Anti-PINP monoclonal antibody or antigen binding fragment thereof and application thereof

Abstract

The invention relates to the field of biological medicine, in particular to an anti-PINP monoclonal antibody or an antigen binding fragment thereof and application thereof. The anti-PINP monoclonal antibody provided by the invention comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 and / or amino acid sequences as shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6; the light chain variable region comprises amino acid sequences as shown in SEQ ID NO: 7, 8 and 9 and / or SEQ ID NO: 10, 11 and 12. The invention provides a pair of kits capable of specifically detecting PINP molecules. The kit can realize high-flux and rapid detection of PINP whole molecules on a full-automatic chemiluminescence analyzer, is simple and convenient to operate, high in sensitivity, strong in specificity, accurate in result and the like, effectively reduces the PINP detection cost, solves the existing technical difficulty, and is beneficial to clinical popularization and use.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to an anti-PINP monoclonal antibody or its antigen-binding fragment and their applications. Background Art

[0002] Osteoporosis is a common bone disease, characterized by reduced bone mass, damaged bone microstructure, and an increased susceptibility to fragility fractures. Its pathogenesis is relatively complex, which can cause osteoporotic fractures, seriously endangering the health of the elderly and increasing the social burden.

[0003] Skeletal type I collagen is the most abundant form of collagen in the human body, accounting for approximately 90% of bone mass. Type I collagen is mainly synthesized in bone tissue. First, procollagen is synthesized, and there is an extension peptide at each of the amino N-terminus and C-terminus of type I procollagen, which are respectively called type I procollagen N-terminal propeptide (PINP) and type I procollagen C-terminal propeptide (PICP); when type I procollagen is secreted by osteoblasts into the extracellular space, the extension peptides at the N-terminus and C-terminus are cleaved by proteases, type I collagen is deposited in the bone matrix, while PINP and PICP are released into the extracellular matrix and eventually enter the blood, and are finally metabolized and cleared by the liver. Since the serum half-life of PICP is less than 10 minutes, this limits its use as a biomarker, while PINP has a longer serum stability. Therefore, excluding the influence of liver diseases, detecting the content of PINP in the blood can accurately reflect the activity of osteoblasts, and thus reflect the intensity of bone metabolism in the body, guiding the diagnosis and treatment of diseases caused by abnormal bone metabolism.

[0004] PINP is composed of two α1 chains and one α2 chain, and is an acidic protein (isoelectric point 6.2) with a molecular weight of 35KD. Its constituent chains are 14250d (α1 chain) and 5500d (α2 chain), containing 139 amino acids (23 - 161) and 57 amino acids (23 - 79) respectively, and are regulated by 17q21.3 and 7q21.3 - q22 respectively. For every 1 mole of type I collagen synthesized, 1 mole of PINP is released. Therefore, PINP can reflect the deposition of type I collagen. Different from most bone metabolism markers, PINP is not affected by in vivo hormone levels, diet, or circadian rhythm. The "Expert Consensus on the Clinical Application of Biochemical Markers of Bone Metabolism (2020)" recommends that it can be used as a bone formation marker, in combination with other biochemical markers of bone metabolism, for the diagnosis and classification of osteoporosis, prediction of fracture risk, evaluation of the efficacy of anti-osteoporosis treatment, and differential diagnosis of metabolic bone diseases, and has important clinical significance in the epidemiology, pathogenesis, and research of osteoporosis drugs.

[0005] Currently, most of the commercially available PINP detection kits are imported reagents. The internationally mainstream representative manufacturers with registered document numbers in China are Roche (electrochemiluminescence method) and IDS (chemiluminescence method). The high cost of commercially available CLIA has restricted the clinical application of these diagnostic tests. SUMMARY OF THE INVENTION

[0006] In view of this, the present invention provides an anti-PINP monoclonal antibody or its antigen-binding fragment and its application. The present invention provides a pair of mouse monoclonal antibodies that can specifically recognize PINP and its preparation method. The present invention also provides a kit for detecting PINP molecules. It can achieve high-throughput and rapid detection of the whole PINP molecule on a fully automated chemiluminescence analyzer, and has the advantages of simple operation, high sensitivity, strong specificity, accurate results, etc., effectively reducing the cost of PINP detection, solving the problems of the prior art, and being conducive to clinical promotion and use.

[0007] In order to achieve the above-mentioned invention purposes, the present invention provides the following technical solutions:

[0008] The present invention provides an anti-PINP monoclonal antibody or its antigen-binding fragment, including: a heavy chain variable region and a light chain variable region, wherein:

[0009] (1), the heavy chain variable region contains HCDR1, HCDR2 and HCDR3 selected from the following group:

[0010] (a1), the amino acid sequences shown in SEQ ID NO: 1, 2 and 3;

[0011] (a2), the amino acid sequences shown in SEQ ID NO: 4, 5 and 6;

[0012] (a3), the amino acid sequences obtained by substituting, deleting or adding one or more amino acids to the amino acid sequences of (a1) or (a2), and the amino acid sequences having the same or similar functions as the amino acid sequences shown in (a1) or (a2);

[0013] (a4), a CDR having at least 85% sequence identity with the amino acid sequences shown in (a1), (a2) or (a3); and

[0014] (2), the light chain variable region contains LCDR1, LCDR2 and LCDR3 selected from the following group:

[0015] (a5), the amino acid sequences shown in SEQ ID NO: 7, 8 and 9;

[0016] (a6), the amino acid sequences shown in SEQ ID NO: 10, 11 and 12;

[0017] (a7), an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence of (a5) or (a6), and having the same or similar function as the amino acid sequence shown in (a5) or (a6);

[0018] (a8), a CDR having at least 85% sequence identity with the amino acid sequence shown in (a5), (a6), or (a7).

[0019] In some embodiments of the present invention, in the above-mentioned anti-PINP monoclonal antibody or its antigen-binding fragment, the sequence of SEQ ID NO:1 is: GYSFTSYW.

[0020] In some embodiments of the present invention, in the above-mentioned anti-PINP monoclonal antibody or its antigen-binding fragment, the sequence of SEQ ID NO:2 is: IHPSDSET.

[0021] In some embodiments of the present invention, in the above-mentioned anti-PINP monoclonal antibody or its antigen-binding fragment, the sequence of SEQ ID NO:3 is: ARTGLRYAMDY.

[0022] In some embodiments of the present invention, in the above-mentioned anti-PINP monoclonal antibody or its antigen-binding fragment, the sequence of SEQ ID NO:4 is: GFTFSSFG.

[0023] In some embodiments of the present invention, in the above-mentioned anti-PINP monoclonal antibody or its antigen-binding fragment, the sequence of SEQ ID NO:5 is: ISNGSRTI.

[0024] In some embodiments of the present invention, in the above-mentioned anti-PINP monoclonal antibody or its antigen-binding fragment, the sequence of SEQ ID NO:6 is: ARLYFDY.

[0025] In some embodiments of the present invention, in the above-mentioned anti-PINP monoclonal antibody or its antigen-binding fragment, the sequence of SEQ ID NO:7 is: SSISY.

[0026] In some embodiments of the present invention, in the above-mentioned anti-PINP monoclonal antibody or its antigen-binding fragment, the sequence of SEQ ID NO:8 is: DTS.

[0027] In some embodiments of the present invention, in the above-mentioned anti-PINP monoclonal antibody or its antigen-binding fragment, the sequence of SEQ ID NO:9 is: HQRSSYPWT.

[0028] In some embodiments of the present invention, in the above-mentioned anti-PINP monoclonal antibody or its antigen-binding fragment, the sequence of SEQ ID NO:10 is: QSLLFSNGKTY.

[0029] In some embodiments of the present invention, in the above-mentioned anti-PINP monoclonal antibody or its antigen-binding fragment, the sequence of SEQ ID NO: 11 is: LVS.

[0030] In some embodiments of the present invention, in the above-mentioned anti-PINP monoclonal antibody or its antigen-binding fragment, the sequence of SEQ ID NO: 12 is: VQGTHFPHT.

[0031] In some embodiments of the present invention, in an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged in a relative position to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of each heavy chain and light chain are referred to as "complementary determining regions" or "CDRs". The assignment of amino acids to each domain is defined according to Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Maryland (1987 and 1991)) or Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987), Chothia et al., Nature 342: 878-883 (1989).

[0032] The "antigen-binding fragment" as described in the present invention refers to a Fab fragment, a Fab' fragment, an F(ab')2 fragment having antigen-binding activity, and an Fv fragment, an scFv fragment that binds to human CD47. The Fv fragment contains the variable region of the heavy chain and the variable region of the light chain of the antibody, but no constant region, and is the smallest antibody fragment having all antigen-binding sites. Generally, the Fv antibody also contains a polypeptide linker between the VH and VL domains and is capable of forming the structure required for antigen binding. Two antibody variable regions can also be linked into a single polypeptide chain with different linkers, called a single-chain antibody or single-chain Fv (scFv).

[0033] In some embodiments of the present invention, the above-mentioned anti-PINP monoclonal antibody or its antigen-binding fragment,

[0034] (3), its heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 13 and / or SEQ ID NO: 14; and

[0035] (4), its light chain variable region has the amino acid sequence shown in SEQ ID NO: 15 and / or SEQ ID NO: 16; or

[0036] (5), an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (3) or (4), and an amino acid sequence that is functionally the same or similar to the amino acid sequence shown in (3) or (4); or

[0037] (6) An amino acid sequence having at least 80% homology with the sequence described in (3), (4), or (5).

[0038] In some embodiments of the present invention, in the above anti-PINP monoclonal antibody or its antigen-binding fragment, the sequence of SEQ ID NO: 13 is: EVQLKESGAELVRPGASVKLSCKASGYSFTSYWMNWVKQRPGQGLEWIGMIHPSDSETRLNQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYCARTGLRYAMDYWGQGTSVTVSS.

[0039] In some embodiments of the present invention, in the above anti-PINP monoclonal antibody or its antigen-binding fragment, the sequence of SEQ ID NO: 14 is: QVQLKESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEKGLEWVAYISNGSRTIYYADTVKGRFTVSRDNPKNTLFLQMTSLRSEDTAMYYCARLYFDYWGQGTTLTVSS.

[0040] In some embodiments of the present invention, in the above anti-PINP monoclonal antibody or its antigen-binding fragment, the sequence of SEQ ID NO: 15 is: ENVLTQSPAIMSASPGEKVTMTCSASSSISYMHWYRQKPGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQRSSYPWTFGGGTKLELK.

[0041] In some embodiments of the present invention, in the above anti-PINP monoclonal antibody or its antigen-binding fragment, the sequence of SEQ ID NO: 16 is: DVLMTQTPLTLSVTIGQPASISCTSSQSLLFSNGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRYTGSGSGTDFTLKISRVEAEDLGVYYCVQGTHFPHTFGGGTKLEIK.

[0042] The present invention also provides a nucleic acid molecule encoding the above anti-PINP monoclonal antibody or its antigen-binding fragment.

[0043] In some embodiments of the present invention, the above nucleic acid molecule,

[0044] (7) Its heavy chain has the nucleotide sequence shown in SEQ ID NO: 17 and / or SEQ ID NO: 18; and

[0045] (8) Its light chain has the nucleotide sequence shown in SEQ ID NO: 19 and / or SEQ ID NO: 20; or

[0046] (9) A nucleotide sequence obtained by modifying, substituting, deleting, or adding one or more bases to the nucleotide sequence described in (7) or (8); or

[0047] (10) A sequence having at least 80% homology with the nucleotide sequence described in (7), (8), or (9); or

[0048] (11) The complementary sequence of the nucleotide sequence described in (7), (8), (9), or (10).

[0049] In some embodiments of the present invention, in the above anti-PINP monoclonal antibody or its antigen-binding fragment, the sequence of SEQ ID NO: 17 is: GAGGTGCAGCTGAAGGAGTCTGGGGCTGAGCTGGTGAGGCCTGGAGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACTCCTTCACCAGCTACTGGATGAACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGCATGATTCATCCTTCCGATAGTGAAACTAGGTTAAATCAGAAGTTCAAGGACAAGGCCACATTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCCGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGAACCGGATTACGCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA.

[0050] In some embodiments of the present invention, in the above-mentioned anti-PINP monoclonal antibody or its antigen-binding fragment, the sequence of SEQ ID NO: 18 is: CAGGTGCAGCTGAAGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGGTCCCGGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTTTGGAATGCACTGGGTTCGTCAGGCTCCAGAGAAGGGGCTGGAGTGGGTCGCATACATTAGTAATGGCAGTCGTACCATCTACTATGCAGACACAGTGAAGGGCCGATTCACCGTCTCCAGAGACAATCCCAAGAACACCCTGTTCCTGCAAATGACCAGTCTGAGGTCTGAGGACACGGCCATGTATTACTGTGCAAGACTGTATTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA.

[0051] In some embodiments of the present invention, in the above-mentioned anti-PINP monoclonal antibody or its antigen-binding fragment, the sequence of SEQ ID NO: 19 is: GAAAATGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAAGTATAAGTTACATGCACTGGTACCGGCAGAAGCCAGGCACCTCCCCCAAAAGATGGATTTATGACACATCCAAACTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTATTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCATCAGCGGAGTAGTTACCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAGCTGAAA.

[0052] In some embodiments of the present invention, in the above-mentioned anti-PINP monoclonal antibody or its antigen-binding fragment, the sequence of SEQ ID NO: 20 is: GACGTTTTGATGACCCAAACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCTATCTCTTGCACGTCAAGTCAGAGCCTCTTATTTAGTAATGGAAAAACCTATTTGAATTGGTTATTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTACACTGGCAGTGGATCAGGAACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTACTGCGTGCAAGGTACACATTTTCCTCACACGTTCGGAGGGGGGACCAAACTGGAAATAAAA。

[0053] In some embodiments of the present invention, the above-mentioned "at least 80% sequence identity" means at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity. The sequence comparison and determination of the percentage of identity between two sequences can be carried out by the BLASTN / BLASTP algorithm on the website of the National Center For Biotechnology Instutute.

[0054] The present invention also provides an expression vector, including the above-mentioned nucleic acid molecule.

[0055] The present invention also provides a host, which is transformed and / or transfected with the above-mentioned expression vector.

[0056] The present invention also provides the application of the above-mentioned anti-PINP monoclonal antibody or its antigen-binding fragment, the above-mentioned nucleic acid molecule, the above-mentioned expression vector and / or the above-mentioned host in any of the following items;

[0057] ( )、Preparing a product for detecting PINP antigen; and / or

[0058] ( )、Preparing a product for detecting the content of PINP antigen; and / or

[0059] ( )、Preparing a product for detecting osteoporosis.

[0060] The present invention also provides a drug, comprising the above-mentioned anti-PINP monoclonal antibody or its antigen-binding fragment, the above-mentioned nucleic acid molecule, the above-mentioned expression vector and / or the above-mentioned host, and a pharmaceutically acceptable excipient.

[0061] The present invention also provides a detection reagent, comprising the above-mentioned anti-PINP monoclonal antibody or its antigen-binding fragment, the above-mentioned nucleic acid molecule, the above-mentioned expression vector and / or the above-mentioned host, and an acceptable adjuvant.

[0062] The present invention also provides a kit, comprising the above-mentioned anti-PINP monoclonal antibody or its antigen-binding fragment, the above-mentioned nucleic acid molecule, the above-mentioned expression vector, the above-mentioned host and / or the above-mentioned detection reagent, and an acceptable adjuvant.

[0063] The present invention uses PINP recombinant protein as an immunogen to prepare a group of murine monoclonal antibodies specific to the whole molecule of PINP. A double-antibody sandwich ELISA method for detecting the whole molecule of PINP is established using the murine monoclonal antibodies prepared by the present invention, and its analytical performance in the laboratory is evaluated. The results show that it has strong specificity, high affinity, and good correlation with Roche PINP reagent, and can effectively meet the requirements of clinical detection reagents. It can provide reliable clinical reference value for the early diagnosis, early intervention, and prognosis of osteoporosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art.

[0065] Figure 1 Show the SDS-PAGE diagrams of the 16H9 and 11B5 antibodies of the present invention;

[0066] Figure 2 Show the correlation between the PINP antibody pairs of the present invention and Roche. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0067] The present invention discloses an anti-PINP monoclonal antibody or its antigen-binding fragment and its application.

[0068] It should be understood that the expression "one or more of..." individually includes each of the objects recited after the expression and various different combinations of two or more of the recited objects, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited objects should be understood to have the same meaning, unless otherwise understood from the context.

[0069] The terms "comprising", "having" or "including", including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unrecited elements or steps, unless specifically stated otherwise or understood from the context.

[0070] It should be understood that as long as the present invention is still operable, the order of steps or the order of performing certain actions is not important. In addition, two or more steps or actions can be carried out simultaneously.

[0071] The use of any and all examples or exemplary language in this document, such as "for example" or "including", is merely intended to better illustrate the present invention and does not limit the scope of the present invention unless a claim is made. No language in this specification should be construed as indicating that any unclaimed element is essential for the practice of the present invention.

[0072] In addition, the numerical ranges and parameters used to define the present invention are approximate values. The relevant values in the specific embodiments have been presented as precisely as possible herein. However, any numerical value inherently and inevitably contains standard deviations due to individual testing methods. Therefore, unless otherwise clearly stated, it should be understood that all ranges, amounts, numerical values and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0073] The present invention provides an anti-PINP monoclonal antibody, named 16H9 and 11B5 respectively, wherein 16H9 has a heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 13 and a light chain variable region with the amino acid sequence shown in SEQ ID NO: 15; 11B5 has a heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 14 and a light chain variable region with the amino acid sequence shown in SEQ ID NO: 16.

[0074] Specifically, the amino acid sequence of SEQ ID NO: 13 is as follows:

[0075] EVQLKESGAELVRPGASVKLSCKASGYSFTSYWMNWVKQRPGQGLEWIGMIHPSDSETRLNQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYCARTGLRYAMDYWGQGTSVTVSS. (CDR: GYSFTSYW....___IHPSDSET..___ARTGLRYAMDY)

[0076] The amino acid sequence of SEQ ID NO: 14 is as follows:

[0077] QVQLKESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEKGLEWVAYISNGSRTIYYADTVKGRFTVSRDNPKNTLFLQMTSLRSEDTAMYYCARLYFDYWGQGTTLTVSS. (CDR: GFTFSSFG....___ISNGSRTI..___ARLYFDY)

[0078] The amino acid sequence of SEQ ID NO:15 is as follows:

[0079] ENVLTQSPAIMSASPGEKVTMTCSASSSISYMHWYRQKPGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQRSSYPWTFGGGTKLELK. (CDR: SSISY.......___DTS.......___HQRSSYPWT)

[0080] The amino acid sequence of SEQ ID NO:16 is as follows:

[0081] DVLMTQTPLTLSVTIGQPASISCTSSQSLLFSNGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRYTGSGSGTDFTLKISRVEAEDLGVYYCVQGTHFPHTFGGGTKLEIK. (CDR: QSLLFSNGKTY.___LVS.......___VQGTHFPHT)

[0082] Furthermore, the monoclonal antibody also includes a heavy chain constant region and a light chain constant region.

[0083] Even further, the heavy chain constant region and the light chain constant region are from a mouse.

[0084] The anti-PINP monoclonal antibody of the present invention is prepared by first immunizing Balb / c mice with the prokaryotically expressed α1 chain recombinant antigen as an immunogen, then fusing the spleen with myeloma cells NS1, and obtaining the PINP antibody through clone screening, ascites preparation, and antibody purification. The prepared anti-PINP monoclonal antibody has good reactivity with both recombinant and native PINP antigens.

[0085] Using the anti-PINP monoclonal antibody prepared by the present invention, a double-antibody sandwich ELISA detection kit was established. The mouse monoclonal antibody 16H9 against PINP was coated on magnetic beads as the capture antibody, and the mouse monoclonal antibody 11B5 against PINP was labeled with HRP as the detection antibody. The PINP protein in serum was detected by the double-antibody sandwich method, which has extremely high sensitivity and specificity and can be applied to clinical detection.

[0086] In Examples 1 to 3 of the present invention, the raw materials and reagents used can be purchased from the market.

[0087] The present invention will be further described below in conjunction with examples:

[0088] Example 1 Preparation of PINP recombinant antigen

[0089] Select the N-terminal gene segment of type I collagen α1 chain, with the sequence SEQ ID No: 27 (atgttcagctttgtggacctccggctcctgctcctcttagcggccaccgccctcctgacgcacggccaagaggaaggccaagtcgagggccaagacgaagacatcccaccaatcacctgcgtacagaacggcctcaggtaccatgaccgagacgtgtggaaacccgagccctgccggatctgcgtctgcgacaacggcaaggtgttgtgcgatgacgtgatctgtgacgagaccaagaactgccccggcgccgaagtccccgagggcgagtgctgtcccgtctgccccgacggctcagagtcacccaccgaccaagaaaccaccggcgtcgaaggacccaagggagacactggcccccgaggcccaaggggacccgcaggcccccctggccgagatggcatccctggacagcctggacttcccggaccccccggaccccccggacctcccggaccccctggcctcggaggaaactttgctccc), and design primers to amplify the target fragment. The upstream primer of the fragment has a BamHI site, and the downstream primer has a HindIII restriction site. The PCR fragment is recovered and digested with the corresponding restriction enzyme, and the digested fragment is ligated into the expression vector pET-32a(+) digested with BamHI and HindIII to obtain the recombinant plasmid pET-32a(+)-PINP. The above recombinant plasmid is transferred into the Escherichia coli expression strain BL21(DE3)pLysS, and cultured with shaking in LB medium containing ampicillin at 37 °C until OD 600≈1.0. Induce overnight with IPTG. Centrifuge at 7000 rpm for 3 minutes at 4°C to collect the bacterial cells. Resuspend the bacterial cells from each liter of the bacterial solution in 20 mL of lysis buffer (50 mM Tris-HCl, pH 8.0, 500 mM NaCl), sonicate, centrifuge at 12000 g for 20 minutes at 4°C, and collect the supernatant to pass through an NI column (Buffer A: 50 mM Tris-HCl, 500 mM NaCl, pH 8.0; Buffer B: 50 mM Tris-HCl, 500 mM NaCl, 200 mM imidazole, pH 8.0). After equilibrating the Ni-NTA affinity column with 10 column volumes of Buffer A, add the protein sample, wash away the unbound proteins with 10 medium volumes of Buffer A, then wash away the contaminating proteins with 25% Buffer B, and elute the target protein with 100% Buffer B. This protein is named the PINP recombinant antigen and stored at -20°C for later use. Transform and express the PET-32a(+) plasmid by the same method, induce expression with IPTG and purify with an NI column to obtain the PET32a-tagged protein, and store it at -20°C for later use.

[0090] Example 2 Preparation of Mouse Monoclonal Antibody Against PINP

[0091] 2.1 Immunization of Mice

[0092] After fully emulsifying the PINP recombinant antigen with Freund's complete adjuvant, immunize female Balb / c mice at 5 weeks of age intraperitoneally. The initial immunization dose is 100 μg / mouse; the second and third immunizations are carried out 21 days and 42 days respectively after the first immunization, and the immunization doses are both 50 μg / mouse. Collect blood from the tail about 10 days after the third immunization, and detect the serum titer by the indirect method using a 96-well plate coated with the PINP recombinant antigen.

[0093] 2.2 Preparation of Hybridoma Cells

[0094] Select mice with an indirect method-detected serum titer greater than 10 4 for intrasplenic booster immunization, and the immunization dose is 100 μg / mouse. Take the spleens of the mice 3 days after the booster immunization and fuse them with mouse myeloma cells NS1 at a ratio of 10:1 using PEG. The fused cells are cultured in DMEM medium (Gibco) containing HAT.

[0095] About 6 - 7 days after fusion, detect the content of specific antibodies in the cell culture supernatant by the indirect method using a 96-well plate coated with the PINP recombinant antigen. Select positive wells with an OD value not lower than 0.5 and perform 3 rounds of subcloning by the limiting dilution method. Finally, obtain 5 hybridoma cell lines that can stably secrete anti-PINP, named 8A5, 13C1, 19F3, 16H9, and 11B5 respectively.

[0096] 2.3 Purification of Mouse Monoclonal Antibody Against PINP

[0097] The obtained mouse hybridoma cells that can stably secrete anti-PINP were injected into the abdominal cavity of mice, and the ascites were collected and purified by SPA, and then the mouse monoclonal antibody against PINP with a purity exceeding 90% was obtained.

[0098] 2.4 Specificity Detection of Anti-PINP Monoclonal Antibody

[0099] The PINP recombinant antigen obtained in Example 1 was diluted to 0.5 μg / mL with 0.05 mM CB buffer at pH = 9.6, added to a 96-well ELISA plate, 50 μg / well, and coated overnight at 4°C. The next day, it was washed 3 times with PBST, and then blocked with 1% Casein at 100 μL / well for 2 hours at 37°C. The PET32a tag protein was coated in the same way. The purified monoclonal antibodies 16H9 and 11B5 (both at a concentration of 5 mg / mL) were diluted to 1 μg / mL with PBS buffer. The diluted antibodies were respectively added to the ELISA plate coated with PINP recombinant antigen / PET32a tag protein, and PBS buffer was added to the negative control wells, 50 μL / well. After reacting at 37°C for 30 minutes, the plate was washed 5 times with PBST, patted dry, and then 1 / 4k diluted HRP-goat anti-mouse IgG (SIGMA) was added, 100 μL / well, and reacted at 37°C for 30 minutes. The plate was washed 5 times with PBST, patted dry, and then ordinary ELISA substrate + chromogenic solution was added, 100 μL / well, and reacted in the dark at room temperature for 10 minutes. Then 50 μL of sulfuric acid at a concentration of 0.1 mol / L was added to terminate the reaction, and the absorbance at 450 nm was measured. The detection results are shown in Table 1 below.

[0100] Table 1 Determination of PINP Antibody Reactivity

[0101]

[0102] As can be seen from Table 1, the reactivity of the 5 monoclonal antibodies with the PINP recombinant antigen is good, and they are all PINP-specific antibodies.

[0103] 2.5 Affinity Determination

[0104] The PINP recombinant antigen was coated on a luminescence plate, and the 5 antibodies were diluted with PBS at different concentration gradients respectively, and the luminescence values were detected by the indirect method.

[0105] Table 2 Determination of PINP Antibody Affinity

[0106]

[0107] Among the 5 antibodies, the EC50 values of 4 antibodies except 8A5 are relatively low, and the affinities are relatively high, and 16H9 and 11B5 are the highest.

[0108] 2.6 Monoclonal Antibody Sequencing

[0109] The following primers were synthesized based on the constant region sequence of the antibody gene:

[0110] LF1 5′-GAAAATGTTCTCACCCAGTCTCCA-3′; (as shown in SEQ ID NO: 21)

[0111] LF2 5′-GACGTTTTGATGACCCAAACTCCA-3′; (as shown in SEQ ID NO: 22)

[0112] LR 5′-TGGACACTGTTGGGGCCGCATCGGCCCT-3′; (as shown in SEQ ID NO: 23)

[0113] HF1 5′-GAGGTGCAGCTGAAGGAGTCTGGG-3′; (as shown in SEQ ID NO: 24)

[0114] HF2 5′-CAGGTGCAGCTGAAGGAGTCTGGG-3′; (as shown in SEQ ID NO: 25)

[0115] HR 5′-GATAGACAGATGGGGGTGTCGTTTTGGC-3′ (as shown in SEQ ID NO: 26)

[0116] 3 × 10 6 Total RNA of hybridoma cells 16H9 and 11B5 was used to reverse transcribe mRNA into cDNA. HF1, HF2 and HR were used as primers for PCR amplification of the heavy chain variable region of the two monoclonal antibodies of 16H9 and 11B5, and LF1, LF2 and LR were used as primers for PCR amplification of the light chain variable region of the two monoclonal antibodies of 16H9 and 11B5. The PCR reactions were all hot-started, and the reaction conditions were: 95℃ for 5 minutes; 95℃ for 15 seconds, 55℃ for 45 seconds, 72℃ for 30 seconds, 30 cycles; 72℃ for 7 minutes. The PCR products were separated by 1% agarose gel electrophoresis and the target fragments were recovered and purified. They were cloned into PM18-T vector, transformed into Escherichia coli DH5α cells, and screened on LB solid plates. White plaques were inoculated in LB liquid medium containing ampicillin for amplification. Positive clones were screened, and plasmids were extracted using QIAGEN's plasmid extraction kit and sequenced to determine the heavy chain and light chain variable region sequences of monoclonal antibodies 16H9 and 11B5.

[0117] The heavy chain variable region of monoclonal antibody 16H9 has the amino acid sequence shown in SEQ ID NO:13, and the light chain variable region has the amino acid sequence shown in SEQ ID NO:15; the heavy chain variable region of monoclonal antibody 11B5 has the amino acid sequence shown in SEQ ID NO:14, and the light chain variable region has the amino acid sequence shown in SEQ ID NO:16.

[0118] Example 3 Establishment of ELISA method for detecting PINP by double antibody sandwich method

[0119] 3.1 PINP monoclonal antibody coated plate

[0120] The PINP antibody obtained in Example 2 was diluted to 2 μg / mL with 0.05 mM CB buffer at pH = 9.6, added to the costar luminescent plate, 100 μL / well, and coated overnight at 4°C. The next day, the plate was washed three times with PBST, and then blocked with 1% Casein, 100 μL / well, at 37°C for 2 hours.

[0121] 3.2 Preparation of PINP enzyme conjugate

[0122] The anti-PINP mouse monoclonal antibody and HRP (Roche) were labeled by sodium periodate method at a mass ratio of 1.5:1. The HRP-labeled anti-PINP mouse monoclonal antibody was diluted with diluent 1 / 4k to prepare the enzyme conjugate.

[0123] 3.3 Optimal pairing

[0124] The PINP recombinant antigen prepared in Example 1 was diluted with PBS to 1000ng / mL, 500ng / mL, 250ng / mL, and 0, and added to the antibody plate prepared in 3.1, 50μL / well, and reacted at 37°C for 30min. The plate was washed 5 times with PBST, and the PINP enzyme conjugate prepared in 3.2 was added after patting dry, 100μL / well, and reacted at 37°C for 30 minutes. The plate was washed 5 times with PBST, and the luminescent substrate was added after patting dry, 100μL / well, and reacted at room temperature in the dark for 5 minutes, and the luminescence value was measured by the luminometer. The test results are shown in Table 3 below. The sensitivity, gradient, and background of 16H9 coating + 11B5 labeling are the best, and this pairing is preferred.

[0125] Table 3 PINP antibody cross-matching

[0126]

[0127] 3.4 Clinical sample testing

[0128] Eight patient samples with constant PINP values ​​were tested using the double antibody sandwich method of the present invention. The results are shown in Table 4, and the correlation with the test results of the Roche kit is shown in Table 4. Figure 2As shown, the overall correlation R between the kit of the present invention and the Roche kit 2 > 0.98.

[0129] Table 4 Detection results of antibody pairs in clinical samples of the present invention

[0130]

[0131] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. An anti-PINP monoclonal antibody or an antigen-binding fragment thereof, characterized in that: include: A heavy chain variable region and a light chain variable region, wherein: (1) The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 selected from the following group: (a1), the amino acid sequences shown in SEQ ID NOs: 1, 2 and 3; (a2), the amino acid sequences shown in SEQ ID NOs: 4, 5 and 6; (a3), an amino acid sequence obtained by substitution, deletion or addition of one or more amino acids in the amino acid sequence described in (a1) or (a2), and an amino acid sequence having the same or similar function as the amino acid sequence described in (a1) or (a2); (a4), a CDR having at least 85% sequence identity with the amino acid sequence shown in (a1), (a2) or (a3); and (2) The light chain variable region comprises LCDR1, LCDR2 and LCDR3 selected from the following group: (a5), the amino acid sequences shown in SEQ ID NOs: 7, 8 and 9; (a6), the amino acid sequences shown in SEQ ID NOs: 10, 11 and 12; An amino acid sequence obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence of (a7), (a5) or (a6), and having the same or similar function as the amino acid sequence shown in (a5) or (a6); (a8), a CDR having at least 85% sequence identity with the amino acid sequence shown in (a5), (a6) or (a7).

2. The anti-PINP monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that: (3) Its heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 13 and / or SEQ ID NO: 14; and (4) whose light chain variable region has the amino acid sequence shown in SEQ ID NO: 15 and / or SEQ ID NO: 16; or (5), an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (3) or (4), and having the same or similar function as the amino acid sequence described in (3) or (4); or (6) An amino acid sequence that is at least 80% homologous to the sequence described in (3), (4) or (5).

3. A nucleic acid molecule encoding the anti-PINP monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2.

4. The nucleic acid molecule according to claim 3, characterized in that (7) Its heavy chain has the nucleotide sequence shown in SEQ ID NO: 17 and / or SEQ ID NO: 18; and (8) whose light chain has the nucleotide sequence shown in SEQ ID NO: 19 and / or SEQ ID NO: 20; or A nucleotide sequence obtained by modifying, replacing, deleting or adding one or more bases of the nucleotide sequence described in (9), (7) or (8); or (10) a sequence having at least 80% homology to the nucleotide sequence described in (7), (8) or (9); or (11) A complementary sequence of the nucleotide sequence described in (7), (8), (9) or (10).

5. An expression vector, characterized in that Comprising the nucleic acid molecule according to claim 3 or 4.

6. A host, characterized in that Transformation and / or transfection is carried out using the expression vector according to claim 5.

7. Use of the anti-PINP monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, the nucleic acid molecule according to claim 3 or 4, the expression vector according to claim 5 and / or the host according to claim 6 in any of the following items; ( ), preparing products for detecting PINP antigen; and / or ( ), preparing products for detecting PINP antigen content; and / or ( ) and preparing products for detecting osteoporosis.

8. A drug, characterized in that The invention comprises the anti-PINP monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, the nucleic acid molecule according to claim 3 or 4, the expression vector according to claim 5 and / or the host according to claim 6, and pharmaceutically acceptable excipients.

9. A detection reagent, characterized in that: The method comprises the anti-PINP monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, the nucleic acid molecule according to claim 3 or 4, the expression vector according to claim 5 and / or the host according to claim 6 and an acceptable auxiliary agent.

10. A kit, characterized in that The method comprises the anti-PINP monoclonal antibody or antigen-binding fragment thereof as claimed in claim 1 or 2, the nucleic acid molecule as claimed in claim 3 or 4, the expression vector as claimed in claim 5, the host as claimed in claim 6 and / or the detection reagent as claimed in claim 9 and an acceptable auxiliary agent.

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