region excluding CDR; 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.

[0059] 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.

[0060] In an alternative embodiment, the antibody further has at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4;

[0061] The HFR1 comprises / is as SEQ ID NO: 7 or an amino acid sequence having at least 80% homology thereto;

[0062] The HFR2 comprises / is as SEQ ID NO: 8 or an amino acid sequence having at least 80% homology thereto;

[0063] The HFR3 comprises / is as SEQ ID NO: 9 or an amino acid sequence having at least 80% homology thereto;

[0064] The HFR4 comprises / is as SEQ ID NO: 10 or an amino acid sequence having at least 80% homology thereto;

[0065] The LFR1 comprises / is SEQ ID NO: 11 or an amino acid sequence having at least 80% homology thereto;

[0066] The LFR2 comprises / is SEQ ID NO: 12 or an amino acid sequence having at least 80% homology thereto;

[0067] The LFR3 comprises / is shown in SEQ ID NO: 13 or an amino acid sequence having at least 80% homology thereto;

[0068] The LFR4 comprises / is SEQ ID NO: 14 or an amino acid sequence having at least 80% homology thereto;

[0069] It should be noted that, in other embodiments, the amino acid sequences of the framework regions of the antibodies provided herein 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 corresponding framework regions (SEQ ID NO: 7, 8, 9, 10, 11, 12, 13 or 14).

[0070] In an alternative embodiment, the antibody has a KD < 9.06 × 10 -9 M binds to P24 with high affinity.

[0071] In an alternative embodiment, the antibody has a KD ≤ 10 -9 M or KD≤10 -10 M binds to P24 with high affinity.

[0072] In an alternative embodiment, the antibody has a KD ≤ 2.09 × 10 -10 M binds to P24 with high affinity.

[0073] There are many methods for determining antibody affinity (KD). Based on the detection principle, they can be divided into thermodynamic detection methods, kinetic detection methods, and dynamic equilibrium detection methods. Among them, thermodynamic detection methods such as isothermal titration calorimetry (ITC) are common; kinetic detection methods such as surface plasmon resonance (SPR) and biofilm interferometry (BLI) are common; and dynamic equilibrium detection methods such as enzyme-linked immunosorbent assay (ELISA) are common.

[0074] In an alternative embodiment, KD is determined using a kinetic assay; preferably, a surface plasmon resonance assay, for example, by using a surface plasmon resonance assay such as System of biosensor systems.

[0075] In a third aspect, an embodiment of the present invention provides 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 shown in SEQ ID NO: 17, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 18.

[0076] In an alternative embodiment, the antibody further comprises a constant region.

[0077] In alternative embodiments, the constant region comprises a heavy chain constant region and / or a light chain constant region.

[0078] In an optional embodiment, the heavy chain constant region is selected from the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD, and the light chain constant region is selected from the κ type or λ type light chain constant region.

[0079] In an alternative embodiment, the species origin of the constant region is cow, horse, dairy cow, pig, sheep, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose, turkey, fighting cock or human.

[0080] In an alternative embodiment, the species origin of the constant region is rabbit.

[0081] In this article, the division of variable and constant region sequences refers to the IMGT division method, see Lefranc, and Martinez-Jean C. and Bosc N. or Ehrenmann,Patrice Duroux,Chantal Ginestoux,Gene table:house mouse(Musmusculus)IGHC,IMGT Repertoire. the international ImMunoGeneticsinformation http: / / www.imgt.org .Created:16 / 03 / 2011.Version:17 / 01 / 2020.or Ehrenmann,Patrice Duroux,Chantal Ginestoux,Gene table:house mouse(Musmusculus)IGLC,IMGT Repertoire. the international ImMunoGeneticsinformation http: / / www.imgt.org .Created: 16 / 03 / 2011.Version: 17 / 01 / 2020.. Variable regions divided by different methods may differ from the variable region C-terminus or constant region N-terminus divided by IMGT. Variable regions or constant regions divided by other methods known in the art are also within the scope of protection of the present invention.

[0082] In an optional embodiment, the heavy chain constant region sequence (CH) is shown as SEQ ID NO:15, and the light chain constant region (CL) sequence is shown as SEQ ID NO:16.

[0083] 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% homology with the above-mentioned constant region (SEQ ID NO: 15 or 16).

[0084] In an alternative embodiment, the antibody comprises any one of F(ab')2, Fab', Fab, Fv and scFv.

[0085] In a fourth aspect, the present invention provides an anti-P24 antibody comprising a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO: 19, and the amino acid sequence of the light chain is shown in SEQ ID NO: 20.

[0086] In a fifth aspect, the present invention provides an antibody conjugate comprising the above-mentioned antibody.

[0087] In an optional embodiment, the above-mentioned antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody.

[0088] In an optional embodiment, the antibody conjugate further comprises a label coupled to the antibody.

[0089] 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.

[0090] In an alternative embodiment, the label includes but is not limited to fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents and nanoparticle labels.

[0091] 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 falls within the scope of protection of the present invention.

[0092] In an optional embodiment, the fluorescent dyes include but are 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, C 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), peridinin-chlorophyll protein (preCP), etc.).

[0093] In alternative embodiments, the enzyme includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxidase.

[0094] 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.

[0095] In an optional embodiment, the chemiluminescent reagent includes but is not limited to luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and their derivatives, dioxetanes and their derivatives, lophanes and their derivatives, and peroxalates and their derivatives.

[0096] 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.

[0097] In alternative embodiments, the colloid includes, but is not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.

[0098] In an alternative embodiment, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0099] In an optional embodiment, the colloidal metal is colloidal gold.

[0100] In an optional embodiment, the above-mentioned antibody conjugate further includes a solid phase carrier coupled to the antibody.

[0101] In an alternative embodiment, the solid support is selected from microspheres, plates, and membranes.

[0102] In an optional embodiment, the solid phase carrier includes but is not limited to magnetic microspheres, plastic microspheres, plastic particles, microplates, glass, capillaries, nylon and nitrocellulose membranes.

[0103] In a sixth aspect, the present invention provides a reagent or a kit, wherein the reagent or the kit comprises the above-mentioned antibody or the above-mentioned antibody conjugate.

[0104] As previously mentioned, the antibodies disclosed in some embodiments or examples of the present invention are capable of effectively binding to the P24 antigen. Therefore, reagents or kits containing these P24 antibodies are capable of effectively performing qualitative or quantitative detection of P24. The reagents or kits provided herein can be used, for example, in immunoblotting, immunoprecipitation, and other assays that utilize the specific binding properties of P24 and its antibodies. As previously mentioned, the antibodies disclosed in some embodiments or examples of the present invention have a higher binding activity or affinity for P24. Therefore, reagents or kits containing these antibodies have a higher detection sensitivity or specificity.

[0105] In a seventh aspect, the present invention provides a method for detecting P24, comprising: a) contacting the above-mentioned antibody, antibody conjugate, reagent or kit with a P24 antigen 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 antigen in the test sample;

[0106] In an alternative embodiment, the immune complex further comprises a second antibody that binds to the antibody.

[0107] In an optional embodiment, the immune complex further comprises a second antibody, which binds to P24.

[0108] In an eighth aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned antibody.

[0109] In a ninth aspect, the present invention provides a vector containing the above-mentioned nucleic acid molecule.

[0110] In a tenth aspect, the present invention provides a cell containing the above-mentioned vector.

[0111] In an eleventh aspect, the present invention provides a method for preparing an antibody, comprising: culturing the cell as described above.

[0112] In a twelfth aspect, the present invention provides use of the above-mentioned antibody, antibody conjugate, or the above-mentioned reagent or kit in detecting P24 or preparing a product for detecting P24.

[0113] Based on the amino acid sequence of the antibody disclosed in the present invention, those skilled in the art can easily conceive of using genetic engineering technology or other technologies (chemical synthesis, recombinant expression) to prepare the antibody. For example, the antibody can be isolated and purified from the culture product of recombinant cells that can recombinantly express the antibody as described in any of the above items. This is easy for those skilled in the art to achieve. Based on this, regardless of the technology used to prepare the antibody of the present invention, it falls within the scope of protection of the present invention.

[0114] Rabbit monoclonal antibodies can be prepared by a variety of production techniques, including but not limited to hybridoma technology, phage display technology or single B cell cloning technology. Exemplary production methods are described below:

[0115] Method 1: Hybridoma technology

[0116] (1) Rabbit immunization

[0117] An emulsified preparation of P24 recombinant protein prepared with incomplete Freund's adjuvant was subcutaneously injected into 4- to 6-week-old New Zealand white rabbits to stimulate immune response. Pre- and post-immunization sera were collected on days 0, 14, 28, 42, and 69, respectively. The spleens of the rabbits were removed and spleen cell suspensions were prepared.

[0118] (2) Cell fusion

[0119] The spleen cell suspension and rabbit fusion partner cells were electrofused to prepare rabbit hybridoma cells, which were then added to HAT hybridoma screening medium for culture.

[0120] (3) Hybridoma screening

[0121] After the hybridoma cells were cultured for 7 days, the hybridoma cell supernatant was identified by ELISA screening method to screen out the hybridoma cell lines that could specifically recognize and bind to the P24 protein.

[0122] The hybridoma cell line resistant to P24 was subcloned by the limiting dilution method. Seven days later, the supernatant of the monoclonal hybridoma cell was screened and identified by the ELISA screening method to obtain the positive monoclonal hybridoma cell line resistant to P24.

[0123] (4) Hybridoma antibody gene sequencing

[0124] RNA of the anti-P24 monoclonal hybridoma cell line was extracted and reverse transcribed into cDNA. Then, the antibody gene fragment was PCR amplified. Next, the antibody gene fragment was connected and inserted into a sequencing T vector. Finally, the antibody gene was sequenced to obtain the gene sequence of the antibody variable region.

[0125] Method 2: Phage display technology

[0126] (1) Immunization of rabbits: see Method 1.

[0127] (2) Construction of phage library

[0128] RNA was extracted from rabbit splenocytes and reverse-transcribed into cDNA. Specifically designed rabbit antibody gene amplification primers were used, using the cDNA as a template, to amplify the VH and VL gene fragments, respectively. These fragments were then sequentially inserted into the phage vector V02 using enzyme digestion and ligation. Finally, the ligated phage plasmids were electroporated into TG1 competent cells. The following day, single colonies were selected for PCR identification and antibody gene sequencing to assess the quality of the phage library. Only qualified clones were screened for phage library screening.

[0129] (3) Screening of phage libraries

[0130] (4) The phage library TG1 bacterial solution was inoculated into a shake flask. 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 bacterial solution was collected the next day and centrifuged. The supernatant was taken and purified by salting out precipitation to obtain the displayed phage library. The phage library was selected for 3-4 rounds of panning using magnetic beads. Then, monoclonal phage-infected colonies were selected for antibody supernatant expression. The monoclonal phage antibody expression supernatant was then screened and identified using ELISA screening to obtain monoclonal phage against P24 protein.

[0131] (5) Phage antibody gene sequencing

[0132] The antibody gene of the monoclonal phage against P24 protein was sequenced, and duplicate and invalid sequences were removed through sequence analysis to obtain a unique rabbit monoclonal antibody sequence.

[0133] Method 3: Single B cell cloning technology

[0134] (1) Rabbit immunization

[0135] See Method 1: Isolate fresh single splenocytes and culture overnight in B cell culture medium.

[0136] (2) Prepare a fresh single-cell suspension by diluting spleen cells with PBS containing 2-3% fetal bovine serum and 1 mM EDTA.

[0137] (3) Single B cells were sorted for P24 antigen specificity using a Sony MA900 flow cytometer and placed in each well of a 96-well plate.

[0138] (4) Primary B cells with P24 specificity were added to B cell culture medium and then cultured at 37°C and 5.5% CO2 for 7-10 days.

[0139] (5) At the end of the primary B cell culture, the B cell culture supernatant was screened and identified by ELISA to obtain anti-P24 B cell positive clones.

[0140] (6) B cell antibody gene sequencing

[0141] RNA from anti-P24 B cell positive clones was extracted and reverse transcribed into cDNA, and then PCR amplification of the antibody gene fragment was performed. Next, the antibody gene fragment was inserted and connected to a sequencing T vector. Finally, the antibody gene was sequenced to obtain the gene sequence of the antibody variable region.

[0142] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0143] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the preparations or unit doses herein, some methods and materials are now described. Unless otherwise indicated, the techniques employed or contemplated herein are standard methods. Materials, methods, and examples are illustrative and non-limiting only.

[0144] 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 person skilled in the art. The technique is fully explained in the literature, for example, in Molecular Cloning: A Laboratory Manual, 2nd ed. (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); and PCR: The Polymerase Chain Reaction. 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.

[0145] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0146] Example 1 Preparation of Anti-P24 17D2 Monoclonal Antibody

[0147] 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 pMD-18T vector was also purchased from Takara. The plasmid extraction kit was purchased from Tiangen. Primer synthesis and gene sequencing were performed by Invitrogen.

[0148] 1. Construction of recombinant antibody expression plasmid

[0149] pcDNA TM 3.4 Vector is a recombinant antibody eukaryotic expression vector constructed, which has introduced multiple cloning restriction sites such as HindIII, BamHI, and EcoRI, and is named pcDNA3.4A expression vector, hereinafter referred to as 3.4A expression vector; based on the antibody variable region gene obtained by any of the above-mentioned hybridoma technology, phage display technology or single B cell cloning technology, the VL and VH gene-specific primers of the antibody are designed, with HindIII and EcoRI restriction sites and protection bases at both ends, respectively, and a 0.71kb Light Chain gene fragment and a 1.40kb Heavy Chain gene fragment are amplified by PCR amplification.

[0150] The Heavy Chain and Light Chain gene fragments were digested with HindIII / EcoRI, and the 3.4A vector was digested with HindIII / EcoRI. After the fragments and vectors were purified and recovered, the Heavy Chain gene and Light Chain gene were respectively connected to the 3.4A expression vector to obtain the recombinant expression plasmids of Heavy Chain and Light Chain, respectively.

[0151] 2. Recombinant Antibody Production

[0152] HEK293 cells were revived in advance and subcultured in 200 ml of culture medium to a cell density of 3-5 cells / mL, which was sufficient for the selected antibody concentration and cell viability >95%. Cells were washed by centrifugation and reconstituted with culture medium. The cell density was adjusted to 2.9, and the cells were washed and reconstituted with culture medium. This was also used as a cell dilution. Plasmid DNA and transfection reagent dilutions were prepared separately using culture medium. The transfection reagent dilution was added to the plasmid DNA dilution, mixed, and allowed to stand at room temperature for 15 minutes. This mixture was slowly added to the cell dilution over 1 minute. After mixing, samples were collected and counted. Cell viability after transfection was recorded and observed. Cells were then incubated in a 35°C incubator at 120 rpm and 8% CO2. After 13 days, samples were collected by centrifugation. The supernatant was affinity purified using a protein A affinity column. Six layers of purified antibody were subjected to reducing SDS-PAGE. The electropherogram is shown in the figure. After reducing SDS-PAGE, two bands were shown, one with a Mr of 50 KD (heavy chain) and the other with a Mr of 28 KD (light chain).

[0153] Example 2 Affinity and activity optimization

[0154] Although the Anti-P24 17D2 monoclonal antibody obtained in Example 1 has the ability to bind to P24, its affinity and antibody activity are not ideal. Therefore, the applicant conducted targeted mutagenesis on the variable region of the antibody. That is, a computer was used to simulate the structure of the antibody variable region, the structure of the complex interacting with the antigen and the antibody variable region, analyze the key amino acids of the antibody, and design mutations. According to the mutation scheme, bidirectional primers covering the mutation site were designed and synthesized. Primers at both ends of the target DNA were synthesized, and a high-fidelity PCR reaction was performed. The PCR product was cloned into a vector, and the mutant antibody was prepared according to the method described in Example 1. After screening, a monoclonal antibody with significantly improved affinity and antibody activity was obtained and named: Anti-P24 17D2mut. The heavy chain and light chain amino acid sequences of Anti-P24 17D2mut are shown in SEQ ID NO: 19 and SEQ ID NO: 20, respectively.

[0155] Example 3 Performance testing of antibodies

[0156] 1. Affinity Analysis

[0157] Purified antibodies were diluted in advance, and P24 recombinant antigen (purchased from Feipeng Bio) was serially diluted. Binding and dissociation curves were measured on a Biacore 8K+ instrument using a CM5 chip pre-coupled with goat anti-rabbit IgG. The instrument automatically fitted the affinity constant, association rate, and dissociation rate. (KD represents the equilibrium dissociation constant, also known as the affinity constant; ka represents the association rate; and kd represents the dissociation rate.)

[0158] Table 2: Affinity data

[0159] Sample name KD(M) Ka(1 / Ms) Kd(1 / s) comparison 9.06E-09 4.47E+04 4.05E-04 Anti-P24 17D2mut 2.09E-10 3.04E+05 6.34E-05

[0160] 2. Activity Identification

[0161] The coating solution (main component NaHCO3) was diluted with P24 recombinant antigen (purchased from Feipeng Bio) to 3ug / ml, 100uL per well, and incubated at 4°C overnight; the next day, the wells were washed twice with washing solution (main component Na2HPO4+NaCl) and patted dry; blocking solution (20% BSA+80% PBS) was added, 120uL per well, 37°C, 1h, and patted dry; diluted purified antibody and control antibody were added, 100uL / well, 37°C, 30min; washing solution was washed 5 times, and patted dry; goat anti-rabbit IgG-HRP was added, 100uL per well, 37°C, 30min; washing solution was washed 5 times, and patted dry; color development solution A (50uL / well, containing 1.05g / L citric acid, 0.186g / L ETA, 0.45g / L TMB and 0.2 Then, add chromogenic buffer B (50 μL / well, containing 1.05 g / L citric acid, 0.186 g / L EDTA, 0.45 g / L TMB, and 0.2 ml / L concentrated HCl) for 10 min. Add stop buffer (50 μL / well, containing 0.75 g / L EDTA and 10.2 ml / L concentrated H2SO4). Read the OD value at 450 nm (reference 630 nm) on a microplate reader. The results are shown in the table below:

[0162] Table 3: Activity data

[0163] Sample concentration (ng / ml) 1.95 0.98 0.49 0.24 0.12 0.00 comparison 1.446 1.003 0.559 0.331 0.187 0.012 Anti-P24 17D2mut 1.732 1.243 0.783 0.519 0.320 0.019

[0164] 3. Stability assessment

[0165] The above-mentioned antibodies were placed at 4°C (refrigerator), -80°C (freezer), and 37°C (incubator) for 21 days. Samples were collected at 7, 14, and 21 days for status observation, and the 21-day sample was tested for activity. The results showed that no significant changes in protein status were observed under the three test conditions for 21 days, and the activity did not show a downward trend with increasing test temperature, indicating that the above-mentioned antibodies are stable. Table 4 below shows the OD results of the enzyme immunoassay activity assay after 21 days of antibody testing.

[0166] Table 4: Stability data

[0167] Sample concentration (ng / ml) 0.98 0.49 0 4℃, 21-day samples 1.244 0.753 0.022 -80℃, 21-day sample 1.285 0.778 0.021 37℃, 21-day samples 1.236 0.741 0..023

[0168] 4. Application of antibodies in colloidal gold detection

[0169] 4.1 Preparation of colloidal gold test paper

[0170] 4.1.1 Preparation of nitrocellulose membrane

[0171] Prepare nitrocellulose membrane: Dilute anti-P24 antibody (Phipeng Biotechnology) to 1-5 mg / ml in coating buffer. Draw a line (T), the test line, near the colloidal gold end. Dilute goat anti-mouse IgG antibody (Phipeng Biotechnology Co., Ltd., Cat. No. BA-PAB-MU0001) to 1-5 mg / ml in coating buffer. Draw a line (C), the control line, near the absorbent pad. Dry at 37°C and seal for later use.

[0172] 4.2 Preparation of colloidal gold and gold-labeled monoclonal antibodies

[0173] 4.2.1 Preparation of colloidal gold

[0174] Dilute 1% chloroauric acid to 0.01% with double-distilled deionized water. Bring to a boil on an electric stove. Add 2ml of 1% trisodium citrate per 100ml of 0.01% chloroauric acid. Continue boiling until the liquid turns bright red. Remove from heat and allow to cool to room temperature before adding water. The prepared colloidal gold should be pure, translucent, and free of precipitates and floating matter. It is valid for one week.

[0175] 4.2.2 Preparation of colloidal gold-labeled recombinant antibodies

[0176] Adjust the pH of the colloidal gold to 8.2 with 0.1M potassium carbonate. Add Anti-P2417D2mut or a mainstream P24 antibody at a concentration of 8-10 μg / ml of colloidal gold. Mix using a magnetic stirrer for 30 minutes. Add BSA to a final concentration of 1% while stirring and let stand for 1 hour. Centrifuge at 13,000 rpm and 4°C for 30 minutes. Discard the supernatant and wash the pellet twice with Labeling Wash Storage Solution. Resuspend the pellet in Labeling Wash Storage Solution at one-tenth the initial volume of colloidal gold and store at 4°C until ready for use. The expiration date is one week.

[0177] 4.3 Preparation of gold label pad

[0178] Soak the gold-labeled pad in blocking solution for 30 minutes and then dry it at 37°C. Then evenly spread the prepared gold-labeled antibody on the gold-labeled pad, with 20 square centimeters per milliliter of solution, freeze-dry, package, and store at 4°C for later use.

[0179] 4.4 Preparation of test strip sample pad

[0180] The sample pad was immersed in blocking solution (containing BSA) for 30 min, dried at 37°C, sealed, and stored at 4°C for later use.

[0181] 4.5 Assembly of test strips

[0182] An absorption pad (purchased from Millipore), a nitrocellulose membrane, a gold-labeled pad, and a sample pad were placed on a non-absorbent support sheet and cut into 3 mm wide strips. Ten strips were packed into a pack, a desiccant was added, and the packing was vacuum-packed to obtain the test paper.

[0183] 4.6 Colloidal gold platform test

[0184] The assembled test strips were used to detect the presence of P24 protein in the test material, thereby confirming the effectiveness of the P24 antibody in detecting P24 protein. During the test, P24 protein first binds to the colloidal gold-labeled P24 antibody to form a P24-colloidal gold-labeled P24 antibody complex. Due to capillary action, the P24-colloidal gold-labeled P24 antibody complex migrates along the nitrocellulose membrane. Upon reaching the test line, the P24-colloidal gold-labeled P24 antibody complex binds to the P24-coated antibody on the marked line, forming a P24 antibody-P24-colloidal gold-labeled P24 antibody complex. This complex is then enriched on the test line, forming a red precipitate line. The test results are shown in Table 5 below, demonstrating that the Anti-P24 17D2mut antibody has superior sensitivity to mainstream P24 antibodies on the market.

[0185] Note: The gold label colorimetric assay consists of C plus a number. The smaller the number after C, the stronger the color and the higher the activity; the higher the number after C, the weaker the color and the lower the activity. A “+” after the number indicates a slightly stronger color of 0.5C, a “-” after the number indicates a slightly weaker color of 0.5C, and “B” represents negative.

[0186] Table 5: Performance evaluation data

[0187]

[0188] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

[0189] Some of the amino acid sequences involved in this application are shown in Table 6 below:

[0190]

[0191]

Claims

1. An anti-P24 antibody comprising HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, characterized in that: The HCDR1, HCDR2, and HCDR3 have amino acid sequences identical to those of the heavy chain variable region HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 17; the LCDR1, LCDR2, and LCDR3 have amino acid sequences identical to those of the light chain variable region LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 18; The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 are defined by the Kabat, Chothia, IMGT, AbM or Contact system and numbered by the Kabat numbering system.

2. An anti-P24 antibody, characterized in that The antibody comprises HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3: The HCDR1 is the amino acid sequence shown in SEQ ID NO: 1; The HCDR2 is the amino acid sequence shown in SEQ ID NO: 2; The HCDR3 is the amino acid sequence shown in SEQ ID NO: 3; The LCDR1 is the amino acid sequence shown in SEQ ID NO: 4; The LCDR2 is the amino acid sequence shown in SEQ ID NO: 5; The LCDR3 has an amino acid sequence as shown in SEQ ID NO:

6.

3. The antibody according to claim 1 or 2, characterized in that The antibody further comprises HFR1, HFR2, HFR3, HFR4 and LFR1, LFR2, LFR3, LFR4, wherein HFR1 comprises SEQ ID NO: 7 or an amino acid sequence having at least 80% homology thereto; The HFR2 comprises SEQ ID NO: 8 or an amino acid sequence having at least 80% homology thereto; The HFR3 comprises SEQ ID NO: 9 or an amino acid sequence having at least 80% homology thereto; The HFR4 comprises SEQ ID NO: 10 or an amino acid sequence having at least 80% homology thereto; The LFR1 comprises SEQ ID NO: 11 or an amino acid sequence having at least 80% homology thereto; The LFR2 comprises SEQ ID NO: 12 or an amino acid sequence having at least 80% homology thereto; The LFR3 comprises SEQ ID NO: 13 or an amino acid sequence having at least 80% homology thereto; The LFR4 comprises SEQ ID NO: 14 or an amino acid sequence having at least 80% homology thereto.

4. The antibody according to claim 1 or 2, characterized in that The antibody has a KD of <9.06×10 -9 M binds to P24 with high affinity.

5. An anti-P24 antibody comprising a heavy chain variable region and a light chain variable region, characterized in that: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 17; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

18.

6. The antibody according to any one of claims 1 to 2 or 5, characterized in that The antibody also comprises a constant region.

7. The antibody according to claim 6, characterized in that The constant region includes a heavy chain constant region and a light chain constant region.

8. The antibody according to claim 7, characterized in that The heavy chain constant region is selected from the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; the light chain constant region is selected from the κ type or λ type light chain constant region.

9. The antibody according to claim 6, characterized in that The species origin of the constant region is cow, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human.

10. The antibody according to claim 6, characterized in that The species origin of the constant region is rabbit.

11. The antibody according to claim 7, characterized in that The heavy chain constant region sequence is as shown in SEQ ID NO: 15 or has at least 80% identity thereto.

12. The antibody according to claim 7, characterized in that The light chain constant region sequence is as shown in SEQ ID NO: 16 or has at least 80% identity thereto.

13. The antibody according to any one of claims 1 to 2 and 5, characterized in that The antibody includes any one of F(ab')2, Fab', Fab, Fv and scFv.

14. An anti-P24 antibody comprising a heavy chain and a light chain, characterized in that: The amino acid sequence of the heavy chain is shown in SEQ ID NO: 19; the amino acid sequence of the light chain is shown in SEQ ID NO:

20.

15. An antibody conjugate, characterized in that The antibody conjugate comprises the antibody according to any one of claims 1 to 14.

16. The antibody conjugate according to claim 15, characterized in that The antibody conjugate further includes biotin conjugated to the antibody.

17. The antibody conjugate according to claim 15, characterized in that The antibody conjugate further includes a label coupled to the antibody.

18. The antibody conjugate according to claim 17, characterized in that The label is selected from fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents and nanoparticle labels.

19. The antibody conjugate according to claim 15, characterized in that The antibody conjugate further includes a solid phase carrier coupled to the antibody.

20. The antibody conjugate according to claim 19, characterized in that The solid support is selected from microspheres, plates and membranes.

21. A reagent or kit, characterized in that The reagent or kit comprises the antibody according to any one of claims 1 to 14 or the antibody conjugate according to any one of claims 15 to 20.

22. Use of the antibody according to any one of claims 1 to 14, or the antibody conjugate according to any one of claims 15 to 20, or the reagent or kit according to claim 21 in the preparation of a product for detecting P24, characterized in that: include: a) contacting the antibody of any one of claims 1 to 14, the antibody conjugate of any one of claims 15 to 20, or the reagent or kit of claim 21 with a P24 antigen in a sample to be tested 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 antigen in the test sample.

23. The use according to claim 22, characterized in that The immune complex further includes a second antibody that binds to the antibody.

24. The use according to claim 22, characterized in that The immune complex further includes a second antibody that binds to P24.

25. A nucleic acid, characterized in that The nucleic acid encodes the antibody according to any one of claims 1 to 14.

26. A carrier, characterized in that The vector contains a nucleic acid encoding the antibody according to any one of claims 1 to 14.

27. A cell, characterized in that The cell contains the nucleic acid of claim 25 or the vector of claim 26.

28. A method for preparing the antibody according to any one of claims 1 to 14, characterized in that: The method comprises culturing the cell of claim 27.

29. Use of the antibody according to any one of claims 1 to 14, the antibody conjugate according to any one of claims 15 to 20, or the reagent or kit according to claim 21 in the preparation of a product for detecting P24 or in detecting P24 for non-diagnostic purposes.

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