WO2025113565A1 - Anti-p24 antibody and use thereof - Google Patents

Abstract

An anti-P24 antibody and a use thereof. The anti-P24 antibody comprises a heavy chain complementarity determining region and a light chain complementarity determining region. The antibody provides an important raw material source for P24 detection, and has good affinity or activity.

Description

Anti-P24 antibody and its application

CROSS-REFERENCE TO RELATED APPLICATIONS

The present disclosure claims priority to a Chinese patent application with application number 202311646314.4 filed with the Chinese Patent Office on November 30, 2023, entitled “An anti-P24 antibody and its application”, the entire contents of which are incorporated by reference into the present disclosure.

Technical Field

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

Background Art

Human immunodeficiency virus (HIV), also known as AIDS, is a virus that causes human immune system defects. Human immunodeficiency virus was first discovered in the United States in 1981. It is a lentivirus that infects human immune system cells and is a type of retrovirus.

HIV is the pathogen of AIDS, which is mainly transmitted through sexual contact, blood and mother-to-child transmission. 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 after birth, 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.

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.

HIV-1 P24 antigen detection adopts serological diagnosis methods, mainly including double antibody sandwich ELISA, immune complex lysis detection, ultra-sensitive EIA, enzyme-linked immunofluorescence, 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 need in the art for anti-P24 antibodies with good performance.

Summary of the invention

The present application provides an anti-P24 antibody, which provides an important source of raw materials for the detection of P24 and has good activity or affinity.

In order to achieve the above-mentioned purpose, according to one aspect of the present disclosure, an anti-P24 antibody is provided, which comprises three complementary determining regions of a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 19 and three complementary determining regions of a light chain variable region having an amino acid sequence as shown in any one of SEQ ID NO: 21, 22, 23, and 24.

In order to achieve the above object, according to the second aspect of the present disclosure, an anti-P24 antibody is provided, wherein the antibody comprises the following complementary 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;

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

In order to achieve the above-mentioned purpose, according to the third aspect of the present disclosure, 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 as shown in SEQ ID NO: 19; and the amino acid sequence of the light chain variable region is as shown in any one of SEQ ID NO: 21, 22, 23, and 24.

In order to achieve the above-mentioned purpose, according to the fourth aspect of the present disclosure, there is provided an anti-P24 antibody, comprising a heavy chain and/or a light chain, wherein the amino acid sequence of the heavy chain is as shown in any one of SEQ ID NO: 20 and 30; and the amino acid sequence of the light chain is as shown in any one of SEQ ID NO: 25, 26, 27 and 28.

In order to achieve the above object, according to the fifth aspect of the present disclosure, an antibody conjugate is provided, wherein the antibody conjugate comprises the above antibody.

In order to achieve the above-mentioned object, according to the sixth aspect of the present disclosure, a reagent or a kit is provided, wherein the reagent or the kit comprises the above-mentioned antibody or the above-mentioned antibody conjugate.

To achieve the above objectives, according to the seventh aspect of the present disclosure, there is provided a use of the above antibodies and antibody conjugates in detecting P24, diagnosing HIV infection in a subject, preparing a product for detecting P24, or preparing a product for diagnosing whether a subject is infected with HIV.

To achieve the above objectives, according to the eighth aspect of the present disclosure, a method for diagnosing whether a subject is infected with HIV is provided, comprising: a) contacting the above-mentioned antibody, antibody conjugate, reagent or kit with an ex vivo sample to be tested 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.

In order to achieve the above-mentioned purpose, according to the ninth aspect of the present disclosure, a method for detecting P24 is provided, comprising: a) contacting the above-mentioned antibody, antibody conjugate, 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 antigen in the test sample.

In order to achieve the above objectives, the present disclosure also provides a nucleic acid, a vector, a cell and a method for preparing the above antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present disclosure 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 paying creative work.

Figure 1 shows the results of reducing SDS-PAGE of Anti-P24 20C5 Rmb1 to Anti-P24 20C5 Rmb8.

DETAILED DESCRIPTION

In a first aspect, the embodiments of the present disclosure provide an anti-P24 antibody, which comprises three complementary determining regions of a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 19 and three complementary determining regions of a light chain variable region having an amino acid sequence of any one of SEQ ID NO: 21, 22, 23, and 24.

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 antibody 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 antibody described in the first aspect.

For example, the HCDR1, HCDR2, and HCDR3 are amino acid sequences consistent with the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region shown in SEQ ID NO:19; the LCDR1, LCDR2, and LCDR3 are amino acid sequences consistent with the LCDR1, LCDR2, and LCDR3 of the light chain variable region shown in SEQ ID NO:21.

In the present disclosure, the term "antibody" is used in the broadest sense, which may include full-length monoclonal antibodies, bispecific antibodies, multispecific antibodies, chimeric antibodies or antigen-binding fragments of antibodies, as long as they exhibit the desired antigen-binding activity. Antigen-binding fragments of antibodies include any one of F(ab)2, F(ab')2, Fab', Fab, Fv and scFv, and generally have the same binding specificity as the antibody from which they are derived. It is easy for those skilled in the art to understand from the contents of the present disclosure that antigen-binding fragments of 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 disclosure, antigen-binding fragments of antibodies can be easily obtained by those skilled in the art.

Antigen-binding fragments of 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.

In the present disclosure, the term "complementarity determining region", "CDR" or "CDRs" refers to the highly variable region 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 play a role in the binding of an antibody or antigen-binding fragment to its recognized antigen or epitope. In a specific embodiment of the present disclosure, CDRs refers to the highly variable region of the heavy and light chains of the antibody.

In the present disclosure, the heavy chain complementarity determining region is denoted by HCDR, which includes HCDR1, HCDR2 and HCDR3; the light chain complementarity determining region is denoted by LCDR, which includes LCDR1, LCDR2 and LCDR3.

Methods for defining CDRs are well known in the art, and 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., U.S. Dept. 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 those in Table 1 also fall within the protection scope of the present disclosure.

Table 1: CDR Definition 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+numbers" and amino acid numbers on the light chain represented by "L+numbers". 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 in Kabat et al., U.S. Patent No. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).
² “AbM” as used in Table 1 with a lowercase “b” refers to CDRs defined by Oxford Molecular’s “AbM” antibody modeling software.
³ If 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.
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.
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.

According to an embodiment of the present disclosure, 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.

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

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

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

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

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

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

In a second aspect, the present disclosure provides an anti-P24 antibody, wherein the antibody comprises the following complementary determining regions:

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

HCDR2 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.

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

According to an embodiment of the present disclosure, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat system.

In the present disclosure, "framework region" or "FR" region includes heavy chain framework region and light chain framework region, and refers to the region other than CDR in the heavy chain variable region and 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.

In the present disclosure, the heavy chain variable region is obtained by 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 connecting the following numbered CDRs and FRs in the following combinations: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

In an alternative embodiment, the antibody of the first aspect or the second aspect further has HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4.

In an optional embodiment, the HFR1 comprises/is such as SEQ ID NO:7 or an amino acid sequence having at least 80% identity thereto;

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

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

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

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

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

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

The LFR4 includes/is such as SEQ ID NO:14 or an amino acid sequence having at least 80% identity thereto.

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

In an optional embodiment, the LFR3 includes/is represented by an amino acid sequence as shown in SEQ ID NO:18.

In an optional embodiment, the LFR2 includes/is represented by an amino acid sequence as shown in SEQ ID NO:17.

In an alternative embodiment, the antibody binds P24 with an affinity of KD < 7.15 x 10 ^-8 M.

In alternative embodiments, the antibody binds P24 with an affinity of KD≤10 ^"8M , KD≤10 ^"9M , KD≤10" ^10M , KD≤10 ^"11M , or KD≤10 ^"12M .

In an alternative embodiment, the antibody binds P24 with an affinity of KD≤1.41×10 ⁻⁹ M.

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).

In an alternative embodiment, KD is determined using a kinetic assay; optionally, surface plasmon resonance, for example by using a kinetic assay such as Biosensor system of the system.

In a third aspect, the embodiments of the present disclosure provide an anti-P24 antibody, comprising a heavy chain variable region and/or a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 19, and the amino acid sequence of the light chain variable region is as shown in any one of SEQ ID NO: 21, 22, 23, and 24.

In an optional embodiment, the antibody described in the first aspect, the second aspect, and the third aspect further comprises a constant region.

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

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.

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.

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

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

In an alternative embodiment, the species of origin of the constant region is cattle, horses, dairy cows, pigs, sheep, rats, mice, dogs, camels, cats, rabbits, donkeys, deer, minks, chickens, ducks, geese, turkeys, fighting cocks or humans.

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

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(Mus musculus)IGHC,IMGT Repertoire. the international ImMunoGenetics information http://www.imgt.org .Created:16/03/2011.Version:17/01/2020.or Ehrenmann,Patrice Duroux,Chantal Ginestoux,Gene table:house mouse(Mus musculus)IGLC,IMGT Repertoire. the international ImMunoGenetics information http://www.imgt.org .Created:16/03/2011.Version:17/01/2020.. The variable regions divided by different methods may have some amino acid differences with the variable region C-terminus or constant region N-terminus divided by IMGT. The variable regions or constant regions divided by other methods known in the art are also within the protection scope of the present disclosure.

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

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

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

In a fourth aspect, the present disclosure 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 any one of SEQ ID NO: 20 and 30, and the amino acid sequence of the light chain is shown in any one of SEQ ID NO: 25, 26, 27 and 28.

In an optional embodiment, the antibody of the first aspect, the second aspect, the third aspect or the fourth aspect comprises any combination of the following heavy chains and light chains:

In a fifth aspect, the present disclosure provides an antibody conjugate, which comprises the above-mentioned antibody.

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

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

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.

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

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 disclosure.

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.).

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.

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.

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.

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.

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

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

In an optional embodiment, the colloidal metal is colloidal gold.

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

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

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.

In a sixth aspect, the present disclosure provides a reagent or a kit, wherein the reagent or the kit comprises the above-mentioned antibody or the above-mentioned antibody conjugate.

As mentioned above, the antibodies in some embodiments or examples of the present disclosure can effectively bind to P24, so the reagents or kits containing the P24 antibodies can effectively detect P24 qualitatively or quantitatively. The reagents or kits provided by the present disclosure can be used, for example, for detections involving the specific binding properties of P24 and its antibodies, such as immunoblotting and immunoprecipitation. As mentioned above, the antibodies in some embodiments or examples of the present disclosure have higher binding activity or affinity with P24, so the reagents or kits containing the antibodies have higher detection sensitivity or specificity.

It should be noted that the products disclosed herein include but are not limited to reagents, test kits, test strips or test plates.

In a seventh aspect, the present disclosure provides a method for detecting P24, comprising: a) contacting the above-mentioned antibody, antibody conjugate, reagent or kit with P24 in a sample to be detected to form an immune complex under conditions sufficient for an antibody/antigen binding reaction to occur; 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;

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

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

In an eighth aspect, the present disclosure provides a method for diagnosing whether a subject is infected with HIV, comprising: a) contacting the above-mentioned antibody, antibody conjugate, reagent or kit with P24 in a sample to be tested to form an immune complex under conditions sufficient for an antibody/antigen binding reaction to occur; 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;

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

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

In a ninth aspect, the present disclosure provides uses of the above-mentioned anti-P24 antibodies and antibody conjugates in detecting P24, diagnosing HIV infection in a subject, preparing products for detecting P24, or preparing products for diagnosing whether a subject is infected with HIV.

In a tenth aspect, the present disclosure provides a nucleic acid molecule encoding the above-mentioned antibody.

In an eleventh aspect, the present disclosure provides a vector containing the above-mentioned nucleic acid molecule.

In a twelfth aspect, the present disclosure provides cells containing the above-mentioned vector.

In a thirteenth aspect, the present disclosure provides a method for preparing an anti-P24 antibody, comprising: culturing the cells as described above.

Based on the amino acid sequence of the anti-P24 antibody disclosed in the present disclosure, 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, for example, separating and purifying the antibody from the culture product of a recombinant cell that can recombinantly express the antibody 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 of the present disclosure, it belongs to the protection scope of the present disclosure.

In order to make the purpose, technical scheme and advantages of the embodiments of the present disclosure clearer, the technical scheme in the embodiments of the present disclosure 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.

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.

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

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

Example 1 Preparation of Anti-P24 20C5 Monoclonal Antibody

The restriction endonucleases and Prime Star DNA polymerase in this example were purchased from Takara. MagExtractor-RNA extraction kit was purchased from TOYOBO. BD SMART ^TM RACE cDNA Amplification Kit was purchased from Takara. pMD-18T vector was purchased from Takara. Plasmid extraction kit was purchased from Tiangen. Primer synthesis and gene sequencing were completed by Invitrogen. The hybridoma cell line secreting mAnti-P24 20C5 monoclonal antibody was prepared in this laboratory and revived for use.

(1) Antibody gene preparation

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

(2) Sequence analysis of the variable region gene of Anti-P24 20C5 antibody

The gene sequences obtained by the above sequencing were placed in the kabat antibody database for analysis, and the VNTI11.5 software was used for analysis to determine that the genes amplified by the heavy chain and light chain primer pairs were correct. Among them, in the gene fragment amplified by the Light Chain, the VL gene sequence was 330bp, and it was preceded by a 57bp leader peptide sequence; in the gene fragment amplified by the Heavy Chain primer pair, the VH gene sequence was 378bp, belonging to the VH1 gene family, and it was preceded by a 57bp leader peptide sequence. The obtained mouse antibodies were then humanized by humanization technology to obtain humanized antibodies and sequences.

(3) Construction of recombinant antibody expression plasmid

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; according to the sequence of the above humanized antibody, the VL and VH gene-specific primers of the antibody were designed, with HindIII, EcoRI restriction sites and protective bases at both ends, respectively, and the 0.71kb Light Chain gene fragment and the 1.42kb Heavy Chain gene fragment were amplified by the PCR amplification method.

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 connected to the 3.4A expression vector, respectively, to obtain the recombinant expression plasmids of Heavy Chain and Light Chain, respectively.

2. Recombinant Antibody Production

HEK293 cells were revived in advance and subcultured to a 200ml system to make the cell density reach 3-5×10 ⁶ cells/ml, the cell density reached the selected antibody concentration and cells, and the cell viability was >95%; the cells were washed by centrifugation, and the cells were re-dissolved with the culture medium. At the same time, the cell density was adjusted to 2.9×10 ⁶ cells/ml to wash the cells, and the cells were re-dissolved with the culture medium. At the same time, it was used as a cell diluent. The plasmid DNA and transfection reagent diluent were prepared with the culture medium. The transfection reagent diluent was added to the plasmid DNA diluent, mixed and left to stand at room temperature for 15 minutes; the mixture was slowly added to the cell diluent within 1 minute, and the samples were counted after mixing, and the viability of the cells after transfection was recorded and observed, and the cells were placed in a 35℃ constant temperature incubator for culture, with a speed of 120rmp and a CO ₂ content of 8%. After 13 days, the samples were collected by centrifugation. The supernatant was affinity purified using a proteinA affinity chromatography column. A 6ug layer of purified antibodies was taken for reducing SDS-PAGE, and the electrophoresis diagram is shown in the figure. After reducing SDS-PAGE, two bands were shown, one with Mr of 50 KD (heavy chain) and the other with Mr of 28 KD (light chain).

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

Table 2: Antibody sequences

Example 2 Performance testing of antibodies

1. Affinity Analysis

The purified antibody was diluted in advance, and the P24 recombinant antigen (from Feipeng Bio) was diluted in a gradient manner; the binding and dissociation curves of the antigen and antibody were tested on the Biacore 8K+ device using the CM5 chip that had been pre-coupled with Protein A. The instrument automatically fitted the affinity constant, binding rate, and dissociation rate. (KD represents the equilibrium dissociation constant, i.e., affinity constant; ka represents binding rate; kd represents dissociation rate)

Table 3: Affinity data

2. Activity identification

The coating solution (main component NaHCO ₃ ) was used to dilute P24 recombinant antigen (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 Na ₂ HPO ₄ +NaCl), and patted dry; blocking solution (20% BSA + 80% PBS) was added, 120uL per well, 37°C, 1h, and patted dry; the diluted purified antibody and control antibody were added, 100uL/well, 37°C, 30min; the wells were washed with washing solution 5 times, and patted dry; goat anti-human IgG-HRP was added, 100uL per well, 37°C, 30min; the wells were washed with washing solution 5 times, and patted dry; color development solution A (50uL/well) and color development solution B (50uL/well) were added, 10min; stop solution was added, 50uL/well; OD value was read at 450nm (reference 630nm) on the microplate reader.

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 H ₂ SO ₄ )

Table 4: Activity data

3. Stability assessment

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. Table 5 below shows the OD results of the enzyme immunoassay activity test of the antibody Anti-P24 20C5Rmb5 for 21 days.

Table 5: Stability data

Example 3: Antibody performance test (exemplary display of the performance test results of the antibody Anti-P24 20C5Rmb4)

1. Antibody performance in chemiluminescent assays

1.1 Antibody coating process

10mg/mL carboxyl magnetic beads, washed 3 times with MES buffer. Resuspend the magnetic beads in MES buffer, add EDC with a final concentration of 1mg/mL, shake at 25℃, and react for 30min. Resuspend the magnetic beads in MES buffer, add antibody Anti-P24-A (from Feipeng Bio) with a final concentration of 0.2mg/mL, use this solution as the magnetic particle working solution, shake at 37℃, and react for 120min. Store in Tris buffer at 2-8℃.

1.2 Antibody labeling process

3 mg/ml antibodies Anti-P24 20C5Rmb4 and Anti-P24-B (control antibody, from Feipeng Bio) were replaced into PBS (100 mM PB, 50 mM sodium chloride, pH 8.0) using a Zeba desalting column (10K MWCO) and labeled with biotin. The biotin-modified antibody was the biotin working solution and was stored at 4°C for future use.

1.3 Detection process

The test was performed on the Yingkai shine i2910 model fully automatic chemiluminescence immunoassay analyzer using the double antibody sandwich method, that is, the instrument sequentially added 100 μL sample, 50 μL magnetic particle working solution and 50 μL biotin working solution, mixed and incubated for 10 minutes, rinsed the reaction mixture after incubation, and then added 100 uL SA-AE working solution. Mix and incubate for 10 minutes, rinsed the reaction mixture after incubation, added pre-excitation solution and excitation solution, and detected the relative luminescence intensity (RLU).

Note: 1) The samples in the experiment refer to: sample diluent, positive test samples of different concentrations, positive clinical samples, negative plasma samples of different populations, and negative serum samples of different populations.

Recombinant positive control containing P24 protein diluted with negative diluent, negative serum matrix, positive NIBSC control, 100 negative clinical samples

1.4 Test results

1.4.1 The sensitivity test results on the chemiluminescence platform are shown in Table 6. The results show that the detection sensitivity of the reagent composed of the antibody Anti-P2420C5Rmb4 to P24 is better than that of the control antibody.

Table 6: Chemiluminescence platform sensitivity test results

1.4.2 953 negative samples were tested on the chemiluminescence platform. The test results are shown in Table 7. The results show that there was only one false positive case and the detection rate was 99.8%, indicating that the reagent composed of the antibody Anti-P24 20C5Rmb4 has good detection specificity for P24.

Table 7: Chemiluminescence platform specific detection results

Some of the amino acid sequences involved in this application are shown in Table 8:

Table 8: Amino Acid Sequence Listing

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

Claims (14)

An anti-P24 antibody, which comprises three complementary determining regions of a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 19 and three complementary determining regions of a light chain variable region having an amino acid sequence as shown in any one of SEQ ID NO: 21, 22, 23, and 24. The antibody according to claim 1, characterized in that the complementarity determining regions of the variable regions are defined by any one system or a combination of multiple systems of Kabat, Chothia, IMGT, AbM or Contact. An anti-P24 antibody, characterized in that the antibody comprises the following complementary 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; LCDR3, which comprises or consists of the amino acid sequence shown in SEQ ID NO:6; 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 antibody binds P24 with an affinity of KD < 7.15 x 10 ^-8 M. An anti-P24 antibody comprising a heavy chain variable region and/or a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 19; the amino acid sequence of the light chain variable region is as shown in any one of SEQ ID NO: 21, 22, 23, and 24; Optionally, the antibody 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, camel, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human; Optionally, the species origin of the constant region is human; Optionally, the heavy chain constant region sequence is as shown in SEQ ID NO: 15 or 29 or has at least 80% identity thereto; Optionally, the light chain constant region sequence is as shown in SEQ ID NO:16 or has at least 80% identity thereto. An anti-P24 antibody, comprising a heavy chain and/or a light chain, characterized in that the amino acid sequence of the heavy chain is shown in any one of SEQ ID NO: 20 and 30; the amino acid sequence of the light chain is shown in any one of SEQ ID NO: 25, 26, 27 and 28. An antibody conjugate, characterized in that the antibody conjugate comprises the antibody according to any one of claims 1 to 5; Optionally, the antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody; Optionally, the antibody conjugate further comprises a label coupled to the antibody; 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 antibody. A reagent or a kit, characterized in that the reagent or the kit comprises the antibody according to any one of claims 1 to 5 or the antibody conjugate according to claim 6. Use of the antibody according to any one of claims 1 to 5 or the antibody conjugate according to claim 6 in detecting P24, diagnosing HIV infection in a subject, preparing a product for detecting P24, or preparing a product for diagnosing whether a subject is infected with HIV. A method for diagnosing whether a subject is infected with HIV, comprising: a) contacting the antibody of any one of claims 1 to 5, the antibody conjugate of claim 6, or the reagent or kit of 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, the presence of the complex indicating the presence of the antigen in the test sample; Optionally, the immune complex further comprises a second antibody, which binds to the antibody; Optionally, the immune complex further comprises a second antibody, which binds to P24. A method for detecting P24, characterized in that the method comprises: a) contacting the antibody 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 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; Optionally, the immune complex further comprises a second antibody, which binds to the antibody; Optionally, the immune complex further comprises a second antibody, which binds to P24. A nucleic acid, characterized in that the nucleic acid encodes the antibody or antigen-binding fragment according to any one of claims 1 to 5. A vector, wherein the vector comprises the nucleic acid according to claim 11. A cell, characterized in that the cell contains the nucleic acid according to claim 11 or the vector according to claim 12. A method for preparing the antibody or antigen-binding fragment of any one of claims 1 to 5, characterized in that the method comprises culturing the cell of claim 13.