Antibodies, kits and uses specific for n-terminal propeptide of procollagen type i

Abstract

The present invention relates to PINP-specific antibodies, kits and uses. In particular, the present invention provides PINP-specific antibodies, as well as kits containing said antibodies, and their use in detecting the presence or level of tPINP in a sample.

Description

Technical Field

[0001] This invention belongs to the field of molecular immunology, specifically relating to a type I procollagen N-terminal propeptide-specific antibody, a kit containing the antibody, and its uses. Background Technology

[0002] Collagen is the most abundant protein in mammals, with 28 types. Type I collagen is a trimer protein primarily found in bone tissue and some soft tissues. The procollagen chain includes N-terminal and C-terminal propeptides, which are cleaved by specific proteases during collagen maturation. The levels of both propeptides are diagnostically significant. Increased levels of the N-terminal propeptide (PINP) in human type I collagen indicate enhanced bone formation, such as during osteoporosis treatment or during bone metastases from cancer.

[0003] A method for measuring the N-terminal propeptide of human type I collagen in human serum has been developed (Melkko et al., 1996, Clinical Chemistry, 42:6, 947-954). This assay uses polyclonal antibodies, but although the monomeric form of PINP does exist in the blood, this method only detects the trimer form, and the sensitivity is insufficient. Therefore, there is still a need for highly sensitive detection of PINP in different forms. Summary of the Invention

[0004] Through extensive experimental research, the inventors have developed high-performance PINP antibodies and unexpectedly discovered antibody pairs particularly suitable for PINP double-antibody sandwich assays. Based on this, the inventors have developed a new PINP quantitative detection kit and method. The antibody (pair), kit, or detection method of this invention achieves a sensitivity of 0.3 ng / ml for tPINP (total type I procollagen N-terminal propeptide), with a detection range of 0.3-2000 ng / ml. In some embodiments, the sensitivity for tPINP reaches 0.03 ng / mg, with a detection range of 0.03-2000 ng / ml. Furthermore, the antibody (pair), kit, or detection method of this invention exhibits strong anti-interference capabilities, effectively shielding against interference from endogenous serum substances and common PINP analogues. Moreover, this invention enables rapid detection with relatively small sample volumes, possessing significant clinical application value.

[0005] Antibody

[0006] The inventors have screened and obtained a class of antibodies that can specifically bind to PINP, exhibiting strong binding activity to PINP with KD values ​​all at the nM level or even lower. This provides a new antibody raw material for the preparation of high-performance PINP detection kits.

[0007] Therefore, in one aspect, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to PINP (type I procollagen N-terminal propeptide) or different forms thereof, comprising:

[0008] The heavy chain variable region contains the following three CDRs: CDR-H1 with amino acid sequences selected from SEQ ID NO:1, 7, 13, 19, 25, 31, 37 and their variants; CDR-H2 with amino acid sequences selected from SEQ ID NO:2, 8, 14, 20, 26, 32, 38 and their variants; and / or CDR-H3 with amino acid sequences selected from SEQ ID NO:3, 9, 15, 21, 27, 33, 39 and their variants; and / or,

[0009] The light chain variable region contains the following three CDRs: CDR-L1 with amino acid sequences selected from SEQ ID NO:4, 10, 16, 22, 28, 34, 40 and their variants; CDR-L2 with amino acid sequences selected from SEQ ID NO:5, 11, 17, 23, 29, 35, 41 and their variants; and / or CDR-L3 with amino acid sequences selected from SEQ ID NO:6, 12, 18, 24, 30, 36, 42 and their variants.

[0010] The variant contains an amino acid mutation compared to the amino acid sequence from which it originates, the amino acid mutation being a substitution, deletion, or addition of one or more amino acids (e.g., a substitution, deletion, or addition of 1, 2, 3, 4, or 5 amino acids).

[0011] In some implementations, the permutation is a conservative permutation.

[0012] In some embodiments, the variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence from which it is derived.

[0013] In some implementations, antibodies containing the aforementioned variant or their antigen-binding fragments can still specifically bind to PINP.

[0014] In some implementations, the three CDRs contained in the heavy chain variable region and / or the three CDRs contained in the light chain variable region are defined by the Kabat, Chothia, or IMGT numbering system.

[0015] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0016] (1a) A heavy chain variable region comprising the following three CDRs: CDR-H1 with the amino acid sequence of SEQ ID NO:1 or a variant thereof, CDR-H2 with the amino acid sequence of SEQ ID NO:2 or a variant thereof, and CDR-H3 with the amino acid sequence of SEQ ID NO:3 or a variant thereof; and a light chain variable region comprising the following three CDRs: CDR-L1 with the amino acid sequence of SEQ ID NO:4 or a variant thereof, CDR-L2 with the amino acid sequence of SEQ ID NO:5 or a variant thereof, and CDR-L3 with the amino acid sequence of SEQ ID NO:6 or a variant thereof;

[0017] (1b) A heavy chain variable region comprising the following three CDRs: CDR-H1 with the amino acid sequence of SEQ ID NO:7 or a variant thereof, CDR-H2 with the amino acid sequence of SEQ ID NO:8 or a variant thereof, and CDR-H3 with the amino acid sequence of SEQ ID NO:9 or a variant thereof; and a light chain variable region comprising the following three CDRs: CDR-L1 with the amino acid sequence of SEQ ID NO:10 or a variant thereof, CDR-L2 with the amino acid sequence of SEQ ID NO:11 or a variant thereof, and CDR-L3 with the amino acid sequence of SEQ ID NO:12 or a variant thereof;

[0018] (1c) A heavy chain variable region comprising the following three CDRs: CDR-H1 with the amino acid sequence of SEQ ID NO:13 or a variant thereof, CDR-H2 with the amino acid sequence of SEQ ID NO:14 or a variant thereof, and CDR-H3 with the amino acid sequence of SEQ ID NO:15 or a variant thereof; and a light chain variable region comprising the following three CDRs: CDR-L1 with the amino acid sequence of SEQ ID NO:16 or a variant thereof, CDR-L2 with the amino acid sequence of SEQ ID NO:17 or a variant thereof, and CDR-L3 with the amino acid sequence of SEQ ID NO:18 or a variant thereof;

[0019] (1d) A heavy chain variable region comprising the following three CDRs: CDR-H1 with the amino acid sequence of SEQ ID NO:19 or a variant thereof, CDR-H2 with the amino acid sequence of SEQ ID NO:20 or a variant thereof, and CDR-H3 with the amino acid sequence of SEQ ID NO:21 or a variant thereof; and a light chain variable region comprising the following three CDRs: CDR-L1 with the amino acid sequence of SEQ ID NO:22 or a variant thereof, CDR-L2 with the amino acid sequence of SEQ ID NO:23 or a variant thereof, and CDR-L3 with the amino acid sequence of SEQ ID NO:24 or a variant thereof;

[0020] (1e) A heavy chain variable region comprising the following three CDRs: CDR-H1 with the amino acid sequence of SEQ ID NO:25 or a variant thereof, CDR-H2 with the amino acid sequence of SEQ ID NO:26 or a variant thereof, and CDR-H3 with the amino acid sequence of SEQ ID NO:27 or a variant thereof; and a light chain variable region comprising the following three CDRs: CDR-L1 with the amino acid sequence of SEQ ID NO:28 or a variant thereof, CDR-L2 with the amino acid sequence of SEQ ID NO:29 or a variant thereof, and CDR-L3 with the amino acid sequence of SEQ ID NO:30 or a variant thereof;

[0021] (1f) Heavy chain variable regions comprising the following three CDRs: CDR-H1 with the amino acid sequence SEQ ID NO:31 or a variant thereof, CDR-H2 with the amino acid sequence SEQ ID NO:32 or a variant thereof, and CDR-H3 with the amino acid sequence SEQ ID NO:33 or a variant thereof; and light chain variable regions comprising the following three CDRs: CDR-L1 with the amino acid sequence SEQ ID NO:34 or a variant thereof, CDR-L2 with the amino acid sequence SEQ ID NO:35 or a variant thereof, and CDR-L3 with the amino acid sequence SEQ ID NO:36 or a variant thereof; or

[0022] (1g) contains heavy chain variable regions of the following three CDRs: CDR-H1 with amino acid sequence of SEQ ID NO:37 or a variant thereof, CDR-H2 with amino acid sequence of SEQ ID NO:38 or a variant thereof, and CDR-H3 with amino acid sequence of SEQ ID NO:39 or a variant thereof; and light chain variable regions of the following three CDRs: CDR-L1 with amino acid sequence of SEQ ID NO:40 or a variant thereof, CDR-L2 with amino acid sequence of SEQ ID NO:41 or a variant thereof, and CDR-L3 with amino acid sequence of SEQ ID NO:42 or a variant thereof;

[0023] The variant contains an amino acid mutation compared to the CDR from which it originates, the amino acid mutation being a substitution, deletion, or addition of one or more amino acids (e.g., a substitution, deletion, or addition of 1, 2, 3, 4, or 5 amino acids).

[0024] In some embodiments, the antibody or its antigen-binding fragment described in (1a) above: (a1) is produced by a hybridoma deposited at the Russian State Industrial Microbiological Collection Center (VKPM) with accession number VKPM H-214; (a2) contains the same amino acid sequence as the three CDRs in the heavy chain variable region and / or the three CDRs in the light chain variable region of the antibody produced by the hybridoma deposited at VKPM with accession number VKPM H-214; (a3) ​​contains the same amino acid sequence as the heavy chain variable region and / or the light chain variable region of the antibody produced by the hybridoma deposited at VKPM with accession number VKPM H-214; or (a4) contains the same amino acid sequence as the heavy chain amino acid sequence and / or the light chain amino acid sequence of the antibody produced by the hybridoma deposited at VKPM with accession number VKPM H-214.

[0025] The hybridoma was deposited on February 21, 2024.

[0026] In some embodiments, the antibody or its antigen-binding fragment described in (1b) above: (b1) comprises an amino acid sequence encoded by an insert fragment of a plasmid deposited in VKPM with accession number VKPM B-14744, and / or by an insert fragment of a plasmid deposited in VKPM with accession number VKPM B-14745; (b2) comprises a heavy chain, the heavy chain comprising an amino acid sequence encoded by an insert fragment of a plasmid deposited in VKPM with accession number VKPM B-14744; and a light chain, the light chain comprising an amino acid sequence encoded by an insert fragment of a plasmid deposited in VKPM with accession number VKPM B-14745; or (b3) comprises an amino acid sequence encoded by an insert fragment of a plasmid deposited in VKPM with accession number VKPM B-14745. The amino acid sequences encoded by the insert fragment of plasmid B-14744 are CDR-H1, CDR-H2, and CDR-H3; and / or are identical amino acid sequences to those encoded by the insert fragment of plasmid B-14745, which is deposited at VKPM with accession number VKPM B-14745.

[0027] In some embodiments, the antibody or its antigen-binding fragment described in (1c) above: (c1) comprises an amino acid sequence encoded by an insert fragment of a plasmid deposited at VKPM, accession number VKPM B-14746, and / or by an insert fragment of a plasmid deposited at VKPM, accession number VKPM B-14747; (c2) comprises a heavy chain, the heavy chain comprising an amino acid sequence encoded by an insert fragment of a plasmid deposited at VKPM, accession number VKPM B-14746; and a light chain, the light chain comprising an amino acid sequence encoded by an insert fragment of a plasmid deposited at VKPM, accession number VKPM B-14747; or (c3) comprises an amino acid sequence encoded by an insert fragment of a plasmid deposited at VKPM, accession number VKPM B-14747; The amino acid sequences encoded by the insert fragment of plasmid B-14746 are CDR-H1, CDR-H2, and CDR-H3; and / or are identical amino acid sequences to those encoded by the insert fragment of plasmid B-14747, which is deposited at VKPM with accession number VKPM B-14747.

[0028] In some embodiments, the antibody or its antigen-binding fragment described in (1d) above: (d1) comprises an amino acid sequence encoded by an insert fragment of a plasmid deposited in VKPM, accession number VKPM B-14748, and / or by an insert fragment of a plasmid deposited in VKPM, accession number VKPM B-14749; (d2) comprises a heavy chain, the heavy chain comprising an amino acid sequence encoded by an insert fragment of a plasmid deposited in VKPM, accession number VKPM B-14748; and a light chain, the light chain comprising an amino acid sequence encoded by an insert fragment of a plasmid deposited in VKPM, accession number VKPM B-14749; or (d3) comprises an amino acid sequence encoded by an insert fragment of a plasmid deposited in VKPM, accession number VKPM B-14749; The amino acid sequences encoded by the insert fragment of plasmid B-14748 are CDR-H1, CDR-H2, and CDR-H3; and / or are identical amino acid sequences to those encoded by the insert fragment of plasmid B-14749, which is deposited at VKPM with accession number VKPM B-14749.

[0029] In some embodiments, the antibody or its antigen-binding fragment described in (1e) above: (e1) comprises an amino acid sequence encoded by an insert fragment of a plasmid deposited at VKPM, accession number VKPM B-14750, and / or by an insert fragment of a plasmid deposited at VKPM, accession number VKPM B-14751; (e2) comprises a heavy chain, the heavy chain comprising an amino acid sequence encoded by an insert fragment of a plasmid deposited at VKPM, accession number VKPM B-14750; and a light chain, the light chain comprising an amino acid sequence encoded by an insert fragment of a plasmid deposited at VKPM, accession number VKPM B-14751; or (e3) comprises an amino acid sequence encoded by an insert fragment of a plasmid deposited at VKPM, accession number VKPM B-14751; The amino acid sequences encoded by the insert fragment of plasmid B-14750 are CDR-H1, CDR-H2, and CDR-H3; and / or are identical amino acid sequences to those encoded by the insert fragment of plasmid B-14751, which is deposited at VKPM with accession number VKPM B-14751.

[0030] In some embodiments, the antibody or its antigen-binding fragment described in (1f) above: (f1) comprises the same amino acid sequence as the amino acid sequence encoded by the insert fragment of the plasmid deposited in VKPM, accession number VKPM B-14752; or (f2) comprises the same amino acid sequence as CDR-H1, CDR-H2 and CDR-H3 as described in the amino acid sequence encoded by the insert fragment of the plasmid deposited in VKPM, accession number VKPM B-14752; and / or comprises the same amino acid sequence as CDR-L1, CDR-L2 and CDR-L3 as described in the amino acid sequence encoded by the insert fragment of the plasmid deposited in VKPM, accession number VKPM B-14752.

[0031] In some embodiments, the antibody or its antigen-binding fragment described in (1g) above: (g1) contains the same amino acid sequence as the amino acid sequence encoded by the insert fragment of the plasmid deposited in VKPM, accession number VKPM B-14753; or (g2) contains the same amino acid sequence as CDR-H1, CDR-H2 and CDR-H3 as described in the amino acid sequence encoded by the insert fragment of the plasmid deposited in VKPM, accession number VKPM B-14753; and / or contains the same amino acid sequence as CDR-L1, CDR-L2 and CDR-L3 as described in the amino acid sequence encoded by the insert fragment of the plasmid deposited in VKPM, accession number VKPM B-14753.

[0032] The plasmids mentioned above were deposited on March 25, 2024.

[0033] In some implementations, the substitution is a conservative substitution.

[0034] In some embodiments, the variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence from which it is derived.

[0035] In some implementations, antibodies containing the aforementioned variant or their antigen-binding fragments can still specifically bind to PINP.

[0036] In some implementations, the three CDRs contained in the heavy chain variable region and / or the three CDRs contained in the light chain variable region are defined by the Kabat, Chothia, or IMGT numbering system.

[0037] In some embodiments, the antibody or its antigen-binding fragment further comprises a heavy chain constant region (CH) and a light chain constant region (CL).

[0038] In some implementations, the heavy chain constant region is the mouse heavy chain constant region, and the light chain constant region is the mouse light chain constant region.

[0039] In some implementations, the heavy chain constant region is a human heavy chain constant region, and the light chain constant region is a human light chain constant region.

[0040] In some embodiments, the antibody or its antigen-binding fragment is an IgG, IgM, IgE, IgD, or IgA antibody.

[0041] In some implementations, the heavy chain constant region is an IgG heavy chain constant region, such as the IgG1, IgG2, IgG3, or IgG4 heavy chain constant region;

[0042] In some implementations, the light chain constant region is a κ or λ light chain constant region (e.g., a human λ light chain constant region).

[0043] In some embodiments, the antigen-binding fragment is selected from scFv, Fab, Fab', (Fab')2, Fv fragment, disulfide-linked Fv (dsFv), diabody, bispecific antibody, and multispecific antibody; and / or, the antibody is a murine antibody, a chimeric antibody, or a humanized antibody.

[0044] In some embodiments, the antibody or its antigen-binding fragment carries a detectable marker.

[0045] In this document, the detectable label can be any substance detectable by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrical, optical, or chemical means. Particularly preferred are those labels suitable for immunological assays (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). Such labels are well known in the art and include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.) and radionuclides (e.g., 3 H, 125 I, 35 S, 14 C or 32 P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., Cy7, Alexa), etc.). 750), chemiluminescent substances (such as acridinium esters), and biotin for binding avidin modified with the above-described markers (e.g., streptavidin). The markers covered in this invention can be detected by methods known in the art. For example, radioactive markers can be detected using photographic film or a scintillation calculator, and fluorescent markers can be detected using a photodetector to detect emitted light. Enzyme markers are generally detected by providing the enzyme with a substrate and detecting the reaction product generated by the enzyme's action on the substrate. Calorimetric markers are detected by simple, visually colored markers. Chemiluminescent substances (such as acridinium esters) are generally detected by providing the luminescent substance with an excitation solution and / or a catalyst. Biotin is generally detected by providing biotin with avidin modified with the above-described markers (e.g., streptavidin) and detecting the marker carried by the avidin linked to biotin. In some embodiments, the detectable markers described above can be linked to the antibodies or antigen-binding fragments of the present invention via connectors of varying lengths to reduce potential steric hindrance.

[0046] In some embodiments, the label is selected from fluorescein, chemiluminescence (e.g., acridine esters), enzymes (e.g., horseradish peroxidase, alkaline phosphatase), radioisotopes, biotin, colloidal gold, and magnetic particles.

[0047] In some embodiments, antibodies against PINP or its different forms of existence, or antigen-binding fragments thereof, or any of the antibodies or antigen-binding fragments thereof described above, specifically bind to epitopes contained in a sequence spanning amino acid residues 23 to 161 of COL1A1 (human type I collagen α1 chain).

[0048] In some implementations, the different forms of PINP include monomeric forms of PINP.

[0049] Isolated nucleic acid molecules, expression vectors and host cells

[0050] The present invention also provides isolated nucleic acid molecules encoding the antibody or an antigen-binding fragment thereof.

[0051] The present invention also provides an expression vector containing the isolated nucleic acid molecules described above. In some embodiments, the vector is a plasmid, virus, bacteriophage, bacteria, or viroid.

[0052] The present invention further provides a host cell comprising the isolated nucleic acid molecules or expression vectors described above.

[0053] In some embodiments, the host cell is a eukaryotic cell, preferably a mammalian cell.

[0054] In some embodiments, the host cell is a prokaryotic cell, preferably Escherichia coli.

[0055] Reagent test kit

[0056] For pregnant women with vitamin D deficiency during pregnancy, tPINP testing in umbilical cord blood is often used to assess the activity of fetal osteoblasts. However, the tPINP content in umbilical cord blood is only about 0.3 ng / ml, and the sensitivity of current technology cannot meet this requirement. For children, especially those under 1 year old, blood tPINP testing is often used to assess bone development. However, the tPINP content in children's blood is often as high as 2000 ng / ml, so the current technology may not be able to meet the needs of these special groups. Whether it is umbilical cord blood testing or testing children's samples, the amount of blood collected clinically is relatively small, while the sample size of current mainstream testing methods is generally greater than 20 μL.

[0057] To address the aforementioned technical problems, based on the monoclonal antibody of this invention, this invention screens and obtains antibody pairs that are particularly suitable for PINP double antibody sandwich detection.

[0058] Therefore, in another aspect, the present invention provides a composition comprising the antibody or antigen-binding fragment thereof described in any of the preceding claims.

[0059] In some embodiments, the composition contains:

[0060] The first antibody is selected from any of the antibodies described above or their antigen-binding fragments, and...

[0061] The second antibody is selected from any of the antibodies or antigen-binding fragments described above;

[0062] The first antibody and the second antibody target different epitopes of PINP.

[0063] In some embodiments, the first antibody is the antibody described in (1a) above or its antigen-binding fragment;

[0064] The second antibody is selected from the antibodies or antigen-binding fragments thereof described in the preceding items: (1b), (1c), (1d), (1e), (1f) and (1g), preferably (1b), (1d), (1e) or (1f), more preferably (1b), (1d) or (1f).

[0065] In some embodiments, the first antibody is selected from any of the antibodies described in (a1)-(a4) above or their antigen-binding fragments; the second antibody is selected from any of the antibodies described in (b1)-(b3), (d1)-(d3), (e1)-(e3) and (f1)-(f2) above or their antigen-binding fragments.

[0066] In some embodiments, the first antibody is the antibody described in (1b) above or its antigen-binding fragment;

[0067] The second antibody is selected from the antibodies described in the preceding items below or their antigen-binding fragments: (1a), (1c), (1d), (1e), (1f) and (1g), preferably (1a), (1c), (1d), (1e) or (1g), more preferably (1g).

[0068] In some embodiments, the first antibody is selected from the antibodies described in any one of claims (b1)-(b3) above or their antigen-binding fragments; the second antibody is selected from the antibodies described in any one of claims 2(a1)-(a4), (c1)-(c3), (d1)-(d3), (e1)-(e3) and (g1)-(g2) or their antigen-binding fragments.

[0069] In some embodiments, the first antibody is the antibody described in (1c) above or its antigen-binding fragment;

[0070] The second antibody is selected from the antibodies described in the preceding items below or their antigen-binding fragments: (1a), (1b), (1d), (1e), (1f) and (1g), preferably (1b), (1e) or (1f).

[0071] In some embodiments, the first antibody is selected from any of the antibodies described in (c1)-(c3) above or their antigen-binding fragments; the second antibody is selected from any of the antibodies described in (b1)-(b3), (e1)-(e3) and (f1)-(f2) above or their antigen-binding fragments.

[0072] In some embodiments, the first antibody is the antibody described in item (1d) above or its antigen-binding fragment;

[0073] The second antibody is selected from the antibodies described in the preceding items below or their antigen-binding fragments: (1a), (1b), (1c), (1e), (1f) and (1g), preferably (1a), (1b) or (1f).

[0074] In some embodiments, the first antibody is selected from any of the antibodies described in (d1)-(d3) above or their antigen-binding fragments; the second antibody is selected from any of the antibodies described in (a1)-(a4), (b1)-(b3) and (f1)-(f2) above or their antigen-binding fragments.

[0075] In some embodiments, the first antibody is the antibody described in item (1e) above or its antigen-binding fragment;

[0076] The second antibody is selected from the antibodies or antigen-binding fragments thereof described in the preceding items: (1a), (1b), (1c), (1d), (1f) and (1g), preferably (1b), (1c) or (1f), more preferably (1b).

[0077] In some embodiments, the first antibody is selected from any of the antibodies described in (e1)-(e3) above or their antigen-binding fragments; the second antibody is selected from any of the antibodies described in (b1)-(b3), (c1)-(c3) and (f1)-(f2) above or their antigen-binding fragments.

[0078] In some embodiments, the first antibody is the antibody described in (1f) above or its antigen-binding fragment;

[0079] The second antibody is selected from the antibodies or antigen-binding fragments thereof described in the preceding items: (1a), (1b), (1c), (1d), (1e) and (1g).

[0080] In some embodiments, the first antibody is the antibody described in the preceding (1g) item or its antigen-binding fragment;

[0081] The second antibody is selected from the antibodies or antigen-binding fragments thereof described in the preceding items: (1a), (1b), (1c), (1d), (1e) and (1f).

[0082] In some embodiments, the composition contains:

[0083] The first antibody shown in paragraph (1a) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (a1)-(a4)) and the second antibody shown in paragraph (1b) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (b1)-(b3));

[0084] The first antibody shown in paragraph (1a) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (a1)-(a4)) and the second antibody shown in paragraph (1d) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (d1)-(d3));

[0085] The first antibody shown in paragraph (1a) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (a1)-(a4)) and the second antibody shown in paragraph (1e) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (e1)-(e3));

[0086] The first antibody shown in item (1a) above (e.g., any one of (a1)-(a4) or an antigen-binding fragment thereof), and the second antibody shown in item (1f) above (e.g., any one of (f1)-(f2) or an antigen-binding fragment thereof); the first antibody shown in item (1b) above (e.g., any one of (b1)-(b3) or an antigen-binding fragment thereof), and the second antibody shown in item (1a) above (e.g., any one of (a1)-(a4) or an antigen-binding fragment thereof);

[0087] The first antibody shown in paragraph (1b) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (b1)-(b3)) above, and the second antibody shown in paragraph (1c) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (c1)-(c3) above);

[0088] The first antibody shown in paragraph (1b) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (b1)-(b3)) above, and the second antibody shown in paragraph (1d) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (d1)-(d3) above);

[0089] The first antibody shown in paragraph (1b) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (b1)-(b3)) above, and the second antibody shown in paragraph (1e) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (e1)-(e3) above);

[0090] The first antibody shown in paragraph (1b) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (b1)-(b3)) above, and the second antibody shown in paragraph (1g) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (g1)-(g2) above);

[0091] The first antibody shown in paragraph (1c) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (c1)-(c3)) above, and the second antibody shown in paragraph (1b) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (b1)-(b3) above);

[0092] The first antibody shown in paragraph (1c) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (c1)-(c3)) above, and the second antibody shown in paragraph (1e) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (e1)-(e3) above);

[0093] The first antibody shown in paragraph (1c) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (c1)-(c3)) above, and the second antibody shown in paragraph (1f) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (f1)-(f2) above);

[0094] The first antibody shown in item (1d) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (d1)-(d3)) and the second antibody shown in item (1a) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (a1)-(a4));

[0095] The first antibody shown in item (1d) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (d1)-(d3)) and the second antibody shown in item (1b) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (b1)-(b3));

[0096] The first antibody shown in item (1d) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (d1)-(d3)), and the second antibody shown in item (1f) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (f1)-(f2));

[0097] The first antibody shown in paragraph (1e) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (e1)-(e3)) above, and the second antibody shown in paragraph (1b) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (b1)-(b3) above);

[0098] The first antibody described in paragraph (1e) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (e1)-(e3)), and the second antibody described in paragraph (1c) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (c1)-(c3)); or

[0099] The first antibody shown in item (1e) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (e1)-(e3)) and the second antibody shown in item (1f) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (f1)-(f2)).

[0100] Building upon this, the present invention further provides reagent kits and detection methods with high sensitivity and a wide detection range to meet specific clinical needs.

[0101] In another aspect, the present invention provides a PINP detection kit, wherein the detection sensitivity of the kit for tPINP is ≤0.3ng / ml, for example ≤0.2ng / ml, ≤0.15ng / ml, ≤0.1ng / ml, ≤0.05ng / ml, or ≤0.03ng / ml.

[0102] In another aspect, the present invention provides a PINP detection kit, wherein the detection range of tPINP is 0.3-2000 ng / ml, for example 0.05-2000 ng / ml, 0.1-2000 ng / ml, or 0.15-2000 ng / ml.

[0103] In another aspect, the present invention provides a PINP detection kit containing the antibody or antigen-binding fragment thereof described in any of the preceding claims.

[0104] In some embodiments, the kit contains:

[0105] The first antibody is selected from any of the antibodies described above or their antigen-binding fragments, and...

[0106] The second antibody is selected from any of the antibodies or antigen-binding fragments described above;

[0107] Preferably, the first antibody and the second antibody target different epitopes of PINP.

[0108] In some embodiments, the first antibody is the antibody described in (1a) above or its antigen-binding fragment;

[0109] The second antibody is selected from the antibodies or antigen-binding fragments thereof described in the preceding items: (1b), (1c), (1d), (1e), (1f) and (1g), preferably (1b), (1d), (1e) or (1f), more preferably (1b), (1d) or (1f).

[0110] In some embodiments, the first antibody is selected from any of the antibodies described in (a1)-(a4) above or their antigen-binding fragments; the second antibody is selected from any of the antibodies described in (b1)-(b3), (d1)-(d3), (e1)-(e3) and (f1)-(f2) above or their antigen-binding fragments.

[0111] In some embodiments, the first antibody is the antibody described in (1b) above or its antigen-binding fragment;

[0112] The second antibody is selected from the antibodies described in the preceding items below or their antigen-binding fragments: (1a), (1c), (1d), (1e), (1f) and (1g), preferably (1a), (1c), (1d), (1e) or (1g), more preferably (1g).

[0113] In some embodiments, the first antibody is selected from the antibodies described in any one of claims (b1)-(b3) above or their antigen-binding fragments; the second antibody is selected from the antibodies described in any one of claims 2(a1)-(a4), (c1)-(c3), (d1)-(d3), (e1)-(e3) and (g1)-(g2) or their antigen-binding fragments.

[0114] In some embodiments, the first antibody is the antibody described in (1c) above or its antigen-binding fragment;

[0115] The second antibody is selected from the antibodies described in the preceding items below or their antigen-binding fragments: (1a), (1b), (1d), (1e), (1f) and (1g), preferably (1b), (1e) or (1f).

[0116] In some embodiments, the first antibody is selected from any of the antibodies described in (c1)-(c3) above or their antigen-binding fragments; the second antibody is selected from any of the antibodies described in (b1)-(b3), (e1)-(e3) and (f1)-(f2) above or their antigen-binding fragments.

[0117] In some embodiments, the first antibody is the antibody described in item (1d) above or its antigen-binding fragment;

[0118] The second antibody is selected from the antibodies described in the preceding items below or their antigen-binding fragments: (1a), (1b), (1c), (1e), (1f) and (1g), preferably (1a), (1b) or (1f).

[0119] In some embodiments, the first antibody is selected from any of the antibodies described in (d1)-(d3) above or their antigen-binding fragments; the second antibody is selected from any of the antibodies described in (a1)-(a4), (b1)-(b3) and (f1)-(f2) above or their antigen-binding fragments.

[0120] In some embodiments, the first antibody is the antibody described in item (1e) above or its antigen-binding fragment;

[0121] The second antibody is selected from the antibodies or antigen-binding fragments thereof described in the preceding items: (1a), (1b), (1c), (1d), (1f) and (1g), preferably (1b), (1c) or (1f), more preferably (1b).

[0122] In some embodiments, the first antibody is selected from any of the antibodies described in (e1)-(e3) above or their antigen-binding fragments; the second antibody is selected from any of the antibodies described in (b1)-(b3), (c1)-(c3) and (f1)-(f2) above or their antigen-binding fragments.

[0123] In some embodiments, the first antibody is the antibody described in (1f) above or its antigen-binding fragment;

[0124] The second antibody is selected from the antibodies or antigen-binding fragments thereof described in the preceding items: (1a), (1b), (1c), (1d), (1e) and (1g).

[0125] In some embodiments, the first antibody is the antibody described in the preceding (1g) item or its antigen-binding fragment;

[0126] The second antibody is selected from the antibodies or antigen-binding fragments thereof described in the preceding items: (1a), (1b), (1c), (1d), (1e) and (1f).

[0127] In some implementations, the kit contains:

[0128] The first antibody shown in paragraph (1a) above (e.g., any of the antibodies described in (a1)-(a4) or their antigen-binding fragments), and the second antibody shown in paragraph (1b) above (e.g., any of the antibodies described in (b1)-(b3) or their antigen-binding fragments);

[0129] The first antibody shown in paragraph (1a) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (a1)-(a4)) and the second antibody shown in paragraph (1d) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (d1)-(d3));

[0130] The first antibody shown in paragraph (1a) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (a1)-(a4)) and the second antibody shown in paragraph (1e) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (e1)-(e3));

[0131] The first antibody (e.g., any one of (a1)-(a4) or its antigen-binding fragment) shown in paragraph (1a) above, and the second antibody (e.g., any one of (f1)-(f2) or its antigen-binding fragment) shown in paragraph (1f) above; the first antibody (e.g., any one of (b1)-(b3) or its antigen-binding fragment) shown in paragraph (1b) above, and the second antibody (e.g., any one of (a1)-(a4) or its antigen-binding fragment) shown in paragraph (1a) above;

[0132] The first antibody shown in paragraph (1b) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (b1)-(b3)) and the second antibody shown in paragraph (1c) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (c1)-(c3));

[0133] The first antibody shown in paragraph (1b) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (b1)-(b3)) and the second antibody shown in paragraph (1d) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (d1)-(d3));

[0134] The first antibody shown in paragraph (1b) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (b1)-(b3)) and the second antibody shown in paragraph (1e) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (e1)-(e3));

[0135] The first antibody shown in paragraph (1b) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (b1)-(b3)) and the second antibody shown in paragraph (1g) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (g1)-(g2));

[0136] The first antibody shown in paragraph (1c) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (c1)-(c3)) and the second antibody shown in paragraph (1b) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (b1)-(b3));

[0137] The first antibody shown in paragraph (1c) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (c1)-(c3)) and the second antibody shown in paragraph (1e) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (e1)-(e3));

[0138] The first antibody shown in paragraph (1c) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (c1)-(c3)) above, and the second antibody shown in paragraph (1f) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (f1)-(f2) above);

[0139] The first antibody shown in item (1d) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (d1)-(d3)) and the second antibody shown in item (1a) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (a1)-(a4));

[0140] The first antibody shown in item (1d) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (d1)-(d3)) and the second antibody shown in item (1b) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (b1)-(b3));

[0141] The first antibody shown in item (1d) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (d1)-(d3)) and the second antibody shown in item (1f) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (f1)-(f2));

[0142] The first antibody shown in paragraph (1e) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (e1)-(e3)) and the second antibody shown in paragraph (1b) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (b1)-(b3));

[0143] The first antibody (e.g., any one of (e1)-(e3) or its antigen-binding fragment) shown in paragraph (1e) above, and the second antibody (e.g., any one of (c1)-(c3) or its antigen-binding fragment) shown in paragraph (1c) above; or

[0144] The first antibody shown in item (1e) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (e1)-(e3)) and the second antibody shown in item (1f) above (e.g., the antibody or antigen-binding fragment thereof described in any one of (f1)-(f2)).

[0145] In some implementations, the first antibody is a capture antibody and the second antibody is a detection antibody.

[0146] In some embodiments, the kit further comprises a solid-phase support. In some embodiments, the solid-phase support comprises a well plate, test tube, beads (e.g., latex particles), or film (e.g., nitrocellulose membrane) made of or coated with a polymeric material (e.g., polyvinyl chloride, polystyrene, polyacrylamide, or cellulose), or magnetic beads pre-coated with functional groups (e.g., amino, carboxyl, biotin, or avidin). In some embodiments, the solid-phase support is selected from magnetic beads or microtiter plates (e.g., microplates or ELISA plates).

[0147] In some implementations, the capture antibody is coated on the surface of a solid-phase support.

[0148] In some implementations, the detection antibody carries a detectable marker.

[0149] In some embodiments, the label is selected from fluorescein, chemiluminescence (e.g., acridine esters), enzymes (e.g., horseradish peroxidase, alkaline phosphatase), radioisotopes, biotin, and colloidal gold.

[0150] In some embodiments, the kit may further include reagents for enabling the detection of the corresponding detectable label. For example, when the detectable label is an enzyme, the kit may also include a chromogenic substrate for the corresponding enzyme, such as o-phenylenediamine (OPD), tetramethylbenzidine (TMB), ABTS, or luminol compounds for horseradish peroxidase, or p-nitrophenyl phosphate (p-NPP) or AMPPD for alkaline phosphatase. For example, when the detectable label is a chemiluminescent reagent (e.g., acrid ester compounds), the kit may also include a pre-excitation solution and / or an excitation solution for chemiluminescence.

[0151] In some embodiments, the kit further comprises a coating reagent for coating the first antibody (capture antibody) onto the solid-phase support, such as a coating buffer (e.g., carbonate buffer, phosphate buffer, Tris-HCl buffer, or borate buffer). Methods for coating proteins or peptides onto solid-phase supports are well known in the art, such as physical adsorption, covalent coupling via aminated or carboxylated surfaces, or mediated binding via avidin-biotin systems, polylysine pre-coated surfaces, or protein A or protein G pre-coated surfaces.

[0152] In some implementations, the kit includes a capture antibody diluent and a detection antibody diluent.

[0153] In some embodiments, the capture antibody diluent contains: a 10-500 mM buffer system, 10-500 mM NaCl, 0.01%-0.1% Proclin 300, 0.05%-0.5% Tween-20, and 0.1%-5% BSA, with a pH of 6.0-9.0.

[0154] In some implementations, the buffer system is selected from Tris, Hepes, and PBS buffer systems.

[0155] In some embodiments, the pH of the capture antibody diluent is 6.0-6.5, 6.0-7.0, 6.0-7.5, 6.0-8.0, 6.0-8.5, 6.0-9.0, 6.5-7.0, 6.5-7.5, 6.5-8.0, 6.5-8.5, 6.5-9.0, 7.0-7.5, 7.0-8.0, 7.0-8.5, 7.0-9.0, 7.5-8.0, 7.5-8.5, 7.5-9.0, 8.0-8.5, 8.0-9.0, or 8.5-9.0.

[0156] In some embodiments, the capture antibody diluent contains: a 50-200 mM buffer system, 50-200 mM NaCl, 0.02%-0.06% Proclin 300, 0.05%-0.2% Tween-20, and 0.5%-2% BSA, with a pH of 6.0-9.0.

[0157] In some embodiments, the capture antibody diluent consists of: 50 mM Tris, 0.9% NaCl, 0.048% Proclin 300, 0.1% Tween 20, 1% BSA, and pH 8.

[0158] 100 mM Hepes, 0.9% NaCl, 0.048% Proclin 300, 0.1% Tween 20, 1% BSA, pH 7.4; or

[0159] 100mM PBS, 0.9% NaCl, 0.048% Proclin 300, 0.1% Tween 20, 1% BSA, pH 7.4.

[0160] In some embodiments, the detection antibody diluent contains: a 10-500 mM buffer system, 10-500 mM NaCl, 0.01%-0.1% Proclin 300, 0.05%-0.5% Tween-20, and 0.1%-5% BSA, with a pH of 6.0-9.0.

[0161] In some implementations, the buffer system is selected from Tris, Hepes, and MES buffer systems.

[0162] In some embodiments, the pH of the detection antibody diluent is 6.0-6.5, 6.0-7.0, 6.0-7.5, 6.0-8.0, 6.0-8.5, 6.0-9.0, 6.5-7.0, 6.5-7.5, 6.5-8.0, 6.5-8.5, 6.5-9.0, 7.0-7.5, 7.0-8.0, 7.0-8.5, 7.0-9.0, 7.5-8.0, 7.5-8.5, 7.5-9.0, 8.0-8.5, 8.0-9.0, or 8.5-9.0.

[0163] In some embodiments, the detection antibody diluent also contains 1-5 mM MgCl2 and 0.05-0.5 mM ZnCl2.

[0164] In some embodiments, the detection antibody diluent contains: a 50-200 mM buffer system, 50-200 mM NaCl, 0.02%-0.06% Proclin 300, 0.05%-0.2% Tween-20, and 0.5%-2% BSA, and optionally 1-3 mM MgCl2 and 0.05-0.2 mM ZnCl2, with a pH of 6.0-9.0.

[0165] In some embodiments, the detection antibody diluent consists of: 50 mM Tris, 0.9% NaCl, 0.048% Proclin 300, 0.1% Tween 20, 1% BSA, and pH 8.

[0166] 100 mM Hepes, 0.9% NaCl, 0.048% Proclin 300, 0.1% Tween 20, 1% BSA, pH 7.4;

[0167] 50 mM MES, 0.9% NaCl, 0.048% Proclin 300, 0.1% Tween 20, 1% BSA, pH 6.0; or

[0168] 50 mM MES, 0.9% NaCl, 0.048% Proclin 300, 0.1% Tween 20, 1% BSA, 2 mM MgCl2, 0.1 mM ZnCl2, pH 6.0.

[0169] In some embodiments, the PINP detection kit includes: a capture antibody immobilized on magnetic particles, a detection antibody labeled with alkaline phosphatase, a capture antibody diluent, and a detection antibody diluent, wherein...

[0170] The capturing antibody is the antibody described in (1a) above or its antigen-binding fragment (e.g., the antibody described in any one of (a1)-(a4) above or its antigen-binding fragment), and the detection antibody is the antibody described in (1d) above or its antigen-binding fragment (e.g., the antibody described in any one of (d1)-(d3) above or its antigen-binding fragment); or

[0171] The capturing antibody is the antibody described in (1c) above or its antigen-binding fragment (e.g., the antibody described in any one of (c1)-(c3) above or its antigen-binding fragment), and the detection antibody is the antibody described in (1f) above or its antigen-binding fragment (e.g., the antibody described in any one of (f1)-(f2) above or its antigen-binding fragment); or

[0172] The capturing antibody is the antibody described in (1e) above or its antigen-binding fragment (e.g., the antibody described in any one of (e1)-(e3) above or its antigen-binding fragment), and the detection antibody is the antibody described in (1b) above or its antigen-binding fragment (e.g., the antibody described in any one of (b1)-(b3) above or its antigen-binding fragment); or

[0173] The capturing antibody is the antibody described in (1a) above or its antigen-binding fragment (e.g., the antibody described in any one of (a1)-(a4) above or its antigen-binding fragment), and the detection antibody is the antibody described in (1f) above or its antigen-binding fragment (e.g., the antibody described in any one of (f1)-(f2) above or its antigen-binding fragment); or

[0174] The capturing antibody is the antibody described in (1a) above or its antigen-binding fragment (e.g., the antibody described in any one of (a1)-(a4) above or its antigen-binding fragment), and the detection antibody is the antibody described in (1b) above or its antigen-binding fragment (e.g., the antibody described in any one of (b1)-(b3) above or its antigen-binding fragment); or

[0175] The capture antibody is the antibody described in (1c) above or its antigen-binding fragment (e.g., the antibody described in any one of (c1)-(c3) above or its antigen-binding fragment), and the detection antibody is the antibody described in (1b) above or its antigen-binding fragment (e.g., the antibody described in any one of (b1)-(b3) above or its antigen-binding fragment).

[0176] In some embodiments, the capture antibody is selected from any of the antibodies described in (a1)-(a4) above or their antigen-binding fragments; the detection antibody is selected from any of the antibodies described in (b1)-(b3) above or their antigen-binding fragments.

[0177] In some embodiments, the capture antibody is selected from any of the antibodies described in (a1)-(a4) above or their antigen-binding fragments; the detection antibody is selected from any of the antibodies described in (d1)-(d3) above or their antigen-binding fragments.

[0178] In some embodiments, the capture antibody is selected from any of the antibodies described in (a1)-(a4) above or their antigen-binding fragments; the detection antibody is selected from any of the antibodies described in (f1)-(f3) above or their antigen-binding fragments.

[0179] In some embodiments, the capture antibody is selected from any of the antibodies described in (c1)-(c3) above or their antigen-binding fragments; the detection antibody is selected from any of the antibodies described in (b1)-(b3) above or their antigen-binding fragments.

[0180] In some embodiments, the capture antibody is selected from any of the antibodies described in (c1)-(c3) above or their antigen-binding fragments; the detection antibody is selected from any of the antibodies described in (f1)-(f2) above or their antigen-binding fragments.

[0181] In some embodiments, the capture antibody is selected from any of the antibodies described in (e1)-(e3) above or their antigen-binding fragments; the detection antibody is selected from any of the antibodies described in (b1)-(b3) above or their antigen-binding fragments.

[0182] In some embodiments, the PINP detection kit includes: a capture antibody immobilized on magnetic particles, a detection antibody labeled with alkaline phosphatase, a capture antibody diluent, and a detection antibody diluent, wherein...

[0183] The capture antibody is the antibody or its antigen-binding fragment described in section (1a) above, and the detection antibody is the antibody or its antigen-binding fragment described in section (1d) above. The diluent for the capture antibody consists of 50 mM Tris, 0.9% NaCl, 0.048% Proclin 300, 0.1% Tween 20, and 1% BSA, with a pH of 8. The diluent for the detection antibody consists of 50 mM Tris, 0.9% NaCl, 0.048% Proclin 300, 0.1% Tween 20, and 1% BSA, with a pH of 8.

[0184] The capture antibody is the antibody described in section (1c) above or its antigen-binding fragment, and the detection antibody is the antibody described in section (1f) above or its antigen-binding fragment. The capture antibody diluent consists of: 100 mM Hepes, 0.9% NaCl, 0.048% Proclin 300, 0.1% Tween 20, and 1% BSA, with a pH of 7.4. The detection antibody diluent consists of: 100 mM Hepes, 0.9% NaCl, 0.048% Proclin 300, 0.1% Tween 20, and 1% BSA, with a pH of 7.4; or

[0185] The capture antibody is the antibody or its antigen-binding fragment described in section (1e) above, and the detection antibody is the antibody or its antigen-binding fragment described in section (1b) above. The capture antibody diluent consists of: 100 mM PBS, 0.9% NaCl, 0.048% Proclin 300, 0.1% Tween 20, and 1% BSA, with a pH of 7.4. The detection antibody diluent consists of: 50 mM PBS, 0.9% NaCl, 0.048% Proclin 300, 0.1% Tween 20, and 1% BSA, with a pH of 6.0.

[0186] In some embodiments, the kit further includes one or more of the following: PINP calibrator, chemiluminescent substrate, washing solution, stop solution, and instructions for use.

[0187] In some embodiments, the PINP calibrator contains an amino acid sequence as shown in SEQ ID NO: 43.

[0188] In some implementations, it is used to detect tPINP, in particular the presence or level of PINP in a sample.

[0189] In some implementations, the sample is derived from a human being, such as human bodily fluids (e.g., blood, serum, or plasma).

[0190] Detection methods and applications

[0191] In another aspect, the present invention provides a method for detecting the presence or level (concentration) of tPINP (especially PINP) in a sample, comprising the steps of using an antibody or its antigen-binding fragment as described in any of the preceding claims or a PINP detection kit.

[0192] In some embodiments, the method includes contacting the sample with two or more antibodies or antigen-binding fragments selected from any of the antibodies or antigen-binding fragments described above, and qualitatively or quantitatively detecting the binding of the two or more antibodies or antigen-binding fragments to PINP and its different forms of existence (e.g., monomeric PINP), the binding indicating the presence or concentration of PINP and its different forms of existence in the sample, wherein,

[0193] The two or more antibodies or their antigen-binding fragments target two or more epitopes of PINP.

[0194] In some embodiments, at least one of the two or more antibodies or their antigen-binding fragments is a capture antibody, and the other or some are detection antibodies.

[0195] In some embodiments, the capture antibody and detection antibody are selected from groups 1-7:

[0196] Group 1: The capture antibody is the antibody or its antigen-binding fragment described in section (1a) above;

[0197] The detection antibody is selected from the following antibodies or their antigen-binding fragments as defined in the preceding items: (1b), (1c), (1d), (1e), (1f) and (1g), preferably (1b), (1d), (1e) or (1f), more preferably (1b), (1d) or (1f);

[0198] Group 2: The capture antibody is the antibody described in section (1b) above or its antigen-binding fragment;

[0199] The detection antibody is selected from the following antibodies or antigen-binding fragments thereof as defined in the preceding items: (1a), (1c), (1d), (1e), (1f) and (1g), preferably (1a), (1c), (1d), (1e) or (1g), more preferably (1g);

[0200] Group 3: The capture antibody is the antibody or its antigen-binding fragment described in section (1c) above;

[0201] The detection antibody is selected from the following antibodies or antigen-binding fragments thereof as defined in the preceding items: (1a), (1b), (1d), (1e), (1f) and (1g), preferably (1b), (1e) or (1f);

[0202] Group 4: The capture antibody is the antibody or its antigen-binding fragment described in item (1d) above;

[0203] The detection antibody is selected from the following antibodies or antigen-binding fragments thereof as defined in the preceding items: (1a), (1b), (1c), (1e), (1f) and (1g), preferably (1a), (1b) or (1f);

[0204] Group 5: The capture antibody is the antibody or its antigen-binding fragment described in section (1e) above;

[0205] The detection antibody is selected from the following antibodies or their antigen-binding fragments as defined in the preceding items: (1a), (1b), (1c), (1d), (1f) and (1g), preferably (1b), (1c) or (1f), more preferably (1b);

[0206] Group 6: The capture antibody is the antibody or its antigen-binding fragment described in section (1f) above;

[0207] The detection antibody is selected from the following antibodies or their antigen-binding fragments as defined in the preceding items: (1a), (1b), (1c), (1d), (1e), and (1g); and

[0208] Group 7: The capture antibody is the antibody or its antigen-binding fragment described in the preceding section (1g);

[0209] The detection antibody is selected from the following antibodies or their antigen-binding fragments as defined in the preceding items: (1a), (1b), (1c), (1d), (1e) and (1f).

[0210] In some implementations, the capture antibody and detection antibody are selected from:

[0211] The capture antibody is the antibody described in (1a) above or its antigen-binding fragment (e.g., the antibody described in any one of (a1)-(a4) or its antigen-binding fragment), and the detection antibody is the antibody described in (1b) above or its antigen-binding fragment (e.g., the antibody described in any one of (b1)-(b3) or its antigen-binding fragment);

[0212] The capture antibody is the antibody described in (1a) above or its antigen-binding fragment (e.g., the antibody described in any one of (a1)-(a4) or its antigen-binding fragment), and the detection antibody is the antibody described in (1d) above or its antigen-binding fragment (e.g., the antibody described in any one of (d1)-(d3) or its antigen-binding fragment);

[0213] The capture antibody is the first antibody in paragraph (1a) above (e.g., any one of (a1)-(a4) or its antigen-binding fragment), and the second antibody in paragraph (1e) above (e.g., any one of (e1)-(e3) or its antigen-binding fragment);

[0214] The capture antibody is the antibody described in (1a) above or its antigen-binding fragment (e.g., the antibody described in any one of (a1)-(a4) or its antigen-binding fragment), and the detection antibody is the antibody described in (1f) above or its antigen-binding fragment (e.g., the antibody described in any one of (f1)-(f2) or its antigen-binding fragment);

[0215] The capture antibody is the first antibody shown in item (1b) above (e.g., any of the antibodies described in (b1)-(b3) or their antigen-binding fragments), and the detection antibody is the second antibody in item (1a) above (e.g., any of the antibodies described in (a1)-(a4) or their antigen-binding fragments);

[0216] The capture antibody is the first antibody shown in item (1b) above (e.g., the antibody or antigen-binding fragment of any one of (b1)-(b3) above), and the detection antibody is the second antibody in item (1c) above (e.g., the antibody or antigen-binding fragment of any one of (c1)-(c3) above);

[0217] The capture antibody is the first antibody shown in item (1b) above (e.g., the antibody or antigen-binding fragment of any one of (b1)-(b3) above), and the detection antibody is the second antibody in item (1d) above (e.g., the antibody or antigen-binding fragment of any one of (d1)-(d3) above);

[0218] The capture antibody is the first antibody shown in item (1b) above (e.g., any of the antibodies described in (b1)-(b3) or their antigen-binding fragments), and the detection antibody is the second antibody shown in item (1e) above (e.g., any of the antibodies described in (e1)-(e3) or their antigen-binding fragments).

[0219] The capture antibody is the antibody described in (1b) above or its antigen-binding fragment (e.g., the antibody described in any one of (b1)-(b3) or its antigen-binding fragment), and the detection antibody is the antibody described in (1g) above or its antigen-binding fragment (e.g., the antibody described in any one of (g1)-(g2) or its antigen-binding fragment);

[0220] The capture antibody is the antibody described in (1c) above or its antigen-binding fragment (e.g., the antibody described in any one of (c1)-(c3) or its antigen-binding fragment), and the detection antibody is the antibody described in (1b) above or its antigen-binding fragment (e.g., the antibody described in any one of (b1)-(b3) or its antigen-binding fragment);

[0221] The capture antibody is the antibody described in (1c) above or its antigen-binding fragment (e.g., the antibody described in any one of (c1)-(c3) or its antigen-binding fragment), and the detection antibody is the antibody described in (1e) above or its antigen-binding fragment (e.g., the antibody described in any one of (e1)-(e3) or its antigen-binding fragment);

[0222] The capturing antibody is the antibody described in (1c) above or its antigen-binding fragment (e.g., the antibody described in any one of (c1)-(c3) or its antigen-binding fragment), and the detection antibody is the antibody described in (1f) above or its antigen-binding fragment (e.g., the antibody described in any one of (f1)-(f2) or its antigen-binding fragment); and

[0223] The capture antibody is the antibody described in (1e) above or its antigen-binding fragment (e.g., the antibody described in any one of (e1)-(e3) or its antigen-binding fragment), and the detection antibody is the antibody described in (1b) above or its antigen-binding fragment (e.g., the antibody described in any one of (b1)-(b3) or its antigen-binding fragment).

[0224] In some embodiments, the capture antibody is selected from any of the antibodies described in (a1)-(a4) above or their antigen-binding fragments; the detection antibody is selected from any of the antibodies described in (b1)-(b3), (d1)-(d3), (e1)-(e3) and (f1)-(f2) above or their antigen-binding fragments.

[0225] In some embodiments, the capture antibody is selected from the antibodies described in any one of claims (b1)-(b3) above or their antigen-binding fragments; the detection antibody is selected from the antibodies described in any one of claims 2(a1)-(a4), (c1)-(c3), (d1)-(d3), (e1)-(e3) and (g1)-(g2) or their antigen-binding fragments.

[0226] In some embodiments, the capture antibody is selected from any of the antibodies described in (c1)-(c3) above or their antigen-binding fragments; the detection antibody is selected from any of the antibodies described in (b1)-(b3), (e1)-(e3) and (f1)-(f2) above or their antigen-binding fragments.

[0227] In some embodiments, the capture antibody is selected from any of the antibodies described in (d1)-(d3) above or their antigen-binding fragments; the detection antibody is selected from any of the antibodies described in (a1)-(a4), (b1)-(b3) and (f1)-(f2) above or their antigen-binding fragments.

[0228] In some embodiments, the capture antibody is selected from any of the antibodies described in (e1)-(e3) above or their antigen-binding fragments; the detection antibody is selected from any of the antibodies described in (b1)-(b3), (c1)-(c3) and (f1)-(f2) above or their antigen-binding fragments.

[0229] In some embodiments, the capture antibody is selected from any of the antibodies described in (a1)-(a4) above or their antigen-binding fragments; the detection antibody is selected from any of the antibodies described in (b1)-(b3) above or their antigen-binding fragments.

[0230] In some embodiments, the capture antibody is selected from any of the antibodies described in (a1)-(a4) above or their antigen-binding fragments; the detection antibody is selected from any of the antibodies described in (d1)-(d3) above or their antigen-binding fragments.

[0231] In some embodiments, the capture antibody is selected from any of the antibodies described in (a1)-(a4) above or their antigen-binding fragments; the detection antibody is selected from any of the antibodies described in (f1)-(f3) above or their antigen-binding fragments.

[0232] In some embodiments, the capture antibody is selected from any of the antibodies described in (c1)-(c3) above or their antigen-binding fragments; the detection antibody is selected from any of the antibodies described in (b1)-(b3) above or their antigen-binding fragments.

[0233] In some embodiments, the capture antibody is selected from any of the antibodies described in (c1)-(c3) above or their antigen-binding fragments; the detection antibody is selected from any of the antibodies described in (f1)-(f2) above or their antigen-binding fragments.

[0234] In some embodiments, the capture antibody is selected from any of the antibodies described in (e1)-(e3) above or their antigen-binding fragments; the detection antibody is selected from any of the antibodies described in (b1)-(b3) above or their antigen-binding fragments.

[0235] In some implementations, the method includes the following steps:

[0236] (1) The sample is contacted with the capture antibody to form an antibody-antigen complex;

[0237] (2) Contact the antibody-antigen complex with the detection antibody to form an antibody-antigen-antibody complex; and

[0238] (3) Determine the amount of the antibody-antigen-antibody complex.

[0239] In some implementations, the capture antibody is coated on the surface of a solid-phase support.

[0240] In some embodiments, the solid support is selected from magnetic particles or microtiter plates (e.g., microplates or ELISA plates);

[0241] The detection antibody carries a detectable marker.

[0242] In some embodiments, the label is selected from fluorescein, chemiluminescence (e.g., acridine esters), enzymes (e.g., horseradish peroxidase, alkaline phosphatase), radioisotopes, biotin, and colloidal gold.

[0243] In some implementations, the method is an ELISA immunoassay, such as a double-antibody sandwich ELISA immunoassay.

[0244] In some implementations, the sample is derived from a human being, such as human bodily fluids (e.g., blood, serum, or plasma).

[0245] In some implementations, the method includes the following steps:

[0246] The capture antibody coated on the surface of the solid support is diluted with the capture antibody diluent to obtain a capture antibody reagent; preferably, the concentration of the capture antibody in the capture antibody reagent is 0.001%-0.005%, for example 0.001%-0.003%;

[0247] The detection antibody with the detectable label is diluted with the detection antibody diluent to obtain the detection antibody reagent; preferably, the mass concentration of the labeled detection antibody in the detection antibody reagent is 1-5 μg / mL, for example 1-3 μg / mL;

[0248] 1-20 μL of the sample is reacted with the capture antibody reagent and the detection antibody reagent, and chemiluminescent immunoassay is performed to obtain the presence or concentration of PINP and its different forms in the sample.

[0249] Preferably, the sample volume is less than 15 μL, more preferably less than 10 μL, and even more preferably less than 5 μL.

[0250] In some implementations, the method further includes a calibration operation using a PINP calibrator.

[0251] In some embodiments, the PINP standard contains an amino acid sequence as shown in SEQ ID NO: 43.

[0252] In some implementations, the sample detection time using the method is less than 20 minutes, preferably less than 15 minutes.

[0253] In some implementations, the detection range for tPINP in samples is 0.03 ng / mL to 2000 ng / mL.

[0254] In another aspect, the present invention provides the use of PINP as a quality control or calibrator, said PINP having the amino acid sequence shown in SEQ ID NO: 43.

[0255] In some implementations, the detection is an ELISA immunoassay, such as a double-antibody sandwich ELISA immunoassay.

[0256] In another aspect, the present invention provides the use of the antibody or antigen-binding fragment thereof described in any of the preceding claims in the preparation of a kit for detecting the presence or level of tPINP (especially PINP) in a sample.

[0257] In another aspect, the present invention provides the use of the antibody or antigen-binding fragment thereof described in any of the preceding claims, the composition thereof, or the detection kit or method for detecting the presence or level of tPINP (especially PINP) in a sample.

[0258] Terminology Definition

[0259] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the virological, biochemical, and immunological laboratory procedures used herein are all standard procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0260] Type I collagen constitutes over 90% of the organic components of the bone matrix. Fibroblasts and osteoblasts first synthesize type I procollagen, which then forms type I collagen. Type I procollagen has extended peptide chains at its N-terminus and C-terminus. These extended peptide chains (propeptides) are cleaved by specific proteases during the conversion of procollagen to collagen. Once mature collagen is formed, it is deposited in the bone matrix. As used herein, the term "PINP" specifically refers to the N-terminal propeptide of type I procollagen. PINP reflects the deposition of type I collagen and can therefore serve as a marker of bone formation. During the formation of type I collagen, PINP is released into the extracellular space and eventually enters the bloodstream. PINP exists in a trimer form (derived from trimer collagen) but quickly degrades into a monomeric form due to thermal degradation. The kits and detection methods of this invention can be used to detect all forms of PINP in the blood, i.e., tPINP, including both monomeric and trimeric forms.

[0261] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules (i.e., a binding molecule and a target molecule), such as the reaction between an antibody and its target antigen. The binding affinity between two molecules can be measured using Kx. D Value description. K D The value refers to the dissociation constant obtained by the ratio of kd (the dissociation rate of a specific binding molecule-target molecule interaction; also known as koff) to ka (the association rate of a specific binding molecule-target molecule interaction; also known as kon), or kd / ka expressed as molar concentration (M). D The smaller the value, the tighter the binding between the two molecules, and the higher the affinity. In some embodiments, an antibody that specifically binds to a certain antigen (or an antibody that is specific to a certain antigen) refers to an antibody with a binding affinity of less than approximately 10. -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or lower affinity (K) D ) binds to this antigen. K D The value can be determined by methods well known in the art, such as using surface plasmon resonance (SPR) in a BIACORE instrument.

[0262] As used herein, the term "immunologic assay" refers to a determination that utilizes the specific interaction / binding affinity between an antigen and antibody, and is generally used to detect the presence or level of a specific antigen or antibody in a sample. Such immunological assays are well known to those skilled in the art and include, but are not limited to, enzyme immunoassay (EIA), chemiluminescent immunoassay (CLIA), radioimmunoassay (RIA), fluorescence immunoassay (FIA), Western blotting, immunoturbidimetry, and surface plasmon resonance assays. For a detailed description of immunological assays, see, for example, Fundamental Immunology, Ch. 7, Paul W., ed., 2nd edition, Raven Press, NY (1989).

[0263] As used herein, the terms “antibody” and “monoclonal antibody” are used interchangeably and refer to immunoglobulin molecules typically composed of two pairs of polypeptide chains (each pair consisting of one light chain (LC) and one heavy chain (HC)). Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both light and heavy chains, variable and constant regions are linked by “J” regions of approximately 12 or more amino acids, and heavy chains also contain “D” regions of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. Constant domains do not directly participate in antibody-antigen binding, but exhibit various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)), interspersed with more conserved regions called framework regions (FRs). H and V LIt consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable regions (VH and VL) of each heavy / light chain pair form the antigen-binding sites. The allocation of amino acids in each region or domain can follow the definitions in Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883.

[0264] As used herein, the term “complementarity-determining region” or “CDR” refers to the amino acid residue in the antibody variable region responsible for antigen binding. Each of the heavy and light chain variable regions contains three CDRs, designated CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883), or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003). For a given antibody, those skilled in the art will readily identify the CDR as defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (see, for example, Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0265] In this invention, the CDR contained in the antibody or antigen-binding fragment thereof can be determined according to various numbering systems known in the art. In some embodiments, the CDR contained in the antibody or antigen-binding fragment thereof is preferably determined by the Kabat, Chothia, or IMGT numbering system.

[0266] As used herein, the term "framework region" or "FR" residues refer to the amino acid residues in the antibody variable region other than the CDR residues as defined above.

[0267] The term "antibody" is not limited to any particular method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0268] As used herein, the term “antigen-binding fragment” of an antibody refers to a polypeptide containing a fragment of the full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen; it is also referred to as the “antigen-binding moiety”. See also Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, complementarity-determining region (CDR) fragments, scFv, diabody, single-domain antibody, chimeric antibody, linear antibody, nanobody (technology from Domantis), and peptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the peptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136.

[0269] As used herein, the term “Fd” refers to an antibody fragment consisting of VH and CH1 domains; the term “dAb fragment” refers to an antibody fragment consisting of VH domains (Ward et al., Nature 341:544 546 (1989)); the term “Fab fragment” refers to an antibody fragment consisting of VL, VH, CL and CH1 domains; the term “F(ab')2 fragment” refers to an antibody fragment containing two Fab fragments connected by disulfide bridges on the hinge region; the term “Fab' fragment” refers to the fragment obtained by reducing the disulfide bonds connecting the two heavy chain fragments in the F(ab')2 fragment, consisting of a complete light chain and heavy chain Fd fragment (consisting of VH and CH1 domains).

[0270] As used herein, the term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody. Fv fragments are generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. It is generally believed that six CDRs confer antigen-binding specificity to the antibody. However, even a variable region (such as an Fd fragment, which contains only three antigen-specific CDRs) can recognize and bind to the antigen, although its affinity may be lower than that of a complete binding site.

[0271] As used herein, the term “scFv” refers to a single polypeptide chain containing VL and VH domains linked by a linker (see, for example, Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Roseburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of a repeating GGGGS amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers that can be used in this invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may also exist between VH and VL of scFv.

[0272] As used herein, the term “biantibody” means that its VH and VL domains are expressed on a single polypeptide chain, but the linker is too short to allow pairing between the two domains on the same chain, thus forcing the domain to pair with the complementary domain of another chain and creating two antigen-binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak RJ et al., Structure 2:1121-1123 (1994)).

[0273] As used herein, the term "single-domain antibody (sdAb)" has the meaning commonly understood by those skilled in the art as an antibody fragment consisting of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that maintains the ability to specifically bind to the same antigen bound by a full-length antibody. Single-domain antibodies are also known as nanobodies.

[0274] Each of the above antibody fragments retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen.

[0275] Antigen-binding fragments (e.g., the antibody fragments described above) of a given antibody (e.g., the antibody provided in this invention) can be obtained using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation methods), and the antigen-binding fragments of the antibody can be specifically screened in the same manner as those used for intact antibodies.

[0276] In this article, unless the context clearly indicates otherwise, when referring to the term "antibody," it includes not only the complete antibody but also the antigen-binding fragment of the antibody.

[0277] As used herein, the term "chimeric antibody" refers to an antibody whose light chain and / or heavy chain portion is derived from one antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and whose light chain and / or heavy chain portion is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but which retains its binding activity to the target antigen in any case (USP4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:68516855 (1984)). For example, the term "chimeric antibody" may include antibodies (e.g., human-mouse chimeric antibodies) in which the variable regions of the heavy and light chains of the antibody are derived from a first antibody (e.g., a murine antibody), while the variable regions of the heavy and light chains of the antibody are derived from a second antibody (e.g., a human antibody).

[0278] As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase sequence homology with human antibodies. Typically, all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., the variable region FR and / or constant region) is derived from a human immunoglobulin (receptor antibody). Humanized antibodies generally retain the intended properties of the donor antibody, including but not limited to antigen specificity, affinity, and reactivity. Donor antibodies can be mouse, rat, rabbit, or non-human primate (e.g., cynomolgus monkey) antibodies with the intended properties (e.g., antigen specificity, affinity, reactivity).

[0279] The chimeric or humanized antibodies of the present invention can be prepared based on the sequence of the mouse monoclonal antibody prepared above. The DNA encoding the heavy and light chains can be obtained from the target mouse hybridoma and engineered using standard molecular biology techniques to contain non-mouse (e.g., human) immunoglobulin sequences.

[0280] To prepare chimeric antibodies, the variable region of a mouse immunoglobulin can be ligated to the constant region of a human immunoglobulin using methods known in the art. For example, DNA encoding VH can be operatively ligated to another DNA molecule encoding the heavy chain constant region to obtain a full-length heavy chain gene. The sequences of human heavy chain constant region genes are known in the art (see, for example, Kabat, E.A. et al. (1991), Sequences of Proteins of Immunological Interest, Fifth Edition, Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but is generally preferred to be an IgG1 or IgG4 constant region. For example, DNA encoding VL can be operatively ligated to another DNA molecule encoding the light chain constant region CL to obtain a full-length light chain gene (and a Fab light chain gene). The sequences of human light chain constant regions are known in the art (see, for example, Kabat, E.A. et al. (1991), Sequences of Proteins of Immunological Interest, Fifth Edition, Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. Light chain constant regions can be κ or λ constant regions, but κ constant regions are generally preferred.

[0281] To prepare humanized antibodies, mouse CDR regions can be inserted into human frame sequences using methods known in the art (see Winter’s U.S. Patent No. 5,225,539; Queen et al.’s U.S. Patent Nos. 5,530,101, 5,585,089, 5,693,762 and 6,180,370; and Lo, Benny, KC, editor, in Antibody Engineering: Methods and Protocols, volume 248, Humana Press, New Jersey, 2004).

[0282] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0283] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0284] As used herein, the term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. Two compared sequences are identical at a position when the same base or amino acid monomeric subunit occupies the same location (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine). The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (three out of six positions match). Typically, two sequences are compared to produce the maximum identity. Such comparisons can be made using methods readily available, for example, computer programs such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48: 443-453. The percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0), which uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J MoIBiol. 48:444-453 (1970)) in the GAP program integrated into the GCG software package (available at www.gcg.com), which uses a Blossum 62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0285] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the intended properties of a protein / peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0286] The twenty common amino acids discussed in this article were written in accordance with standard usage. See, for example, Immunology-ASynthesis (2nd Edition, ESGolub and DRGren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference.

[0287] In this invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Furthermore, in this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0288] As used herein, the term “subject” includes, but is not limited to, various animals, particularly mammals such as humans.

[0289] In this article, unless otherwise taught, "%" refers to mass percentage. Attached Figure Description

[0290] Figure 1 The display shows the antibody binding curves obtained by biomembrane interferometry, where A, B, C, D, E, F and G correspond to antibodies PINP01, hPINP02, hPINP03, hPINP04, hPINP05, hPINP06 and hPINP07, respectively.

[0291] Figure 2 This demonstrates the detection range and linearity of the kit in Example 6.

[0292] Figure 3 This shows the detection range and linearity of the kit in Example 7.

[0293] Figure 4 This demonstrates the detection range and linearity of the kit in Example 8.

[0294] Figure 5 This demonstrates the detection range and linearity of the kit in Example 9.

[0295] Figure 6 This demonstrates the detection range and linearity of the kit from Example 10.

[0296] Figure 7 This demonstrates the detection range and linearity of the kit from Example 11. Detailed Implementation

[0297] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0298] Example 1: Generation of a human type I procollagen N-terminal propeptide (PINP)-specific monoclonal antibody

[0299] Mice and rats were immunized with recombinant A1-PINP, the sequence of which (HyTest Cat#8PIN7) corresponds to region 23-161 (N-terminal propeptide) of the human type I procollagen α1 chain (UniProt ID P02452). Anti-A1-PINP mouse antibodies PINP01, PINP02, PINP03, PINP04, PINP05, PINP06, and PINP07 were obtained through hybridoma screening, with PINP01 being a mouse antibody and the others being rat antibodies. These seven antibodies were sequenced, and the results are shown in Tables 1 and 2.

[0300] Table 1. Antibody heavy chain sequencing results

[0301]

[0302]

[0303] Table 2. Antibody light chain sequencing results

[0304]

[0305]

[0306]

[0307] Example 2: Preparation and affinity purification of antibodies

[0308] The heavy chain variable regions of murine antibodies PINP02, PINP03, PINP04, PINP05, PINP06, and PINP07 were combined with the human IgG1 heavy chain constant region, and the light chain variable regions of the above antibodies were combined with the human λ light chain constant region, forming gene constructs of six recombinant antibodies. These constructs were expressed in mammalian cells to obtain recombinant antibodies hPINP02, hPINP03, hPINP04, hPINP05, hPINP06, and hPINP07, which were then purified.

[0309] Expression vectors containing the gene sequences of the aforementioned recombinant antibodies can be obtained through bacterial culture and plasmid purification, and then transfected into the mammalian cell line Expi293F.

[0310] For example, light chain and heavy chain gene sequences are transferred into the open reading frame (ORF) of an expression vector through homologous recombination or restriction enzyme digestion, respectively. This results in the formation of "promoter-antibody light chain gene-terminator" and "promoter-antibody heavy chain gene-terminator" structures on the two expression vectors, respectively. Then, using biological, physical, and chemical methods, the two expression vectors (antibody light chain expression vector and heavy chain expression vector) are simultaneously transformed into the mammalian cell line Expi293F for expression. Finally, the two light chains and two heavy chains are reassembled into a complete antibody in the cell and secreted into the culture supernatant. Alternatively, the expression vector can be modified to contain two open reading frames (ORFs). Then, through homologous recombination or restriction enzyme digestion, the light chain and heavy chain gene sequences are inserted into the two ORFs of the expression vector, forming a structure of "promoter-antibody light chain gene-terminator-vector sequence-promoter-antibody heavy chain gene-terminator" or "promoter-antibody heavy chain gene-terminator-vector sequence-promoter-antibody light chain gene-terminator". This expression vector containing both antibody light chain and heavy chain gene sequences is then transformed into the mammalian cell line Expi293F for expression using biological, physical, and chemical methods. Finally, the two light chains and two heavy chains reassemble into a complete antibody in the cell and are secreted into the culture supernatant.

[0311] Antibodies (PINP01, hPINP02, hPINP03, hPINP04, hPINP05, hPINP06, hPINP07) were purified from conditioned medium using protein A affinity chromatography. Affinity chromatography packing material was from GE Healthcare Life Sciences (Piscataway, NJ), and purification methods followed the manufacturer's instructions. The purified monoclonal antibodies were stored in suspension in 50% ammonium sulfate at 4°C.

[0312] Preservation information:

[0313] PINP01: The hybridoma cell line PINP01 was deposited at VKPM on February 21, 2024, with accession number VKPM H-214.

[0314] hPINP02: Escherichiacoli Rosetta plasmid containing an insert fragment encoding the hPINP02 VH amino acid sequence. TM(DE3)pLysS hPINP02 VH was deposited at VKPM on March 25, 2024, with accession number VKPM B-14744; the plasmid containing the insert encoding the amino acid sequence of hPINP02 VL is an Escherichia coli Rosetta arabic plasmid. TM (DE3)pLysS hPINP02VL was deposited in VKPM on March 25, 2024, with accession number VKPM B-14745;

[0315] hPINP03: Escherichiacoli Rosetta plasmid containing an insert fragment encoding the hPINP03 VH amino acid sequence. TM (DE3)pLysS hPINP03 VH was deposited at VKPM on March 25, 2024, with accession number VKPM B-14746; the plasmid containing the insert encoding the amino acid sequence of hPINP03 VL is an Escherichia coli Rosetta oleifera plasmid. TM (DE3)pLysS hPINP03VL was deposited in VKPM on March 25, 2024, with accession number VKPM B-14747;

[0316] hPINP04: Escherichiacoli Rosetta plasmid containing an insert fragment encoding the hPINP04 VH amino acid sequence. TM (DE3)pLysS hPINP04 VH was deposited at VKPM on March 25, 2024, with accession number VKPM B-14748; the plasmid containing the insert encoding the amino acid sequence of hPINP04 VL is an Escherichia coli Rosetta oleifera plasmid. TM (DE3)pLysS hPINP04VL was deposited in VKPM on March 25, 2024, with accession number VKPM B-14749;

[0317] hPINP05: Escherichiacoli Rosetta plasmid containing an insert fragment encoding the hPINP05 VH amino acid sequence. TM (DE3)pLysS hPINP05 VH was deposited at VKPM on March 25, 2024, with accession number VKPM B-14750; the plasmid containing the insert encoding the amino acid sequence of hPINP05 VL is an Escherichia coli Rosetta lysate. TM(DE3)pLysS hPINP05VL was deposited in VKPM on March 25, 2024, with accession number VKPM B-14751;

[0318] hPINP06: Escherichia coli Rosetta plasmid containing an insert fragment encoding the hPINP06 VH amino acid sequence and an insert fragment encoding the hPINP06 VL amino acid sequence. TM (DE3)pLysS hPINP06 was deposited at VKPM on March 25, 2024, with accession number VKPM B-14752;

[0319] hPINP07: Escherichia coli Rosetta plasmid containing an insert fragment encoding the hPINP07 VH amino acid sequence and an insert fragment encoding the hPINP07 VL amino acid sequence. TM (DE3)pLysS hPINP07 was deposited in VKPM on March 25, 2024, with accession number VKPM B-14753.

[0320] Example 3 Determination of Antibody Affinity

[0321] The affinity of the antibody for A1-PINP was obtained by performing biomembrane interference measurements according to the following protocol, specifically as follows: Figure 1 As shown in Table 3.

[0322] The antibody was bound to the sensor in PBS solution (2 μg / mL) to block the sensor. Then, the antigen (A1-PINP) at concentrations of 20, 30, and 50 nM reacted with the antibody on the sensor. The complete experimental procedure was as follows: sensor hydration → antibody binding to sensor → sensor cleaning → sensor blocking → binding (for each concentration of the test antibody) → dissociation → sensor regeneration.

[0323] Table 3 Results of Monoclonal Antibody Affinity Assay

[0324]

[0325]

[0326] Example 4: Development of a chemiluminescent "sandwich" immunoassay

[0327] The sandwich method was used to determine the different pairing combinations of all monoclonal antibodies in order to find the combination with the best properties.

[0328] Immunoassay based on chemiluminescent sandwich particles: Alkaline phosphatase (catalog number 03137031103, Roche Custom Biotech) was used to label and detect monoclonal antibodies, while biotin (catalog number PG82075, Thermo) was used to label and capture monoclonal antibodies. The capture antibodies were incubated together with streptavidin-labeled magnetic beads (MyOne T1 Dynabeads streptavidin PMP hydrophobic particles 100 mg / mL, Cat#35604D, Thermo). The incubated magnetic beads were diluted with a solution containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2% BSA, 0.2% Tween-20, 0.05% ProClin 300, and 0.09% NaN3 to obtain a magnetic bead suspension with a concentration of 0.25 mg / mL. The antibody-alkaline phosphatase conjugate was diluted in a solution containing 50 mM MES, pH 6, 0.9% NaCl, 1% BSA, 0.05% Tween-20, 0.048% ProClin 300, 2 mM MgCl2, and 0.1 mM ZnCl2 to obtain a 0.5 μg / mL antibody-alkaline phosphatase conjugate solution. A1-PINP was used as a calibrator for the immunoassay. A series of dilutions of A1-PINP were prepared using a solution containing 50 mM MES, pH 6, 0.9% NaCl, 1% BSA, 0.05% Tween-20, 0.048% ProClin 300, 2 mM MgCl2, and 0.1 mM ZnCl2. 20 μL of A1-PINP dilution was mixed with 50 μL of magnetic bead suspension and 50 μL of antibody-alkaline phosphatase conjugate solution. A one-step immunoassay was performed according to the instructions for the Minray CL-6000i automated analyzer, using the substrate solution provided by the analyzer supplier. Chemiluminescence is expressed in relative light units (RLU). This method can detect both trimer and monomeric forms of recombinant human type I collagen N-terminal propeptide.

[0329] Determination of the limit of detection (LOD) of antibody

[0330] LOB and LOD were determined according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI) (Clinical Laboratory Measurement Procedures: Assessment of Detection Capabilities; Approved Guideline—Second Edition; EP17-A, Vol. 24, No. 34). Twenty zero analytes (50 mM MES, pH 6, 0.9% NaCl, 1% BSA, 0.05% Tween-20, 0.048% ProClin 300, 2 mM MgCl2, 0.1 mM ZnCl2) were analyzed in the corresponding chemiluminescent sandwich immunoassay.

[0331] Calculate LOB (blank limit) and LOD using the following formula:

[0332] LOB = 1.64 * mean;

[0333] LOD = LOB + 1.65 * SD

[0334] Table 4. LOD of antibody pairs in CLIA

[0335]

[0336]

[0337] Example 5: Sequencing of antigens used for quality control or as calibrators in PINP kits.

[0338] The A1-PINP coding sequence (SEQ ID NO:43) was available from public databases and corresponds to region 23-161 of the human collagen α-1(I) chain (Uniprot P02452, COL1A1-collagen α-1(I) chain-Homo sapiens (human)|UniProtKB|UniProt). The coding nucleotide sequence was synthesized in Genscript, and the gene sequence was subcloned into the pcDNA3.4 expression vector using standard cloning techniques. The gene construct contained an H6 affinity tag at the C-terminus and was separated from the A1-PINP sequence via a GGGSGGGSGGGS adapter. Endotoxin-free plasmid DNA was purified using the Zymo Research DNA Plasmid Purification Kit. The Expi293F cell line was transfected with linear PEI, and the culture medium was collected on day 7. The cell-free medium was further purified by metal chelate chromatography to homogenize.

[0339] SEQ ID NO:43

[0340] MFSFVDLRLLLLLAATALLTHG QEEGQVEGQDEDIPPITCVQNGLRYHDRDVWKPEPCRICVCDNGKVLCDDVICDETKNCPGAEVPEGECCPVCPDGSESPTDQETTGVEGPKGDTGPRGPRGPAGPPGRDGIPGQPGLPGPPGPPGPPGPPGLGGNFAPGGGSGGGSGGGSHHHHHH

[0341] Signal peptides are indicated by an underscore.

[0342] Example 6: Preparation of tPINP reagent kit and calibrators, and detection of tPINP.

[0343] Step 1: Preparation of R1 reagent

[0344] The specific PINP antibody (PINP01) was mixed with superparamagnetic microparticles in TBS buffer. After the reaction was complete, the mixture was placed on a magnetic separator until the supernatant was clear. The supernatant was removed, and the superparamagnetic microparticles coated with PINP antibody were collected. The microparticles were then washed three times with TBS buffer. Finally, the superparamagnetic microparticles coated with PINP antibody were dissolved in R1 diluent. The final content of superparamagnetic microparticles was 0.07%, and the content of PINP antibody was 0.002%. The composition of R1 diluent was: 50 mM Tris, 0.9% NaCl, 0.048% Proclin 300, 0.1% Tween 20, 1% BSA, and pH 8.0. The R1 reagent was then stored at 2-8°C.

[0345] Step 2: Preparation of R2 reagent

[0346] Another specific PINP antibody (hPINP04) was conjugated with alkaline phosphatase at a molar ratio of 15:1 to the antibody. The conjugate was then dissolved in R2 diluent to a final concentration of 2 μg / mL. The R2 diluent consisted of 50 mM Tris, 0.9% NaCl, 0.048% Proclin 300, 0.1% Tween 20, and 1% BSA at pH 8.0. The resulting R2 reagent was then stored at 2–8 °C.

[0347] Step 3: Preparation of PINP calibrators

[0348] The PINP antigen A1-PINP was diluted to concentrations of 0 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL, 300 ng / mL, 500 ng / mL, 1000 ng / mL, 1600 ng / mL, and 2200 ng / mL to prepare PINP calibrators.

[0349] Step 4: Measurement Procedure for the tPINP Kit

[0350] ① Load the tPINP kit (including reagents R1 and R2) into the Mindray CL series fully automated chemiluminescence immunoassay analyzer;

[0351] ② The system requires a sample volume of 5 μL for each measurement, and the total measurement time for each report is 17.5 min;

[0352] The specific sample addition process of the detection system is as follows: The fully automated chemiluminescence immunoassay analyzer adds 5 μL of sample, 50 μL of R1 reagent, and 50 μL of R2 reagent to the reaction tube, respectively. After incubation, PINP in the sample binds to the anti-PINP antibody coated on the magnetic beads, while the anti-PINP antibody-alkaline phosphatase conjugate binds to another site of PINP in the sample, forming a sandwich complex. After the reaction is complete, the magnetic field attracts the magnetic beads, washing away unbound substances.

[0353] The reaction substrate or excitation solution is then added to the reaction tube to generate a light signal. The number of photons generated is measured, and the number of photons generated is proportional to the concentration of PINP in the sample.

[0354] ③ Perform calibration tests using the matching PINP calibrators. Based on the calibration data, the system software uses a weighted four-parameter logarithmic curve (4PLC) mathematical method to fit the luminescence signal and concentration. The final result is given in the form of concentration in ng / mL.

[0355] Example 7: Preparation of tPINP reagent kit and calibrators, and detection of tPINP.

[0356] Step 1: Preparation of R1 reagent

[0357] The specific PINP antibody (hPINP03) was mixed with superparamagnetic microparticles in TBS buffer. After sufficient reaction, the mixture was placed on a magnetic separator until the supernatant was clear. The supernatant was removed, and the superparamagnetic microparticles coated with PINP antibody were collected. The microparticles were then washed three times with TBS buffer. Finally, the superparamagnetic microparticles coated with PINP antibody were dissolved in R1 diluent. The final content of superparamagnetic microparticles was 0.1%, and the content of PINP antibody was 0.003%. The composition of R1 diluent was: 100 mM Heps, 0.9% NaCl, 0.048% Proclin 300, 0.1% Tween 20, 1% BSA, and pH 7.4. The R1 reagent was then stored at 2-8°C.

[0358] Step 2: Preparation of R2 reagent

[0359] Another specific PINP antibody (hPINP06) was conjugated with alkaline phosphatase at a molar ratio of 15:1 to the antibody. The conjugate was then dissolved in R2 diluent to obtain a final concentration of 2.5 μg / mL. The R2 diluent consisted of 100 mM Hepes, 0.9% NaCl, 0.048% Proclin 300, 0.1% Tween 20, and 1% BSA at pH 7.4. The resulting R2 reagent was then stored at 2–8 °C.

[0360] Step 3: Preparation of PINP calibrators

[0361] The PINP antigen A1-PINP was diluted to concentrations of 0 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL, 300 ng / mL, 500 ng / mL, 1000 ng / mL, 1600 ng / mL, and 2200 ng / mL to prepare PINP calibrators.

[0362] Step 4: Measurement Procedure for the tPINP Kit

[0363] ① Load the tPINP kit (including reagents R1 and R2) into the Mindray CL series fully automated chemiluminescence immunoassay analyzer;

[0364] ② The system requires a sample volume of 5 μL for each measurement, and the total measurement time for each report is 17.5 min;

[0365] The specific sample addition process of the detection system is as follows: The fully automated chemiluminescence immunoassay analyzer adds 5 μL of sample, 50 μL of R1 reagent, and 50 μL of R2 reagent to the reaction tube, respectively. After incubation, PINP in the sample binds to the anti-PINP antibody coated on the magnetic beads, while the anti-PINP antibody-alkaline phosphatase conjugate binds to another site of PINP in the sample, forming a sandwich complex. After the reaction is complete, the magnetic field attracts the magnetic beads, washing away unbound substances.

[0366] The reaction substrate or excitation solution is then added to the reaction tube to generate a light signal. The number of photons generated is measured, and the number of photons generated is proportional to the concentration of PINP in the sample.

[0367] ③ Use the matching PINP calibrator to perform calibration tests. Based on the calibration data, the system software uses a weighted four-parameter logarithmic curve (4PLC) mathematical method to fit the luminescence signal and concentration. The final result is given in the form of concentration in ng / mL.

[0368] Example 8: Preparation of tPINP reagent kit and calibrators, and tPINP detection method.

[0369] Step 1: Preparation of R1 reagent

[0370] The specific PINP antibody (hPINP05) was mixed with superparamagnetic microparticles in TBS buffer. After sufficient reaction, the mixture was placed on a magnetic separator until the supernatant was clear. The supernatant was removed, and the superparamagnetic microparticles coated with PINP antibody were collected. The microparticles were then washed three times with TBS buffer. Finally, the superparamagnetic microparticles coated with PINP antibody were dissolved in R1 diluent. The final content of superparamagnetic microparticles was 0.05%, and the content of PINP antibody was 0.0015%. The composition of R1 diluent was: 100mM PBS, 0.9% NaCl, 0.048% Proclin 300, 0.1% Tween 20, 1% BSA, and pH 7.4. The R1 reagent was then stored at 2-8℃.

[0371] Step 2: Preparation of R2 reagent

[0372] Another specific PINP antibody (hPINP02) was conjugated with alkaline phosphatase at a molar ratio of 15:1 to the antibody. The conjugate was then dissolved in R2 diluent to obtain a final concentration of 1.5 μg / mL. The R2 diluent consisted of 50 mM MES, 0.9% NaCl, 0.048% Proclin 300, 0.1% Tween 20, and 1% BSA at pH 6.0. The resulting R2 reagent was then stored at 2–8 °C.

[0373] Step 3: Preparation of PINP calibrators

[0374] The PINP antigen A1-PINP was diluted to concentrations of 0 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL, 300 ng / mL, 500 ng / mL, 1000 ng / mL, 1600 ng / mL, and 2200 ng / mL to prepare PINP calibrators.

[0375] Step 4: Measurement Procedure for the tPINP Kit

[0376] ① Load the tPINP kit (including reagents R1 and R2) into the Mindray CL series fully automated chemiluminescence immunoassay analyzer;

[0377] ② The system requires a sample volume of 5 μL for each measurement, and the total measurement time for each report is 17.5 min;

[0378] The specific sample addition process of the detection system is as follows: The fully automated chemiluminescence immunoassay analyzer adds 5 μL of sample, 50 μL of R1 reagent, and 50 μL of R2 reagent to the reaction tube, respectively. After incubation, PINP in the sample binds to the anti-PINP antibody coated on the magnetic beads, while the anti-PINP antibody-alkaline phosphatase conjugate binds to another site of PINP in the sample, forming a sandwich complex. After the reaction is complete, the magnetic field attracts the magnetic beads, washing away unbound substances.

[0379] The reaction substrate or excitation solution is then added to the reaction tube to generate a light signal. The number of photons generated is measured, and the number of photons generated is proportional to the concentration of PINP in the sample.

[0380] ③ Perform calibration tests using the matching PINP calibrators. Based on the calibration data, the system software uses a weighted four-parameter logarithmic curve (4PLC) mathematical method to fit the luminescence signal and concentration. The final result is given in the form of concentration in ng / mL.

[0381] Example 9: Preparation of tPINP reagent kit and calibrators, and detection of tPINP.

[0382] Step 1: Preparation of R1 reagent

[0383] The specific PINP antibody (PINP01) was mixed with superparamagnetic microparticles in TBS buffer. After the reaction was complete, the mixture was placed on a magnetic separator until the supernatant was clear. The supernatant was removed, and the superparamagnetic microparticles coated with PINP antibody were collected. The microparticles were then washed three times with TBS buffer. Finally, the superparamagnetic microparticles coated with PINP antibody were dissolved in R1 diluent. The final content of superparamagnetic microparticles was 0.05%, and the content of PINP antibody was 0.0015%. The composition of R1 diluent was: 50 mM Tris, 0.9% NaCl, 0.048% Proclin 300, 0.1% Tween 20, 1% BSA, and pH 7.4. The R1 reagent was then stored at 2-8℃.

[0384] Step 2: Preparation of R2 reagent

[0385] Another specific PINP antibody (hPINP06) was conjugated with alkaline phosphatase at a molar ratio of 15:1 to the antibody. The conjugate was then dissolved in R2 dilution buffer to achieve a final concentration of 1.5 μg / mL. The R2 dilution buffer consisted of 50 mM MES, 0.9% NaCl, 0.048% Proclin 300, 0.1% Tween 20, 1% BSA, 2 mM MgCl2, 0.1 mM ZnCl2, and pH 6.0. The resulting R2 reagent was then stored at 2–8 °C.

[0386] Step 3: Preparation of PINP calibrators

[0387] The PINP antigen A1-PINP was diluted to concentrations of 0 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL, 300 ng / mL, 500 ng / mL, 1000 ng / mL, 1600 ng / mL, and 2200 ng / mL to prepare PINP calibrators.

[0388] Step 4: Measurement Procedure for the tPINP Kit

[0389] ① Load the tPINP kit (including reagents R1 and R2) into the Mindray CL series fully automated chemiluminescence immunoassay analyzer;

[0390] ② The system requires a sample volume of 5 μL for each measurement, and the total measurement time for each report is 17.5 min;

[0391] The specific sample addition process of the detection system is as follows: The fully automated chemiluminescence immunoassay analyzer adds 5 μL of sample, 50 μL of R1 reagent, and 50 μL of R2 reagent to the reaction tube, respectively. PINP in the sample binds to the anti-PINP antibody coated on the magnetic beads, while the anti-PINP antibody-alkaline phosphatase conjugate binds to another PINP site in the sample, forming a sandwich complex. After the reaction is complete, the magnetic field attracts the magnetic beads, washing away unbound substances.

[0392] The reaction substrate or excitation solution is then added to the reaction tube to generate a light signal. The number of photons generated is measured, and the number of photons generated is proportional to the concentration of PINP in the sample.

[0393] ③ Perform calibration tests using the matching PINP calibrators. Based on the calibration data, the system software uses a weighted four-parameter logarithmic curve (4PLC) mathematical method to fit the luminescence signal and concentration. The final result is given in the form of concentration in ng / mL.

[0394] Example 10: Preparation of tPINP reagent kit and calibrators, and detection of tPINP.

[0395] Step 1: Preparation of R1 reagent

[0396] The specific PINP antibody (PINP01) was mixed with superparamagnetic microparticles in TBS buffer. After the reaction was complete, the mixture was placed on a magnetic separator until the supernatant was clear. The supernatant was removed, and the superparamagnetic microparticles coated with tPINP antibody were collected. The microparticles were then washed three times with TBS buffer. Finally, the superparamagnetic microparticles coated with PINP antibody were dissolved in R1 diluent. The final content of superparamagnetic microparticles was 0.05%, and the content of PINP antibody was 0.0015%. The composition of R1 diluent was: 50 mM Tris, 0.9% NaCl, 0.048% Proclin 300, 0.1% Tween 20, 1% BSA, and pH 7.4. The R1 reagent was then stored at 2-8°C.

[0397] Step 2: Preparation of R2 reagent

[0398] Another specific PINP antibody (hPINP02) was conjugated with alkaline phosphatase at a molar ratio of 15:1 to the antibody. The conjugate was then dissolved in R2 dilution buffer to achieve a final concentration of 1.5 μg / mL. The R2 dilution buffer consisted of 50 mM MES, 0.9% NaCl, 0.048% Proclin 300, 0.1% Tween 20, 1% BSA, 2 mM MgCl2, 0.1 mM ZnCl2, and pH 6.0. The resulting R2 reagent was then stored at 2–8 °C.

[0399] Step 3: Preparation of PINP calibrators

[0400] PINP antigen was diluted to concentrations of 0 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL, 300 ng / mL, 500 ng / mL, 1000 ng / mL, 1600 ng / mL, and 2200 ng / mL to prepare PINP calibrators.

[0401] Step 4: Measurement Procedure for the tPINP Kit

[0402] ① Load the tPINP kit (including reagents R1 and R2) into the Mindray CL series fully automated chemiluminescence immunoassay analyzer;

[0403] ② The system requires a sample volume of 5 μL for each measurement, and the total measurement time for each report is 17.5 min;

[0404] The specific sample addition process of the detection system is as follows: The fully automated chemiluminescence immunoassay analyzer adds 5 μL of sample, 50 μL of R1 reagent, and 50 μL of R2 reagent to the reaction tube, respectively. PINP in the sample binds to the anti-PINP antibody coated on the magnetic beads, while the anti-PINP antibody-alkaline phosphatase conjugate binds to another PINP site in the sample, forming a sandwich complex. After the reaction is complete, the magnetic field attracts the magnetic beads, washing away unbound substances.

[0405] The reaction substrate or excitation solution is then added to the reaction tube to generate a light signal. The number of photons generated is measured, and the number of photons generated is proportional to the concentration of PINP in the sample.

[0406] ③ Perform calibration tests using the matching PINP calibrators. Based on the calibration data, the system software uses a weighted four-parameter logarithmic curve (4PLC) mathematical method to fit the luminescence signal and concentration. The final result is given in the form of concentration in ng / mL.

[0407] Example 11: Preparation of tPINP reagent kit and calibrators, and detection of tPINP.

[0408] Step 1: Preparation of R1 reagent

[0409] The specific PINP antibody (hPINP03) was mixed with superparamagnetic microparticles in TBS buffer. After sufficient reaction, the mixture was placed on a magnetic separator until the supernatant was clear. The supernatant was removed, and the superparamagnetic microparticles coated with tPINP antibody were collected. The microparticles were then washed three times with TBS buffer. Finally, the superparamagnetic microparticles coated with PINP antibody were dissolved in R1 diluent. The final superparamagnetic microparticle content was 0.05%, and the PINP antibody content was 0.0015%. The R1 diluent consisted of 50 mM Tris, 0.9% NaCl, 0.048% Proclin 300, 0.1% Tween 20, and 1% BSA, with a pH of 7.4. The R1 reagent was then stored at 2-8°C.

[0410] Step 2: Preparation of R2 reagent

[0411] Another specific PINP antibody (hPINP02) was conjugated with alkaline phosphatase at a molar ratio of 15:1 to the antibody. The conjugate was then dissolved in R2 dilution buffer to achieve a final concentration of 1.5 μg / mL. The R2 dilution buffer consisted of 50 mM MES, 0.9% NaCl, 0.048% Proclin 300, 0.1% Tween 20, 1% BSA, 2 mM MgCl2, 0.1 mM ZnCl2, and pH 6.0. The resulting R2 reagent was then stored at 2–8 °C.

[0412] Step 3: Preparation of PINP calibrators

[0413] PINP antigen was diluted to concentrations of 0 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL, 300 ng / mL, 500 ng / mL, 1000 ng / mL, 1600 ng / mL, and 2200 ng / mL to prepare PINP calibrators.

[0414] Step 4: Measurement Procedure for the tPINP Kit

[0415] ① Load the tPINP kit (including reagents R1 and R2) into the Mindray CL series fully automated chemiluminescence immunoassay analyzer;

[0416] ② The system requires a sample volume of 5 μL for each measurement, and the total measurement time for each report is 17.5 min;

[0417] The specific sample addition process of the detection system is as follows: The fully automated chemiluminescence immunoassay analyzer adds 5 μL of sample, 50 μL of R1 reagent, and 50 μL of R2 reagent to the reaction tube, respectively. PINP in the sample binds to the anti-PINP antibody coated on the magnetic beads, while the anti-PINP antibody-alkaline phosphatase conjugate binds to another PINP site in the sample, forming a sandwich complex. After the reaction is complete, the magnetic field attracts the magnetic beads, washing away unbound substances.

[0418] The reaction substrate or excitation solution is then added to the reaction tube to generate a light signal. The number of photons generated is measured, and the number of photons generated is proportional to the concentration of PINP in the sample.

[0419] ③ Perform calibration tests using the matching PINP calibrators. Based on the calibration data, the system software uses a weighted four-parameter logarithmic curve (4PLC) mathematical method to fit the luminescence signal and concentration. The final result is given in the form of concentration in ng / mL.

[0420] Test items and results:

[0421] 1. Minimum detection limit

[0422] Using a zero-concentration sample as the sample, the test was repeated 20 times, and the RLU values ​​(relative luminescence values) of the 20 measurements were obtained. The mean (M) and standard deviation (SD) of these values ​​were calculated. The reagent was fitted with a two-point regression equation (E2) based on the concentration and RLU values ​​between the zero-concentration calibrator and adjacent calibrators according to the main calibration curve. The RLU value corresponding to M+2SD was then substituted into equation E2 to calculate the corresponding concentration value, which is the limit of detection. The current kit has a detection capability of 0.03 ng / mL.

[0423]

[0424]

[0425] 2. Detection range and linearity

[0426] A quantitative determination method based on establishing a linear range was employed, requiring the selection of 11 concentration levels within the expected measurement range. High-value samples approaching the upper limit of the linear range were diluted proportionally to several concentrations, while low-concentration samples approached the lower limit of the linear range. The measured results and theoretical results were then linearly fitted, and the correlation coefficient R0 was calculated within the linear range. 2 The results are shown Figures 2-7 .

[0427] The results showed that the current kit can detect concentrations above 2000 ng / mL, and exhibits linearity (R0.03) between 0.03 ng / mL and 2000 ng / mL. 2 Greater than 0.99.

[0428] 3. Specificity

[0429] Specificity evaluation primarily assesses interference from endogenous serum interfering substances: hemolysis (hemoglobin), hyperlipidemia, hyperbilirubinemia, total protein, and common P1NP analogues.

[0430] Interfering drugs were added to serum to prepare separate interfering serum samples. Simultaneously, control serum samples were prepared by mixing the base serum with the solvents for each interfering drug, following the same preparation method as the interfering sample samples. The interfering sample and control serum samples were then tested separately. The mean test result for the interfering sample was denoted as M, and the mean test result for the control serum sample was denoted as T. The relative deviation B of the measured concentration was calculated using the formula B = (MT) / T × 100%. The results are shown in the table below.

[0431]

[0432] The results showed that the specificity deviation of the current kit was within 5%, indicating that the prepared kit has strong anti-interference ability.

[0433] Information about the sequences involved in this application is described in the table below:

[0434]

[0435]

[0436]

[0437]

[0438]

[0439]

[0440]

[0441]

[0442] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details based on all the teachings disclosed, and all such changes are within the scope of protection of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to PINP (type I procollagen N-terminal propeptide), wherein the antibody is selected from antibody 2 or antibody 3 below: Antibody 2: The antibody 2 comprises a heavy chain containing an amino acid sequence identical to that encoded by an insert fragment of a plasmid deposited at VKPM with accession number VKPM B-14744; and a light chain containing an amino acid sequence identical to that encoded by an insert fragment of a plasmid deposited at VKPM with accession number VKPM B-14745; Antibody 3: The antibody 3 comprises a heavy chain containing an amino acid sequence identical to that encoded by an insert fragment of a plasmid deposited at VKPM with accession number VKPM B-14746; and a light chain containing an amino acid sequence identical to that encoded by an insert fragment of a plasmid deposited at VKPM with accession number VKPM B-14747.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region (CH) and a light chain constant region (CL).

3. The antibody or antigen-binding fragment thereof according to claim 2, wherein the heavy chain constant region is the mouse heavy chain constant region and the light chain constant region is the mouse light chain constant region.

4. The antibody or antigen-binding fragment thereof according to claim 2, wherein the heavy chain constant region is a human heavy chain constant region and the light chain constant region is a human light chain constant region.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, wherein the antibody or antigen-binding fragment thereof is an IgG, IgM, IgE, IgD or IgA antibody.

6. The antibody or antigen-binding fragment thereof according to any one of claims 2-4, wherein the heavy chain constant region is the IgG heavy chain constant region.

7. The antibody or antigen-binding fragment thereof according to claim 6, wherein the heavy chain constant region is the IgG1, IgG2, IgG3 or IgG4 heavy chain constant region.

8. The antibody or antigen-binding fragment thereof according to any one of claims 2-4, wherein the light chain constant region is a κ or λ light chain constant region.

9. The antibody or antigen-binding fragment thereof of claim 8, wherein the light chain constant region is the human λ light chain constant region.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, wherein it is labeled with a detectable marker.

11. The antibody or antigen-binding fragment thereof according to claim 10, wherein the label is selected from fluorescein, chemiluminescent labels, enzymes, radioisotopes, biotin, colloidal gold, and magnetic particles.

12. The antibody or antigen-binding fragment thereof of claim 11, wherein the detectable label is an acridinium ester compound, horseradish peroxidase, or alkaline phosphatase.

13. An isolated nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof as described in any one of claims 1-12.

14. An expression vector containing the isolated nucleic acid molecule of claim 13.

15. The expression vector of claim 14, wherein the expression vector is a plasmid, virus, bacteriophage, or viroid.

16. A host cell comprising the isolated nucleic acid molecule of claim 13 or the expression vector of claim 14 or 15.

17. The host cell of claim 16, wherein the host cell is a eukaryotic cell or a prokaryotic cell.

18. The host cell of claim 16, wherein the host cell is bacteria.

19. The host cell of claim 16, wherein the host cell is a mammalian cell or Escherichia coli.

20. A composition or PINP detection kit comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1-12.

21. The composition or PINP detection kit of claim 20, wherein the composition or PINP detection kit contains a first antibody and a second antibody; in, The first antibody and the second antibody target different epitopes of PINP, and: The first antibody is selected from antibody 2 of claim 1 or its antigen-binding fragment; The second antibody is selected from the following antibodies or their antigen-binding fragments: Antibody 1, wherein the antibody 1: (1) is produced by a hybridoma cell line deposited at the Russian State Industrial Microbial Collection Center (VKPM) with accession number VKPM H-214; (2) contains an amino acid sequence identical to the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence of the antibody produced by the hybridoma deposited at VKPM with accession number VKPM H-214; or (3) contains an amino acid sequence identical to the heavy chain amino acid sequence and the light chain amino acid sequence of the antibody produced by the hybridoma deposited at VKPM with accession number VKPM H-214; Antibody 3 comprises a heavy chain containing an amino acid sequence identical to that encoded by an insert fragment of a plasmid deposited at VKPM with accession number VKPM B-14746; and a light chain containing an amino acid sequence identical to that encoded by an insert fragment of a plasmid deposited at VKPM with accession number VKPM B-14747. Antibody 4 comprises a heavy chain containing an amino acid sequence identical to that encoded by an insert fragment of a plasmid deposited at VKPM with accession number VKPM B-14748; and a light chain containing an amino acid sequence identical to that encoded by an insert fragment of a plasmid deposited at VKPM with accession number VKPM B-14749. Antibody 5, comprising a heavy chain containing an amino acid sequence identical to that encoded by an insert fragment of a plasmid deposited at VKPM, accession number VKPM B-14750; and a light chain containing an amino acid sequence identical to that encoded by an insert fragment of a plasmid deposited at VKPM, accession number VKPM B-14751. Antibody 6, wherein antibody 6 comprises the same heavy chain variable region and light chain variable region amino acid sequences as the insert fragment encoded by the plasmid deposited at VKPM, accession number VKPM B-14752; or Antibody 7, wherein the antibody 7 comprises the same heavy chain variable region and light chain variable region amino acid sequences as the insert fragment encoded by the plasmid deposited in VKPM, accession number VKPM B-14753.

22. The composition of claim 21 or the PINP detection kit, wherein the second antibody is selected from antibodies 1, 3, 4, 5, 7 or their antigen-binding fragments.

23. The composition of claim 21 or the PINP detection kit, wherein the second antibody is antibody 7 or its antigen-binding fragment.

24. The composition or PINP detection kit of claim 20, wherein the composition or PINP detection kit contains a first antibody and a second antibody; in, The first antibody and the second antibody target different epitopes of PINP, and: The first antibody is selected from antibody 3 of claim 1 or its antigen-binding fragment; The second antibody is selected from the following antibodies or their antigen-binding fragments: Antibody 1, wherein the antibody 1: (1) is produced by a hybridoma cell line deposited at the Russian State Industrial Microbial Collection Center (VKPM) with accession number VKPM H-214; (2) contains an amino acid sequence identical to the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence of the antibody produced by the hybridoma deposited at VKPM with accession number VKPM H-214; or (3) contains an amino acid sequence identical to the heavy chain amino acid sequence and the light chain amino acid sequence of the antibody produced by the hybridoma deposited at VKPM with accession number VKPM H-214; Antibody 2, comprising a heavy chain containing an amino acid sequence identical to that encoded by an insert fragment of a plasmid deposited in VKPM with accession number VKPM B-14744; and a light chain containing an amino acid sequence identical to that encoded by an insert fragment of a plasmid deposited in VKPM with accession number VKPM B-14745. Antibody 4 comprises a heavy chain containing an amino acid sequence identical to that encoded by an insert fragment of a plasmid deposited at VKPM with accession number VKPM B-14748; and a light chain containing an amino acid sequence identical to that encoded by an insert fragment of a plasmid deposited at VKPM with accession number VKPM B-14749. Antibody 5, comprising a heavy chain containing an amino acid sequence identical to that encoded by an insert fragment of a plasmid deposited at VKPM, accession number VKPM B-14750; and a light chain containing an amino acid sequence identical to that encoded by an insert fragment of a plasmid deposited at VKPM, accession number VKPM B-14751. Antibody 6, wherein antibody 6 comprises the same heavy chain variable region and light chain variable region amino acid sequences as the insert fragment encoded by the plasmid deposited at VKPM, accession number VKPM B-14752; or Antibody 7, wherein the antibody 7 comprises the same heavy chain variable region and light chain variable region amino acid sequences as the insert fragment encoded by the plasmid deposited in VKPM, accession number VKPM B-14753.

25. The composition of claim 24 or the PINP detection kit, wherein the second antibody is selected from antibody 2, 5, 6 or its antigen-binding fragment.

26. The composition or PINP detection kit according to any one of claims 20-25, wherein the first antibody is a capture antibody and the second antibody is a detection antibody.

27. The composition or PINP detection kit according to any one of claims 20-25, wherein the first antibody is a detection antibody and the second antibody is a capture antibody.

28. The composition or PINP detection kit according to any one of claims 20-25, wherein the PINP detection kit is an ELISA kit.

29. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-12 in the preparation of a kit for detecting the presence or concentration of PINP in a sample.

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