Completely carboxylated osteocalcin specific monoclonal antibody and application thereof

Abstract

The invention discloses a complete carboxylation osteocalcin specific monoclonal antibody and an application thereof. In the monoclonal antibody 3A421, the amino acid sequence of a heavy chain variable region is as shown in SEQ ID NO.1, and the amino acid sequence of a light chain variable region is as shown in SEQ ID NO.5. The 3A421 monoclonal antibody is a monoclonal antibody which only specifically recognizes completely carboxylated osteocalcin, does not recognize non-carboxylated osteocalcin or incompletely carboxylated osteocalcin, does not generate cross reaction with the non-carboxylated osteocalcin or incompletely carboxylated osteocalcin, and can be used for accurately quantifying the completely carboxylated osteocalcin.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a monoclonal antibody specifically recognizing fully carboxylated osteocalcin and applications thereof. Background Art

[0002] Osteocalcin (OC), also known as γ-carboxyglutamic acid protein (BGP), is a calcium-binding protein primarily synthesized by osteoblasts, odontoblasts, and proliferating chondrocytes. Osteocalcin is the most abundant non-collagenous protein in bone tissue, accounting for 10-20% of the total non-collagenous protein. Its primary physiological function is to maintain normal bone mineralization, inhibit the formation of abnormal hydroxyapatite crystals, and suppress cartilage mineralization. Changes in its circulating levels are closely correlated with osteoblast function and bone formation, making it a specific marker of bone metabolism.

[0003] The mature osteocalcin molecule consists of 49 amino acids with a molecular mass of approximately 5210 to 5889 Da. This is referred to as full-length osteocalcin. It is unstable in peripheral blood, with a half-life of only 4-5 minutes. The carboxyl-terminal 5-6 amino acids are easily hydrolyzed by proteases, forming a 43-amino acid (1-43aa) or 44-amino acid (1-44aa) osteocalcin macromolecule, referred to as the N-terminal plus middle segment (N-MID). N-MID exhibits excellent stability. Furthermore, glutamic acid residues (Glu) at positions 17, 21, and 24 in the osteocalcin amino acid sequence undergo carboxylation in the presence of vitamin K to form gamma-carboxyglutamate (Gla). Only fully carboxylated osteocalcin (Gla-OC), in which all three glutamic acid residues are carboxylated, has the ability to bind to hydroxyapatite (bone mineral) and promote bone formation. Therefore, fully carboxylated osteocalcin is a true marker of bone formation. Glu21 ​​and Glu24 of osteocalcin are almost entirely carboxylated, while Glu17 is typically uncarboxylated. Incompletely carboxylated osteocalcin (abbreviated as Glu / a-OC) does not participate in bone mineralization and is an indicator of poor bone quality rather than bone formation. Up to 20-30% of osteocalcin in adult blood is in the undercarboxylated form, and its concentration increases significantly with age. In elderly women who are able to move independently, incompletely carboxylated osteocalcin is an independent predictor of hip fracture. Furthermore, studies have shown that incompletely carboxylated osteocalcin acts as a hormone, participating in processes related to glucose metabolism, lipid metabolism, fertility, and aging.

[0004] In summary, circulating osteocalcin molecules are highly heterogeneous, consisting of fragments of varying lengths and multiple forms with varying degrees of carboxylation. Previous studies have shown that in both normal individuals and osteoporotic patients, full-length osteocalcin and osteocalcin N-MID are the most abundant forms of osteocalcin, accounting for approximately two-thirds of the total molecule. The remaining third consists of various smaller fragments that are rapidly cleared by the kidneys. Full-length osteocalcin and osteocalcin N-MID exist in fully carboxylated, incompletely carboxylated, and uncarboxylated forms, respectively, with only fully carboxylated osteocalcin being the true marker of bone formation. However, to date, the precise concentrations of fully and incompletely carboxylated osteocalcin in the circulation have not been clearly reported in the literature. This is primarily due to the lack of monoclonal antibodies that specifically bind to fully carboxylated osteocalcin. Therefore, the purpose of the present invention is to prepare and screen monoclonal antibodies that can specifically recognize only fully carboxylated osteocalcin, but not uncarboxylated osteocalcin or incompletely carboxylated osteocalcin, so as to achieve accurate, simple and rapid quantification of fully carboxylated osteocalcin and promote the widespread clinical application of this detection indicator. Summary of the Invention

[0005] To this end, the object of the present invention is to provide a monoclonal antibody prepared using a fused hybridoma cell line that specifically recognizes only fully carboxylated osteocalcin. The monoclonal antibody obtained experimentally does not recognize non-carboxylated osteocalcin or incompletely carboxylated osteocalcin, does not cross-react with non-carboxylated osteocalcin or incompletely carboxylated osteocalcin, and can be used for the precise quantification of fully carboxylated osteocalcin.

[0006] Therefore, one aspect of the present invention relates to a monoclonal antibody or an antigen-binding fragment thereof that specifically recognizes only fully carboxylated osteocalcin, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, and the light chain variable region comprises a light chain CDR1, a light chain CDR2, and a light chain CDR3, wherein:

[0007] The amino acid sequence of the heavy chain CDR1 is the sequence shown in SEQ ID NO. 2 or an amino acid sequence having one conservative amino acid substitution compared with the sequence shown in SEQ ID NO. 2;

[0008] The amino acid sequence of the heavy chain CDR2 is the sequence shown in SEQ ID NO. 3 or an amino acid sequence having one conservative amino acid substitution compared to the sequence shown in SEQ ID NO. 3;

[0009] The amino acid sequence of the heavy chain CDR3 is the sequence shown in SEQ ID NO. 4 or an amino acid sequence having one conservative amino acid substitution compared with the sequence shown in SEQ ID NO. 4;

[0010] The amino acid sequence of the light chain CDR1 is the sequence shown in SEQ ID NO.6 or an amino acid sequence having one conservative amino acid substitution compared with the sequence shown in SEQ ID NO.6;

[0011] The amino acid sequence of the light chain CDR2 is LVS or an amino acid sequence having one conservative amino acid substitution compared thereto;

[0012] The amino acid sequence of the light chain CDR3 is the sequence shown in SEQ ID NO.7 or an amino acid sequence having one conservative amino acid substitution compared with the sequence shown in SEQ ID NO.7.

[0013] In a further aspect, the present invention also relates to a monoclonal antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO.5.

[0014] The present invention also relates to the above-mentioned monoclonal antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a single-chain antibody or a humanized antibody. These antibodies or antigen-binding fragments can specifically recognize fully carboxylated osteocalcin because they retain the variable regions of the light chain and the heavy chain, or only retain the variable region of the heavy chain.

[0015] In addition, the present invention also relates to a nucleic acid molecule comprising a nucleic acid encoding the above-mentioned antibody or its antigen-binding fragment, and an expression vector comprising the above-mentioned nucleic acid molecule, wherein the expression vector is capable of expressing the above-mentioned antibody or its antigen-binding fragment. At the same time, the present invention also relates to a recombinant comprising the above-mentioned nucleic acid molecule or the above-mentioned expression vector, which can produce the above-mentioned antibody or its antigen-binding fragment. On the other hand, the present invention relates to a monoclonal antibody hybridoma cell line that specifically recognizes fully carboxylated osteocalcin, and the monoclonal antibody hybridoma cell line secretes the above-mentioned monoclonal antibody. Further, the present invention relates to a monoclonal antibody hybridoma cell line that specifically recognizes only fully carboxylated osteocalcin, and the monoclonal antibody hybridoma cell line is a mouse hybridoma cell line 3A421, with a deposit number of CGMCC No.46547.

[0016] In another aspect, the present invention relates to the use of the aforementioned monoclonal antibody or antigen-binding fragment thereof in the preparation of a product for detecting fully carboxylated osteocalcin. Furthermore, the present invention relates to a kit for detecting fully carboxylated osteocalcin, comprising the aforementioned monoclonal antibody or antigen-binding fragment thereof for specifically recognizing and binding to fully carboxylated osteocalcin; preferably, the kit is a double-antibody sandwich detection kit, in which the monoclonal antibody or antigen-binding fragment thereof serves as the capture antibody.

[0017] Description of biological material deposit

[0018] The monoclonal antibody hybridoma cell line of the present invention, mouse hybridoma cell line 3A421, has been deposited with the China General Microbiology Center (CGMCC) under the registration number CGMCC No. 46547, with a deposit date of June 18, 2025. The address of the China General Microbiology Center is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is an SDS-PAGE electrophoresis diagram showing prokaryotic expression of recombinant osteocalcin, where M is a molecular weight standard.

[0020] Figure 2 This is a diagram showing the specificity identification results of the anti-fully carboxylated osteocalcin monoclonal antibody 3A421.

[0021] Figure 3 This is a graph showing the titer test results of rabbit anti-osteocalcin 25-43 segment polypeptide polyclonal antibody.

[0022] Figure 4 This is the result of the double antibody sandwich method for fully carboxylated osteocalcin detection.

[0023] Figure 5 This is the result of clinical sample testing using the double-antibody sandwich method for fully carboxylated osteocalcin.

[0024] Figure 6 This is a diagram showing the subtype identification results of the anti-fully carboxylated osteocalcin monoclonal antibody 3A421. DETAILED DESCRIPTION

[0025] The present invention aims to provide a monoclonal antibody specifically recognizing fully carboxylated osteocalcin (Gla-OC), produced using a fused hybridoma cell line. The specific preparation process involves first synthesizing a 10-25aa segment of the fully carboxylated osteocalcin polypeptide (designated Gla-OC10-25). This segment is then used as an immunogen to immunize mice to produce a monoclonal antibody. Screening was then performed using fully carboxylated osteocalcin, incompletely carboxylated osteocalcin, uncarboxylated osteocalcin, prokaryotically expressed recombinant osteocalcin, and extracted bovine osteocalcin to obtain a highly specific monoclonal antibody that specifically recognizes fully carboxylated osteocalcin. This monoclonal antibody, when used in conjunction with a polyclonal antibody against the 25-43aa segment of osteocalcin, can detect both fully carboxylated full-length osteocalcin and the fully carboxylated osteocalcin N-MID. The mouse hybridoma cell line secreting this monoclonal antibody, named 3A421, specifically recognizes fully carboxylated osteocalcin and does not cross-react with uncarboxylated and incompletely carboxylated osteocalcin, demonstrating very high specificity. The inventors deposited this cell line with the General Microbiology Center of the China Culture Collection Administration on June 18, 2025, with the deposit number CGMCC No. 46547.

[0026] Subsequently, the inventors sequenced the monoclonal antibody secreted by the mouse hybridoma cell line CGMCC No.46547 and analyzed the immunoglobulin domain sequence, and found that its heavy chain variable region amino acid sequence was: DVQLQESGPGLVKPSQTVSLTCTVTGISITTGNYRWSGIRQFPGNKLEWIGYIYYSGTITCNPSLTSRTTITRDTSKNQFFLEMNSLTTEDTATYCARDRSGWTTGVKEPQSPSP (SEQ ID NO.1), wherein the heavy chain CDR1 amino acid sequence was GISITTGNYR (SEQ ID NO.2), the heavy chain CDR2 amino acid sequence was IYYSGTI (SEQ ID NO.3), and the heavy chain CDR3 amino acid sequence was ARDRSGWTT (SEQ ID NO.4). The amino acid sequence of the light chain variable region is: DIVMTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCRHIRELTRSEGHQAGNQ (SEQ ID NO.5), wherein the light chain CDR1 amino acid sequence is KSVSTSGYSY (SEQ ID NO.6), the light chain CDR2 amino acid sequence is LVS, and the light chain CDR3 amino acid sequence is RHIRELTR (SEQ ID NO.7).

[0027] The inventors evaluated the specificity of the monoclonal antibody for fully carboxylated osteocalcin using an enzyme-linked immunosorbent assay (ELISA). They found that the monoclonal antibody specifically recognized only fully carboxylated osteocalcin, but not uncarboxylated and incompletely carboxylated osteocalcin, demonstrating high specificity. ELISA testing of clinical samples demonstrated that the monoclonal antibody of the present invention can be used to prepare products for the detection of fully carboxylated osteocalcin, assisting in the clinical diagnosis and medication management monitoring of osteoporosis.

[0028] It is well known in the art that the heavy chain CDR and light chain CDR regions of antibodies are important amino acid sequence regions for recognizing and binding to corresponding antigens, and that a single conservative amino acid substitution in the amino acid sequence of these CDR regions may not alter the protein structure. Therefore, a single conservative amino acid substitution in these regions may still confer binding properties to the corresponding antigen. Therefore, a monoclonal antibody or antigen-binding fragment thereof obtained by making a single conservative amino acid substitution in the heavy chain CDR1 and / or heavy chain CDR2 and / or heavy chain CDR3 and / or light chain CDR1 and / or light chain CDR2 and / or light chain CDR3 can still specifically recognize fully carboxylated osteocalcin. In the patent application of the present invention, conservative amino acid substitution refers to the replacement of a certain amino acid in a protein by another chemically similar amino acid, such as the mutual replacement between aromatic amino acids Phe, Trp, and Tyr, the mutual replacement between aliphatic amino acids Ala, Gly, Leu, Ile, and Val, the mutual replacement between polar amino acids Gln and Asn, the mutual replacement between basic amino acids Lys, Arg, and His, the mutual replacement between acidic amino acids Asp and Glu, and the mutual replacement between hydroxy amino acids Ser and Thr, etc.

[0029] Those skilled in the art can also use existing techniques to prepare various antibody fragments, i.e., antigen-binding fragments, from the monoclonal antibodies of the present invention that specifically recognize fully carboxylated osteocalcin, such as, but not limited to, Fab, Fab', and F(ab')2. The Fab fragment is the region of the antibody structure capable of binding to an antigen. It consists of a complete light chain, the variable region VH, and the constant region CH1 domain (Fd segment) of the heavy chain. Both the light and heavy chains have a constant and variable region, and the light and heavy chains are linked by disulfide bonds. Antigen-binding fragments can be prepared, for example, by enzymatic degradation of IgG with papain into two Fab fragments and an Fc fragment. Pepsin then degrades the IgG into an F(ab')2 fragment and an Fc fragment. The F(ab')2 fragment is further reduced to form two Fab' fragments. Because these antigen-binding fragments can still bind to the corresponding antigen, they can be used to prepare products for detecting fully carboxylated osteocalcin.

[0030] Those skilled in the art can also prepare single-chain antibodies (scFvs) from the monoclonal antibodies of the present invention using existing techniques. Single-chain antibodies are synthetic antibodies consisting of a single chain, consisting of the heavy and light chain variable regions of an antibody, connected by a short peptide linker consisting of several amino acids. Single-chain antibodies can also consist solely of the heavy chain variable region of an antibody. The length and amino acid composition of short peptide linkers are well known in the art, and suitable peptide linkers for use with the monoclonal antibodies of the present invention can be determined through simple, repeated experiments. Single-chain antibodies can be expressed in organisms such as bacteria, yeast, and cells using genetic engineering techniques. The single-chain antibodies of the present invention prepared in this manner have the property of specifically recognizing fully carboxylated osteocalcin and can be used in the detection of fully carboxylated osteocalcin.

[0031] Those skilled in the art can design and synthesize nucleic acid molecules encoding the above-mentioned monoclonal antibody variable region that specifically recognizes fully carboxylated osteocalcin based on the amino acid sequence, and can also insert the synthesized nucleic acid molecules into nucleic acid vectors to construct expression vectors, which can express monoclonal antibodies or antigen-binding fragments thereof that specifically recognize fully carboxylated osteocalcin. Those skilled in the art can also introduce the synthesized nucleic acid molecules or constructed expression vectors into organisms such as cells, bacteria, yeast, etc. to obtain recombinants, and express and produce antibodies or antigen-binding fragments of the present invention via the above-mentioned recombinant. The antibodies or antigen-binding fragments expressed in this way can specifically recognize fully carboxylated osteocalcin, so the above-mentioned nucleic acid molecules, expression vectors, and recombinants are within the scope of protection of the claims of the present invention. And the above-mentioned technologies are all well-known technologies in the art, and those skilled in the art can carry out without creative work.

[0032] As described above, the antibodies or antigen-binding fragments thereof of the present invention are capable of specifically recognizing fully carboxylated osteocalcin and can therefore be used to prepare a kit for detecting fully carboxylated osteocalcin. The kit can be any kit that utilizes the binding reaction between the antibodies or antigen-binding fragments thereof of the present invention and fully carboxylated osteocalcin, such as, but not limited to, enzyme-linked immunosorbent assay (ELISA), chemiluminescence, fluorescent immunochromatography, colloidal gold immunochromatography, immunoblotting, and immunohistochemistry. Methods used in ELISA, chemiluminescence, fluorescent immunochromatography, and colloidal gold immunochromatography kits include, but are not limited to, double-antibody sandwich methods. In a double-antibody sandwich detection kit, the monoclonal antibody or antigen-binding fragment thereof is preferably used as a capture antibody. The term capture antibody is well known in the art and is responsible for specifically "capturing" the target antigen in the bound sample to form a stable antigen-antibody complex, providing a basis for subsequent detection.

[0033] In order to explain the technical content, achieved objectives and effects of the technical solution in detail, the following is an explanation in conjunction with specific embodiments.

[0034] Example 1: Synthesis and preparation of different epitope peptides of mature osteocalcin

[0035] According to the amino acid sequence of human osteocalcin preproprotein published in the NCBI database (NCBI Reference Sequence: NP_954642.1), the mature osteocalcin molecule is 49 amino acids long: YLYQWLGAPVPYPDPLEPRREVCELNPDCDELADHIGFQEAYRRFYGPV (SEQ ID NO. 8). Glutamic acid (Glu, abbreviated E) at positions 17, 21, and 24 is readily carboxylated in the presence of vitamin K to form gamma-carboxyglutamate (Gla), represented in the sequence as E(Gla). To prepare monoclonal antibodies that specifically recognize fully carboxylated osteocalcin, we first synthesized a fully carboxylated osteocalcin 10-25 segment peptide with the sequence 10-VPYPDPLE(Gla)PRRE(Gla)VCE(Gla)L-25 (SEQ ID NO. 9) and conjugated it to KLH (called Gla-OC10-25-K) or BSA (called Gla-OC10-25-B) for immunization and screening of monoclonal antibodies, respectively. In parallel, an incompletely carboxylated osteocalcin 10-25 segment peptide (10-VPYPDPLEPRRE(Gla)VCE(Gla)L-25 (SEQ ID NO. 10)) and an uncarboxylated osteocalcin 10-25 segment peptide (10-VPYPDPLEPRREVCEL-25 (SEQ ID NO. 11)) were synthesized and conjugated to BSA (referred to as Glu / a-OC10-25-B and Glu-OC10-25-B, respectively) for characterization of monoclonal antibody recognition specificity. Furthermore, an osteocalcin 25-43 segment peptide (25-LNPDCDELADHIGFQEAYR-43 (SEQ ID NO. 12)) was synthesized and conjugated to KLH and BSA (referred to as OC25-43-K and OC25-43-B, respectively) for the generation and characterization of polyclonal antibodies targeting the middle segment of osteocalcin. All synthesized peptides are summarized in Table 1. Peptide synthesis and KLH or BSA coupling were commissioned to Shanghai Dechi Biotechnology Co., Ltd. according to conventional technical methods in the field.

[0036] Table 1 Summary of synthetic peptides with different epitopes of mature osteocalcin

[0037] name sequence Conjugate use Gla-OC10-25-K 10-VPYPDPLE(Gla)PRRE(Gla)VCE(Gla)L-25 KLH Monoclonal antibody preparation Gla-OC10-25-B 10-VPYPDPLE(Gla)PRRE(Gla)VCE(Gla)L-25 BSA Monoclonal antibody screening Glu / a-OC10-25-B 10-VPYPDPLEPRR E(Gla)VCE(Gla)L-25 BSA Monoclonal antibody specificity identification Glu-OC10-25-B 10-VPYPDPLEPRREVCEL-25 BSA Monoclonal antibody specificity identification OC25-43-K 25-LNPDCDELADHIGFQEAYR-43 KLH Polyclonal antibody preparation OC25-43-B 25-LNPDCDELADHIGFQEAYR-43 BSA Polyclonal antibody identification

[0038] Example 2: Prokaryotic expression of osteocalcin

[0039] Based on the genetic code bias of Escherichia coli, the nucleotide sequence of osteocalcin was deduced: 5'-TATCTGTACCAGTGGTTGGGCGCGCCGGTGCCATATCCAGATCCACTGGAACCGCGTCGCGAGGTTTGCGAATTAAACCCAGACTGTGATGAACTGGCCGATCATATTGGTTTTCAAGAGGCGTACCGTCGCTTCTATGGCCCGGTG-3' (SEQ ID NO. 13). A prokaryotic expression recombinant plasmid was constructed using BamHI and EcoRI restriction sites. The upstream primer used was 5'-GCGGATCCTATCTGTACCAGTGGT-3' (SEQ ID NO. 14), and the downstream primer was 5'-GCGAATTCTTACACCGGGCCATAG-3' (SEQ ID NO. 15). The full-length gene was amplified using the optimized synthetic nucleotide sequence as a template. Amplification conditions were: 95°C for 2 minutes, followed by 30 cycles of 95°C for 30 seconds, 58°C for 30 seconds, and 72°C for 30 seconds; followed by extension at 72°C for 5 minutes. 2% agarose gel electrophoresis confirmed the molecular weight of the amplified fragment to be approximately 550 bp. The purified PCR product was double-digested with BamHI and EcoRI restriction sites and ligated into pET-32a plasmid, which had also been double-digested with BamHI and EcoRI, to generate the pET-OC recombinant plasmid. Transform the correctly sequenced recombinant expression plasmid into E. coli BL21 competent cells, pick a single colony and place it in 5 mL of LB liquid medium containing ampicillin sodium, culture it with shaking at 37°C overnight, inoculate it into 250 mL of fresh LB liquid medium the next day, culture it to the logarithmic growth phase, adjust the temperature to 16°C, add 150 μl of 1 mol / L IPTG induction solution after 30 minutes, and induce it at 16°C for 12-14 hours. The induced bacteria were collected by centrifugation, resuspended with 25 mmol / LTris-HCl (pH8.5), ultrasonically disrupted, and centrifuged at 20,000g for 30 minutes at 4°C to collect the supernatant for Ni column purification. First, the Ni column was equilibrated with equilibrium buffer (25 mmol / L TE, 1% β-mercaptoethanol, 6 mol / L urea, pH8.5). The supernatant was added to the Ni column. After the sample was completely entered, the target protein was eluted and collected with a washing solution containing 25 mmol / L imidazole. SDS-PAGE gel electrophoresis was performed. Recombinant osteocalcin (rOC) was expressed in a soluble form with a molecular weight of approximately 27 kDa. The results are as follows. Figure 1 shown.

[0040] Example 3: Preparation of monoclonal antibodies against fully carboxylated osteocalcin

[0041] The fully carboxylated osteocalcin 10-25 segment peptide KLH conjugate Gla-OC10-25-K prepared in Example 1 was used as the immunogen. Six- to eight-week-old female BALB / c mice were immunized with 100 µg / mouse antigen in an equal volume of complete Freund's adjuvant, thoroughly emulsified with a blender, and then injected subcutaneously and intraperitoneally on the back. Three mice were immunized. A second immunization was performed four weeks apart, and a third immunization was performed eight weeks later. The antigen was added to incomplete Freund's adjuvant at a dose of 50 µg / mouse, thoroughly emulsified with a blender, and then injected subcutaneously and intraperitoneally on the back. One week after the third immunization, tail vein blood was collected from the mice to determine the titer of the immune serum. Mice with the highest titer were selected for an intraperitoneal booster (50 µg / mouse). Three days later, spleen cells were harvested for fusion. SP20 myeloma cells were resuscitated and cultured until they reached the logarithmic growth phase. Spleens from the immunized BALB / c mice were harvested to prepare a splenocyte suspension. The above spleen cells and myeloma cells were mixed in a serum-free DMEM medium at a ratio of 9:1, centrifuged at 1500 rpm for 5 minutes, the supernatant was aspirated, and the cells were dispersed by gentle shaking. The cells were fused in a 37°C water bath. Within 1 minute, 1 mL of preheated 50% PEG was added to fuse the cells, and the mixture was gently shaken while adding. After addition, the cells were allowed to stand for 90 seconds. Serum-free DMEM medium was added to terminate the fusion. The cells were allowed to stand at 37°C for 10 minutes and centrifuged at 1500 rpm for 5 minutes. The precipitate was suspended in HAT medium and distributed into 96-well cell plates containing feeder cells. After culturing in a cell culture incubator at 37°C and 5% CO2 for 5 days, the medium was replaced with HAT medium once. On the 10th day, the medium was replaced with HAT medium. When the fused cells covered about 60% of the bottom of the well, the cell culture supernatant was taken and positive clones were screened by indirect ELISA. The specific method is as follows: 150 μl of the fully carboxylated osteocalcin 10-25 peptide conjugate, Gla-OC10-25-B, was diluted in carbonate coating buffer to a concentration of 2.0 μg / ml. The plate was coated with 150 μl per well at 4°C overnight. The plate was washed twice with washing buffer, then blocked with 200 μl / well of blocking buffer at room temperature for 6 hours. The plate was then washed five times with washing buffer. After adding 100 μl of sample diluent to each well, 10 μl of cell culture supernatant was added to each well and incubated at room temperature for 30 minutes. The solution was then discarded. The plate was washed five times. The washed plate was inverted onto absorbent paper, patted dry, and 100 μl / well of HRP-conjugated goat anti-mouse IgG antibody was added and incubated at room temperature for 30 minutes. The plate was washed five times. 50 μl each of TMB colorimetric reagents A and B were added to each well and the color was developed at room temperature in the dark for 15 minutes. The reaction was terminated by adding 50 μl of 2 M H2SO4 stop solution to each well. Set the detection wavelength on a microplate reader to 450 nm and measure the OD value of each well. Read the value within 10 minutes after termination. Hybridoma cell lines identified as strongly positive were cultured in 1640 medium supplemented with 10% fetal bovine serum. Each BALB / c male mouse was intraperitoneally injected with 0.5 mL of liquid paraffin.After 10 days, the cells were collected and resuspended in 10 mL of normal saline to a cell density of 1 × 10. 7 Each mouse was intraperitoneally injected with 0.5 mL of the monoclonal antibody. After 2 weeks, ascites was collected. Antibodies were purified using the Thermo Fisher Scientific Melon Gel Monoclonal IgG Purification Kit (Cat. No. 45214). Purified antibodies were aliquoted and stored at -20°C.

[0042] Example 4: Screening of monoclonal antibodies against fully carboxylated osteocalcin

[0043] The fully carboxylated osteocalcin 10-25 segment polypeptide BSA conjugate Gla-OC10-25-B, the incompletely carboxylated osteocalcin 10-25 segment polypeptide BSA conjugate Glu / a-OC10-25-B, the non-carboxylated osteocalcin 10-25 segment polypeptide BSA conjugate Glu-OC10-25-B, the prokaryotically expressed recombinant osteocalcin rOC in Example 2, and osteocalcin bOC extracted from bovine bones (Cat. No. BP-010, purchased from K-Assay) prepared in Example 1 were diluted with carbonate buffer and coated on an enzyme-linked plate at a concentration of 2.5 μg / ml. 150 μL was coated per well and incubated at 4°C overnight. The plate was washed twice with washing solution, blocked with 200 μL / well of blocking solution at room temperature for 6 hours, and washed five times with washing solution. After adding 100 μL of sample diluent to each well, add 10 μL of cell culture supernatant to each well, incubate at room temperature for 30 minutes, and discard. Wash the plate five times. Invert the washed plate on absorbent paper, pat dry, and add 100 μL / well of HRP-labeled goat anti-mouse IgG antibody. Incubate at room temperature for 30 minutes. Wash the plate five times. Add 50 μL each of TMB colorimetric solution A and B to each well and develop at room temperature in the dark for 15 minutes. Terminate the reaction by adding 50 μL of 2 M H2SO4 stop solution to each well. Set the detection wavelength on a microplate reader to 450 nm and measure the OD value of each well. Read the value within 10 minutes after termination.

[0044] The results are as follows Figure 2 As shown, among the multiple anti-fully carboxylated osteocalcin monoclonal antibodies prepared in Example 3, one monoclonal antibody, 3A421, was screened to recognize only fully carboxylated osteocalcin. This monoclonal antibody binds to the fully carboxylated osteocalcin 10-25 segment polypeptide Gla-OC10-25-B and osteocalcin bOC extracted from bovine bones (containing a fully carboxylated osteocalcin molecule), but does not bind to the incompletely carboxylated osteocalcin 10-25 segment polypeptide Glu / a-OC10-25-B, the uncarboxylated osteocalcin 10-25 segment polypeptide Glu-OC10-25-B, or prokaryotically expressed recombinant osteocalcin rOC (a uncarboxylated osteocalcin molecule).

[0045] Example 5: Preparation of rabbit anti-osteocalcin 25-43 segment polypeptide polyclonal antibodies

[0046] Healthy male white rabbits were selected. 1.0 mg of the osteocalcin 25-43 segment peptide KLH conjugate, OC25-43-K, synthesized in Example 1, was used as the immunogen. The mixture was mixed with 1.0 mL of complete Freund's adjuvant and thoroughly emulsified using a blender. 0.2 mL was then injected subcutaneously at points along the rabbit's spine. Four weeks later, 1.0 mg of the OC25-43-K immunogen was mixed with 1.0 mL of incomplete Freund's adjuvant and thoroughly emulsified using a blender. A second immunization was performed at different points along the same spine. A third booster immunization was performed four weeks later to prepare polyclonal antibody serum. One week later, blood was collected from the heart. After the blood clot had coagulated and retracted, it was centrifuged at 5000 rpm for 15 minutes. The serum was aliquoted and stored at -20°C until further use. The indirect ELISA method for determining the titer of polyclonal antibodies is performed as follows: the enzyme-linked plate is coated with the osteocalcin 25-43 segment peptide BSA conjugate OC25-43-B at a concentration of 2.0 μg / ml, 150 μL per well, and incubated at 4°C overnight; the plate is washed twice with washing solution; 200 μL / well of blocking solution is added and blocked at room temperature for 6 hours; and the plate is washed five times with washing solution. Rabbit serum was diluted with PBS at the following ratios: 1:2000, 1:8000, 1:32000, 1:128000, 1:512000, 1:1024000, 1:2048000 and 1:4096000, with 100 μL added to each well; incubated at 37°C for 45 min; washed the plate 5 times with washing buffer, with 200 μL added to each well; HRP-labeled goat anti-rabbit secondary antibody was incubated at 37°C for 45 min; washed the plate 5 times with washing buffer, with 200 μL added to each well; freshly prepared substrate solution was added, with 100 μL added to each well, and incubated at 37°C for 10 min; 50 μL of 2 MH2SO4 was added to each well to terminate the reaction, and the absorbance of each well was measured at 450 nm using a microplate reader. The values ​​were read within 10 minutes after termination. The results are shown in Table 1. Figure 3 Using serial dilutions of pre-immune rabbit serum as a negative control, the titer of the polyclonal antibody prepared by immunizing rabbits with osteocalcin 25-43 segment peptide can reach 1:1024000.

[0047] Example 6: Establishment of a double antibody sandwich detection method for fully carboxylated osteocalcin

[0048] The monoclonal antibody 3A421 prepared by the present invention, which recognizes only fully carboxylated osteocalcin, was used as a capture antibody to specifically capture fully carboxylated osteocalcin in the sample. In Example 5, a rabbit polyclonal antibody against the osteocalcin 25-43 segment polypeptide was prepared as a detection antibody to establish a method for the specific detection of fully carboxylated osteocalcin (Gla-OC) based on a double-antibody sandwich method. This method can detect all fully carboxylated osteocalcin, including fully carboxylated full-length osteocalcin and fully carboxylated osteocalcin N-MID. The specific steps were as follows: 100 μL of the monoclonal antibody 3A421, which recognizes only fully carboxylated osteocalcin, was coated into an ELISA plate at a concentration of 2.0 μg / mL. The plate was incubated overnight at 4°C and washed twice with washing buffer. 120 μL / well of blocking buffer was added to block the plate for 6 hours at room temperature and washed five times with washing buffer. Osteocalcin (bOC) extracted from bovine bones and the recombinant osteocalcin (rOC) expressed prokaryotically in Example 2 were serially diluted in PBS to concentrations of 2000, 400, 80, 16, 3.2, 0.64, 0.32, and 0.16 ng / mL, respectively. 100 μL of each was added to each well and incubated at 37°C for 60 min. The solution was then discarded. The plate was washed five times with washing buffer. 100 μL of a horseradish peroxidase-labeled rabbit anti-osteocalcin 25-43 segment polyclonal antibody was added to each well and incubated at 37°C for 60 min. Wash the plate five times, pat dry, and add 50 μL each of TMB colorimetric solution A and solution B to each well. Develop at room temperature in the dark for 15 min. Terminate the reaction by adding 50 μL / well of 2 M H₂SO₄ stop solution. Measure the OD value of each well using a microplate reader at a wavelength of 450 nm, reading within 10 minutes after termination.

[0049] The results are as follows Figure 4 As shown, the established double-antibody sandwich assay specifically detects fully carboxylated osteocalcin (bOC) extracted from bovine bones and does not cross-react with uncarboxylated recombinant osteocalcin (rOC), demonstrating that this method can be used for the specific detection of fully carboxylated osteocalcin. Using a cutoff value of three times the negative control detection value (Cut = 0.207), the established double-antibody sandwich assay has a minimum detection limit of 0.32 ng / mL for fully carboxylated osteocalcin.

[0050] Example 7: Specific Detection of Fully Carboxylated Osteocalcin in Blood Samples

[0051] The established double-antibody sandwich assay for fully carboxylated osteocalcin was used to simultaneously assay serum samples from 63 women 5 years postmenopausal and 63 premenopausal women. The following steps were used: 100 μL of the monoclonal antibody 3A421, which recognizes only fully carboxylated osteocalcin, was coated into an enzyme-linked microplate at a concentration of 2.0 μg / mL. The plate was incubated at 4°C overnight and washed twice with washing buffer. Blocking was performed with 120 μL / well of blocking buffer at room temperature for 6 hours, followed by five washes with washing buffer. 100 μL of serum sample was added to each well, incubated at 37°C for 60 minutes, and the buffer was discarded. The plate was washed five times with washing buffer, and 100 μL of horseradish peroxidase-conjugated rabbit anti-osteocalcin 25-43 peptide polyclonal antibody was added to each well and incubated at 37°C for 60 minutes. The plate was washed five times, patted dry, and 50 μL each of TMB colorimetric reagents A and B were added to each well. The plate was developed at room temperature in the dark for 15 minutes. Terminate the reaction by adding 50 μL / well of 2 M H2SO4 stop solution. Measure the OD value of each well using a microplate reader at a wavelength of 450 nm, reading the value within 10 minutes after termination.

[0052] The results are as follows Figure 5 As shown, the average OD value of the postmenopausal group was 0.991±0.082, which was significantly higher than the average OD value of the premenopausal group of 0.479±0.021. The difference between the two groups was significant (p<0.001), indicating that the method of the present invention can be used to prepare products for the detection of fully carboxylated osteocalcin to assist in the diagnosis of osteoporosis patients.

[0053] Example 8: Subtype Analysis of Anti-Fully Carboxylated Osteocalcin Monoclonal Antibody 3A421

[0054] The mouse antibody subtype rapid test card (catalog number THJ-ISO-M8a-10 / 20) from Antaiji (Beijing) Biotechnology Co., Ltd. was used to identify the heavy and light chain subtypes of mouse antibodies. First, the antibody was diluted to 1 μg / mL with PBS, and then 100 μl of the diluted antibody was added to each well. After standing for 5-10 minutes, the results were observed and recorded. Figure 6 As shown, the anti-fully carboxylated osteocalcin monoclonal antibody 3A421 is of mouse IgG2a subtype, and the antibody light chain is of Igκ subtype.

[0055] Example 9: Sequencing of the variable regions of the anti-fully carboxylated osteocalcin monoclonal antibody 3A421

[0056] Mouse hybridoma cell line 3A421 was cultured, and total RNA was extracted from the hybridoma cells using the Trizol method. After reverse transcription of cDNA, PCR amplification was performed using primer sequences for the Fab region of a mouse monoclonal antibody synthesized by Beijing Qingke Biotechnology Co., Ltd. The amplified fragments were preheated at 95°C for 2 minutes, followed by 30 cycles of 95°C for 30 seconds, 58°C for 30 seconds, and 72°C for 30 seconds, and a final extension at 72°C for 5 minutes. The amplified fragments were ligated into the pMD18-T vector and transformed into Escherichia coli JM109. Positive clones were selected for sequencing. The determined sequences were compared with the CDR region sequences of the mouse monoclonal antibody using the IgBLAST module in the NCBI website (https: / / www.ncbi.nlm.nih.gov / igblast / ).

[0057] After sequence analysis, it was found that the amino acid sequence of the heavy chain variable region was 115 amino acids, and its sequence was as follows: DVQLQESGPGLVKPSQTVSLTCTVTGISITTGNYRWSGIRQFPGNKLEWIGYIYYSGTITCNPSLTSRTTITRDTSKNQFFLEMNSLTTEDTATYCARDRSGWTTGVKEPQSPSP (SEQ ID NO.1), among which the heavy chain CDR1 was located at 26-35aa, and the amino acid sequence was GISITTGNYR (SEQ ID NO.2); the heavy chain CDR2 was located at 53-59aa, and the amino acid sequence was IYYSGTI (SEQ IDNO.3); the heavy chain CDR3 was located at 97-105aa, and the amino acid sequence was ARDRSGWTT (SEQ ID NO.4). The amino acid sequence of the light chain variable region is 109 amino acids, and its sequence is as follows: DIVMTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCRHIRELTRSEGHQAGNQ (SEQ ID NO.5), wherein the light chain CDR1 is located at 27-36aa, and the amino acid sequence is KSVSTSGYSY (SEQ ID NO.6); the light chain CDR2 is located at 54-56aa, and the amino acid sequence is LVS; the light chain CDR3 is located at 93-100aa, and the amino acid sequence is RHIRELTR (SEQ ID NO.7).

Claims

1. A monoclonal antibody or antigen-binding fragment thereof that specifically recognizes fully carboxylated osteocalcin, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, and the light chain variable region comprises a light chain CDR1, a light chain CDR2, and a light chain CDR3, characterized in that: The amino acid sequence of the heavy chain CDR1 is the sequence shown in SEQ ID NO. 2 or an amino acid sequence having one conservative amino acid substitution compared with the sequence shown in SEQ ID NO. 2; The amino acid sequence of the heavy chain CDR2 is the sequence shown in SEQ ID NO. 3 or an amino acid sequence having one conservative amino acid substitution compared to the sequence shown in SEQ ID NO. 3; The amino acid sequence of the heavy chain CDR3 is the sequence shown in SEQ ID NO. 4 or an amino acid sequence having one conservative amino acid substitution compared with the sequence shown in SEQ ID NO. 4; The amino acid sequence of the light chain CDR1 is the sequence shown in SEQ ID NO.6 or an amino acid sequence having one conservative amino acid substitution compared with the sequence shown in SEQ ID NO.6; The amino acid sequence of the light chain CDR2 is LVS or an amino acid sequence having one conservative amino acid substitution compared thereto; The amino acid sequence of the light chain CDR3 is the sequence shown in SEQ ID NO.7 or an amino acid sequence having one conservative amino acid substitution compared with the sequence shown in SEQ ID NO.

7.

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO.

5.

3. The monoclonal antibody according to claim 2, characterized in that It is secreted by the mouse hybridoma cell line 3A421 with a deposit number of CGMCC No. 46547.

4. The monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that The monoclonal antibody or antigen-binding fragment is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a single-chain antibody or a humanized antibody.

5. A nucleic acid molecule, characterized in that It comprises a nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

6. An expression vector, characterized in that It comprises the nucleic acid molecule according to claim 5.

7. A cell, bacterium or yeast recombinant, characterized in that It comprises the nucleic acid molecule according to claim 5 or the expression vector according to claim 6.

8. A hybridoma cell line secreting a monoclonal antibody that specifically recognizes fully carboxylated osteocalcin, characterized in that: The cell line is mouse hybridoma cell line 3A421 with a deposit number of CGMCC No. 46547.

9. Use of the monoclonal antibody or antigen-binding fragment thereof that specifically recognizes fully carboxylated osteocalcin according to any one of claims 1 to 4 in the preparation of a product for detecting fully carboxylated osteocalcin.

10. A kit for detecting fully carboxylated osteocalcin, characterized in that: The method comprises the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

11. The kit according to claim 10, characterized in that The invention is a detection kit of double antibody sandwich method, wherein the monoclonal antibody or the antigen binding fragment thereof is used as the capture antibody.

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