Fully carboxylated osteocalcin-specific monoclonal antibodies and uses thereof

Abstract

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

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical technology, and particularly relates to a monoclonal antibody specifically recognizing fully carboxylated osteocalcin and application thereof. BACKGROUND

[0002] Osteocalcin (OC) is also known as bone γ-carboxyglutamic acid protein (BGP), which is a calcium-binding protein mainly synthesized by osteoblasts, odontoblasts and proliferating chondrocytes. Osteocalcin is the most abundant non-collagen protein in bone tissue, accounting for 10-20% of non-collagen protein, and its main physiological function is to maintain the normal mineralization rate of bone, inhibit the formation of abnormal hydroxyapatite crystals, and inhibit the mineralization rate of cartilage. Changes in its circulating level are closely related to osteoblast function and bone formation, and it can be used as a specific marker of bone metabolism.

[0003] The mature osteocalcin molecule has a full length of 49 amino acids and a relative molecular mass of about 5210-5889 Da, which is referred to as full-length osteocalcin. It is unstable in the peripheral blood and has a half-life of only 4-5 min in the blood. The carboxy-terminal 5-6 amino acids are easily hydrolyzed by proteases to form a 43 (1-43 aa) or 44 (1-44 aa) amino acid osteocalcin macromolecule fragment, which is referred to as N-terminal and middle segment osteocalcin macromolecule (abbreviated as N-MID). N-MID has good stability. In addition, the glutamic acid residues (Glu) at positions 17, 21 and 24 in the amino acid sequence of osteocalcin will be carboxylated under the participation of vitamin K to form γ-carboxyglutamic acid (Gla). Only the fully carboxylated osteocalcin (abbreviated as Gla-OC) with all three glutamic acid residues carboxylated has the ability to bind to hydroxyapatite (bone mineral) and promote bone formation, so the fully carboxylated osteocalcin is the real bone formation marker. Glu21 and Glu24 of osteocalcin are almost all carboxylated, while Glu17 is usually non-carboxylated. The incompletely carboxylated osteocalcin (abbreviated as Glu / a-OC) does not participate in bone mineralization and is an indicator of poor bone quality, but not an indicator of bone formation. Up to 20-30% of osteocalcin in adult blood is in the form of low carboxylation, and its concentration increases significantly with age. In the elderly female population who can move independently, the incompletely carboxylated osteocalcin is an independent predictor of hip fracture. In addition, studies have shown that the incompletely carboxylated osteocalcin can act as a hormone and be involved in processes related to glucose metabolism, lipid metabolism, fertility, aging, etc.

[0004] In summary, the osteocalcin molecules in the blood circulation have high heterogeneity, both different length of molecular fragments and different degrees of carboxylation of various forms. Studies have shown that in normal and osteoporosis patients, full-length osteocalcin and osteocalcin N-MID are the most abundant osteocalcin molecular forms, accounting for about two-thirds of all molecules, and the remaining one-third is other various smaller fragments, which can be rapidly cleared by the kidney. And full-length osteocalcin and osteocalcin N-MID have completely carboxylated, incompletely carboxylated and non-carboxylated forms, and only completely carboxylated osteocalcin is the true bone formation marker. However, so far, there is no relevant literature to clearly report the accurate concentration of completely carboxylated osteocalcin and incompletely carboxylated osteocalcin in the blood circulation, the key reason is the lack of a monoclonal antibody that can specifically bind to completely carboxylated osteocalcin. Therefore, the purpose of the present application is to prepare and screen a monoclonal antibody that can specifically recognize only completely carboxylated osteocalcin, but not non-carboxylated osteocalcin or incompletely carboxylated osteocalcin, so as to realize the accurate, simple and rapid quantification of completely carboxylated osteocalcin, and promote the wide application of this detection index in the clinic. SUMMARY

[0005] Therefore, the purpose of the present application is to provide a monoclonal antibody that can specifically recognize only completely carboxylated osteocalcin, and the monoclonal antibody obtained through experiments does not recognize non-carboxylated osteocalcin or incompletely carboxylated osteocalcin, and does not have cross-reaction with non-carboxylated osteocalcin or incompletely carboxylated osteocalcin, and can be used for accurate quantification of completely carboxylated osteocalcin.

[0006] Therefore, one aspect of the present application relates to a monoclonal antibody or an antigen binding fragment thereof that can specifically recognize only completely carboxylated osteocalcin, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3, and the light chain variable region comprising 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 1 amino acid conservative 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 1 amino acid conservative substitution compared with 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 1 amino acid conservative substitution compared with the sequence shown in SEQ ID NO. 4;

[0010] the amino acid sequence of the heavy chain CDR1 is the sequence set forth in SEQ ID NO. 1 or an amino acid sequence with 1 conservative substitution to the sequence set forth in SEQ ID NO. 1;

[0011] the amino acid sequence of the light chain CDR2 is LVS or an amino acid sequence with 1 conservative substitution to LVS;

[0012] the amino acid sequence of the light chain CDR3 is the sequence set forth in SEQ ID NO. 7 or an amino acid sequence with 1 conservative substitution to the sequence set forth in SEQ ID NO. 7.

[0013] In a further aspect, the present application 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 set forth in SEQ ID NO. 1 and the amino acid sequence of the light chain variable region is the sequence set forth in SEQ ID NO. 5.

[0014] The present application 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, and these antibodies or antigen binding fragments can specifically recognize the fully carboxylated osteocalcin due to the retention of the variable regions of the light chain and the heavy chain, or only the heavy chain variable region.

[0015] In addition, the present application also relates to a nucleic acid molecule comprising a nucleic acid encoding the above-mentioned antibody or antigen binding fragment thereof, and an expression vector comprising the above-mentioned nucleic acid molecule, wherein the expression vector can express the above-mentioned antibody or antigen binding fragment thereof. Meanwhile, the present application also relates to a recombinant comprising the above-mentioned nucleic acid molecule or the above-mentioned expression vector, wherein the recombinant can produce the above-mentioned antibody or antigen binding fragment thereof. On the other hand, the present application relates to a monoclonal antibody hybridoma cell strain specifically recognizing the fully carboxylated osteocalcin, wherein the monoclonal antibody hybridoma cell strain secretes the above-mentioned monoclonal antibody. Further, the present application relates to a monoclonal antibody hybridoma cell strain specifically recognizing only the fully carboxylated osteocalcin, wherein the monoclonal antibody hybridoma cell strain is a mouse hybridoma cell strain 3A421, and the preservation number is CGMCC No. 46547.

[0016] In still another aspect, the present application relates to the use of the above-mentioned monoclonal antibody or antigen binding fragment thereof in the preparation of a product for detecting the fully carboxylated osteocalcin. Further, the present application relates to a kit for detecting the fully carboxylated osteocalcin, wherein the kit comprises the above-mentioned monoclonal antibody or antigen binding fragment thereof for specifically recognizing and binding the fully carboxylated osteocalcin; preferably, the kit is a double antibody sandwich detection kit, and the monoclonal antibody or antigen binding fragment thereof serves as a capture antibody.

[0017] Biological material preservation instruction

[0018] The monoclonal antibody hybridoma cell strain of the present application: mouse hybridoma cell strain 3A421, is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the registration number is CGMCC No. 46547, and the preservation date is June 18, 2025. The address of the China General Microbiological Culture Collection Center is No. 1, Xili, Beichen West Road, Chaoyang District, Beijing, China, with a postcode of 100101. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is an SDS-PAGE electrophoretogram showing prokaryotic expression of recombinant osteocalcin, in which M is a molecular weight marker.

[0020] Figure 2 is a specific identification result map of the recognition specificity of the anti-complete carboxylated osteocalcin monoclonal antibody 3A421.

[0021] Figure 3 is a polyclonal antibody titer detection result map of rabbit anti-osteocalcin 25-43 segment polypeptide.

[0022] Figure 4 is a double antibody sandwich method detection result map of complete carboxylated osteocalcin.

[0023] Figure 5 is a double antibody sandwich method clinical sample detection result map of complete carboxylated osteocalcin.

[0024] Figure 6 is a subtype identification result map of the anti-complete carboxylated osteocalcin monoclonal antibody 3A421. DETAILED DESCRIPTION

[0025] The present application aims to provide a monoclonal antibody which specifically recognizes only fully carboxylated osteocalcin (Gla-OC) and is prepared by a fused hybridoma cell strain. The specific preparation process is as follows: first, a fully carboxylated osteocalcin polypeptide 10-25 aa segment (named Gla-OC10-25) is synthesized, then the segment is used as an immunogen to immunize mice to prepare a monoclonal antibody, and the monoclonal antibody is screened by using fully carboxylated osteocalcin, incompletely carboxylated osteocalcin, non-carboxylated osteocalcin, prokaryotic expression recombinant osteocalcin and extracted bovine osteocalcin, so as to obtain a high-specificity monoclonal antibody which only recognizes fully carboxylated osteocalcin. The monoclonal antibody is used together with a polyclonal antibody against the 25-43 aa segment of osteocalcin to detect fully carboxylated full-length osteocalcin and fully carboxylated osteocalcin N-MID. The mouse hybridoma cell strain secreting the monoclonal antibody is named 3A421, which only specifically recognizes fully carboxylated osteocalcin and does not cross-react with non-carboxylated osteocalcin and incompletely carboxylated osteocalcin, and has very high specificity. The present inventors deposited the cell strain in the China General Microbiological Culture Collection Center on June 18, 2025, and the deposit number is CGMCC No. 46547.

[0026] Subsequently, the present inventors sequenced and immunoglobulin domain sequence analyzed the monoclonal antibody secreted by the mouse hybridoma cell strain CGMCC No. 46547, and found that the heavy chain variable region amino acid sequence is: DVQLQESGPGLVKPSQTVSLTCTVTGISITTGNYRWSGIRQFPGNKLEWIGYIYYSGTITCNPSLTSRTTITRDTSKNQFFLEMNSLTTEDTATYCARDRSGWTTGVKEPQSPSP (SEQ ID NO. 1), wherein the heavy chain CDR1 amino acid sequence is GISITTGNYR (SEQ ID NO. 2), the heavy chain CDR2 amino acid sequence is IYYSGTI (SEQ ID NO. 3), and the heavy chain CDR3 amino acid sequence is ARDRSGWTT (SEQ ID NO. 4). The light chain variable region amino acid sequence 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 present inventors evaluated the recognition specificity of the monoclonal antibody to the fully carboxylated osteocalcin by enzyme-linked immunosorbent assay, and found that the monoclonal antibody specifically recognizes only the fully carboxylated osteocalcin, but does not recognize the non-carboxylated osteocalcin and the incompletely carboxylated osteocalcin, and has high specificity. The detection of clinical samples by enzyme-linked immunosorbent assay showed that the monoclonal antibody of the present application can be used to prepare products for detecting the fully carboxylated osteocalcin, assisting the clinical diagnosis of osteoporosis and the monitoring of drug management.

[0028] It is well known in the art that the heavy chain CDR region and the light chain CDR region of an antibody are important amino acid sequence regions for recognizing and binding to the corresponding antigen, and a conservative substitution of one amino acid in the above CDR region amino acid sequence can not change the structure of the protein, so a single amino acid conservative substitution in the above region can still have the property of binding to the corresponding antigen. Therefore, the monoclonal antibody or antigen-binding fragment thereof obtained after a conservative substitution of one amino acid 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 the fully carboxylated osteocalcin. In the present patent application, the conservative substitution of an amino acid refers to the substitution of one amino acid in a protein with another chemically similar amino acid, such as the mutual substitution between aromatic amino acids Phe, Trp, Tyr, the mutual substitution between aliphatic amino acids Ala, Gly, Leu, Ile, Val, the mutual substitution between polar amino acids Gln, Asn, the mutual substitution between basic amino acids Lys, Arg, His, the mutual substitution between acidic amino acids Asp, Glu, and the mutual substitution between hydroxyl amino acids Ser, Thr, etc.

[0029] The skilled person in the art can also prepare various antibody fragments, i.e. antigen-binding fragments, such as but not limited to Fab, Fab', F(ab')2, which can specifically recognize only the fully carboxylated osteocalcin, from the monoclonal antibody of the present application, by the existing technology in the art. The Fab fragment is the region of the antibody structure that can bind to the antigen, and is composed of one complete light chain and the variable region VH and constant region CH1 domain (Fd fragment) of the heavy chain. The light chain and the heavy chain each have one constant region and one variable region, and there is a disulfide bond linkage between the light chain and the heavy chain. The antigen-binding fragment can be prepared, for example, by using papain to degrade the antibody IgG into two Fab fragments and one Fc fragment. Under the action of pepsin, the antibody IgG is degraded into one F(ab')2 fragment and one Fc fragment, and the F(ab')2 fragment is further reduced to form two Fab' fragments. Because the above antigen-binding fragment can still bind to the corresponding antigen, it can be used to prepare products for detecting the fully carboxylated osteocalcin.

[0030] The skilled person in the art can also prepare single chain antibodies (scFv) from the monoclonal antibodies of the present application by using the current techniques in the art. Single chain antibodies are antibodies that have only one chain, which is artificially synthesized by connecting the variable region of the heavy chain and the variable region of the light chain of an antibody through a short peptide linker of several amino acids. The single chain antibody can also contain only the variable region of the heavy chain. The length and amino acid composition of the short peptide linker are well known in the art, and the short peptide linker that can be used for the monoclonal antibodies of the present application can be determined by simple repeated experiments. The single chain antibodies of the present application can be expressed in organisms such as bacteria, yeast, cells, etc. by genetic engineering techniques. The single chain antibodies of the present application thus prepared have the property of specifically recognizing fully carboxylated osteocalcin and can be used in the detection of fully carboxylated osteocalcin.

[0031] The skilled person in the art can design and synthesize nucleic acid molecules encoding the variable region of the monoclonal antibodies of the present application based on the amino acid sequences of the specific recognition of fully carboxylated osteocalcin described above, and can also insert the synthesized nucleic acid molecules into nucleic acid vectors to construct expression vectors that can express monoclonal antibodies or antigen-binding fragments thereof that specifically recognize fully carboxylated osteocalcin. The skilled person in the art can also introduce the synthesized nucleic acid molecules or the constructed expression vectors into organisms such as cells, bacteria, yeast, etc. to obtain recombinants, and produce the antibodies or antigen-binding fragments thereof of the present application through expression by the above-mentioned recombinants. The antibodies or antigen-binding fragments thereof thus expressed can specifically recognize fully carboxylated osteocalcin, and therefore the above-mentioned nucleic acid molecules, expression vectors and recombinants are within the scope of the claims of the present application. Moreover, the above-mentioned techniques are all well known in the art and can be carried out by the skilled person in the art without inventive labor.

[0032] As described above, the antibodies or antigen-binding fragments thereof of the present application can specifically recognize 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 of the antibodies or antigen-binding fragments thereof of the present application with fully carboxylated osteocalcin, such as, but not limited to, enzyme-linked immunosorbent assay, chemiluminescence, fluorescent immunochromatography, colloidal gold immunochromatography, immunoblotting, immunohistochemistry type kits, wherein the methods used in enzyme-linked immunosorbent assay, chemiluminescence, fluorescent immunochromatography, colloidal gold immunochromatography type kits include, but are not limited to, double antibody sandwich method, etc. In the double antibody sandwich detection kit, the monoclonal antibodies or antigen-binding fragments thereof are preferably used as capture antibodies; in the art, the meaning of capture antibodies is well known, which is responsible for specifically "capturing" the target antigen in the sample to form a stable antigen-antibody complex to provide a basis for subsequent detection.

[0033] To make the technical contents of the technical solutions, the purposes and effects achieved more clear, the following will be described in detail 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 database of NCBI (NCBI Reference Sequence: NP_954642.1), the full length of mature osteocalcin molecule is 49 amino acids, i.e. YLYQWLGAPVPYPDPLEPRREVCELNPDCDELADHIGFQEAYRRFYGPV (SEQ ID NO. 8). Among them, glutamic acid (Glu, single letter abbreviation E) at positions 17, 21 and 24 is easily carboxylated under the participation of vitamin K to form gamma carboxyglutamic acid (Gla), which is represented as E(Gla) in the sequence. In order to prepare monoclonal antibodies that can specifically recognize completely carboxylated osteocalcin, we first synthesized a completely carboxylated osteocalcin 10-25 segment polypeptide with the sequence 10-VPYPDPLE(Gla)PRRE(Gla)VCE(Gla)L-25 (SEQ ID NO. 9), and coupled it to KLH (referred to as Gla-OC10-25-K) or BSA (referred to as Gla-OC10-25-B) respectively, which were used for immunization and screening of monoclonal antibodies. At the same time, we synthesized an incompletely carboxylated osteocalcin 10-25 segment polypeptide with the sequence 10-VPYPDPLEPRRE(Gla)VCE(Gla)L-25 (SEQ ID NO. 10) and a non-carboxylated osteocalcin 10-25 segment polypeptide with the sequence 10-VPYPDPLEPRREVCEL-25 (SEQ ID NO. 11), which were coupled to BSA (referred to as Glu / a-OC10-25-B, Glu-OC10-25-B respectively) for identification of the recognition specificity of monoclonal antibodies. In addition, we synthesized a bone osteocalcin 25-43 segment polypeptide with the sequence 25-LNPDCDELADHIGFQEAYR-43 (SEQ ID NO. 12), which was coupled to KLH and BSA (referred to as OC25-43-K, OC25-43-B respectively) for the preparation and identification of polyclonal antibodies against the middle segment of osteocalcin. All the synthetic peptides are summarized in Table 1. The synthesis of polypeptides and the coupling of KLH or BSA were entrusted to Shanghai Dewe Biological Technology Co., Ltd. according to the conventional technical methods in the field.

[0036] Table 1: Summary of synthetic peptides of 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 production Gla-OC10-25-B 10-VPYPDPLE(Gla)PRRE(Gla)VCE(Gla)L-25 BSA Monoclonal antibody screening Glu / a-OC10-25-B 10-VPYPDPLEPRREVCEL-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 production OC25-43-B 25-LNPDCDELADHIGFQEAYR-43 BSA Polyclonal antibody identification

[0038] Example 2: Prokaryotic expression of osteocalcin

[0039] According to the preference of the genetic code of E. coli, the nucleotide sequence was deduced from the amino acid sequence of osteocalcin, which was 5'-TATCTGTACCAGTGGTTGGGCGCGCCGGTGCCATATCCAGATCCACTGGAACCGCGTCGCGAGGTTTGCGAATTAAACCCAGACTGTGATGAACTGGCCGATCATATTGGTTTTCAAGAGGCGTACCGTCGCTTCTATGGCCCGGTG-3' (SEQ ID NO. 13). The prokaryotic expression recombinant plasmid was constructed by using BamH I and EcoR I enzyme cutting 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 synthesized optimized nucleotide sequence as a template, and the amplification conditions were as follows: 95°C for 2 min; 95°C for 30 s, 58°C for 30 s, 72°C for 30 s, for a total of 30 cycles; and then 72°C for 5 min. 2% agarose gel electrophoresis showed that the relative molecular weight of the amplified fragment was about 550 bp. The purified PCR product was double-digested using BamH I and EcoR I enzyme cutting sites, and was ligated into the same pET-32a plasmid that had been double-digested with BamH I and EcoR I, to obtain the pET-OC recombinant plasmid. The recombinant expression plasmid that had been correctly sequenced was transformed into E. coli BL21 competent cells, and a single colony was inoculated in 5 mL of LB liquid medium containing ampicillin sodium and was incubated at 37°C overnight. The next day, it was inoculated in 250 mL of fresh LB liquid medium, and was cultured until the logarithmic growth phase. The temperature was adjusted to 16°C, and 150 μl of 1 mol / L IPTG induction solution was added after 30 min. The induction was performed at 16°C for 12-14 h. The induced bacterial cells were collected by centrifugation, were resuspended with 25 mmol / L Tris-HCl (pH 8.5), were broken by ultrasonic waves, and were collected by high-speed centrifugation at 20000g at 4°C for 30 min. The supernatant was subjected to Ni column purification. First, the Ni column was equilibrated with equilibration buffer (25 mmol / L TE, 1% β-mercaptoethanol, 6 mol / L urea, pH 8.5). The supernatant was added to the Ni column, and after the sample was completely introduced, the target protein was eluted and collected using washing solution containing 25 mmol / L imidazole, and was subjected to SDS-PAGE gel electrophoresis. The recombinant osteocalcin (rOC) was expressed in a soluble form, and the molecular weight was about 27 kDa. The results are shown in Figure 1

[0040] ​Example 3: Preparation of Anti-fully carboxylated osteocalcin monoclonal antibodies

[0041] Take the fully carboxylated osteocalcin 10-25 segment polypeptide KLH conjugate Gla-OC10-25-K prepared in Example 1 as the immunogen, use 6-8 week old BALB / c female mice, 100 μg / one antigen plus an equal amount of Freund's complete adjuvant, stir thoroughly emulsified, subcutaneous and intraperitoneal injection of mice, immunize 3 mice. The second immunization was carried out at an interval of 4 weeks, and the third immunization was carried out 8 weeks later, respectively 50 μg / one antigen plus incomplete Freund's adjuvant, stir thoroughly emulsified, subcutaneous and intraperitoneal injection of mice. One week after the third immunization, the mouse tail vein blood was taken to detect the titer of immune serum, and the mouse with the highest titer was selected for intraperitoneal booster immunization (50 μg / one). Three days later, the spleen cells were taken for fusion. The SP20 myeloma cells were resuscitated and cultured until they were in the logarithmic growth phase. Take the immunized BALB / c mice, take their spleens to prepare spleen cell suspension. Take the above spleen cells and myeloma cells in a ratio of 9:1, mix them in serum-free DMEM medium, centrifuge at 1500 rpm for 5 minutes, aspirate the supernatant, gently shake to disperse the cells, and fuse in a 37°C water bath. Within 1 minute, add 1 mL of preheated 50% PEG to the fused cells, shake gently while adding, and then let stand for 90 seconds. Add serum-free DMEM medium to terminate fusion, stand at 37°C for 10 min, centrifuge at 1500 rpm for 5 min, suspend the precipitate with HAT medium, and distribute it to 96-well cell plates containing feeder cells. After 5 days of culture in a 37°C, 5% CO2 cell incubator, replace the medium with HAT medium once, and replace it with HAT medium on the 10th day. When the fused cells cover about 60% of the well bottom, take the cell culture supernatant and screen for positive clones using indirect ELISA. The specific method is as follows: carbonate coating buffer dilutes fully carboxylated osteocalcin 10-25 segment polypeptide BSA conjugate Gla-OC10-25-B, the concentration is 2.0 μg / ml, each well is coated with 150 μl, 4°C overnight; wash the plate with washing solution for 2 times; add 200 μl / well blocking solution and incubate at room temperature for 6 hours; wash the plate with washing solution for 5 times. Add 100 μl of sample diluent to each well, then add 10 μl of cell culture supernatant, incubate at room temperature for 30 min, and discard the liquid. Wash the plate 5 times, invert the washed enzyme-labeled plate on the absorbent paper and pat dry, add 100 μl / well of HRP-labeled goat anti-mouse IgG antibody, and incubate at room temperature for 30 min. Wash the plate 5 times. Add 50 μL of TMB color developing liquid A and B to each well, and develop color at room temperature for 15 min. Add 50 μL of 2 M H2SO4 termination liquid to each well to terminate the reaction. Set the enzyme marker detection wavelength to 450 nm, and measure the OD value of each well within 10 minutes after termination. The strong positive clones detected are taken as hybridoma cell strains and cultured in 1640 medium containing 10% fetal bovine serum. Each BALB / c male mouse is injected intraperitoneally with 0.5 mL of liquid paraffin.10 days later, the cells were collected, resuspended with 10 mL of normal saline, and the cell density was adjusted to 1 x 10. 7 After 2 weeks, the ascites were collected. The antibodies were purified by using a Thermo Melon Gel Monoclonal IgG Purification Kit (No. 45214), and the purified antibodies were stored at -20°C after being aliquoted.

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

[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 prepared in Example 1, the prokaryotic-expressed recombinant osteocalcin rOC in Example 2, and the osteocalcin bOC extracted from bovine bone (No. BP-010, purchased from K-Assay Company) were each diluted with carbonate buffer to a concentration of 2.5 μg / mL, 150 μL of each was coated on an enzyme-linked plate, and the plate was incubated at 4°C overnight; the plate was washed twice with washing solution; 200 μL / well of blocking solution was added and the plate was blocked at room temperature for 6 hours; the plate was washed 5 times with washing solution. After 100 μL of sample diluent was added to each well, 10 μL of cell culture supernatant was added to each well, and the plate was incubated at room temperature for 30 min, and the liquid was discarded. The plate was washed 5 times, and the washed plate was inverted on absorbent paper and dried, 100 μL / well of HRP-labeled goat anti-mouse IgG antibody was added, and the plate was incubated at room temperature for 30 min. The plate was washed 5 times. 50 μL of TMB color developing solution A and B was added to each well, and the plate was color developed at room temperature for 15 min in the dark. 50 μL of 2 M H2SO4 was added to each well to terminate the reaction. The detection wavelength of the microplate reader was set to 450 nm, and the OD value of each well was determined within 10 min after termination.

[0044] The results are shown in Table 1. Figure 2 Among the multiple anti-fully-carboxylated osteocalcin monoclonal antibodies prepared in Example 3, one monoclonal antibody that only recognizes fully-carboxylated osteocalcin, i.e., 3A421, was screened. This monoclonal antibody can bind to the fully-carboxylated osteocalcin 10-25 segment polypeptide Gla-OC10-25-B and the osteocalcin bOC extracted from bovine bone (containing a fully-carboxylated osteocalcin molecule), but cannot bind to the incompletely-carboxylated osteocalcin 10-25 segment polypeptide Glu / a-OC10-25-B, the non-carboxylated osteocalcin 10-25 segment polypeptide Glu-OC10-25-B, and the prokaryotic-expressed recombinant osteocalcin rOC (a non-carboxylated osteocalcin molecule).

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

[0046] A healthy male rabbit was selected, and 1.0 mg of the osteocalcin 25-43 segment polypeptide KLH conjugate OC25-43-K prepared in Example 1 was mixed with 1.0 mL of Freund's complete adjuvant, and emulsified thoroughly with a stirrer. Then, 0.2 mL was injected subcutaneously at both sides of the spine of the rabbit. Four weeks later, 1.0 mg of the OC25-43-K immunogen was mixed with 1.0 mL of Freund's incomplete adjuvant, and emulsified thoroughly with a stirrer. Then, the second immunization was performed at different points of the above-mentioned parts. Four weeks later, the third immunization was performed for the preparation of polyclonal antibody serum. One week later, the blood was collected from the heart, and the serum was separated by centrifugation at 5000 rpm for 15 minutes, and then stored in a refrigerator at -20°C for standby. The indirect ELISA method for determining the titer of the polyclonal antibody was performed as follows: the osteocalcin 25-43 segment polypeptide BSA conjugate OC25-43-B was used to coat the enzyme-linked plate, and the concentration was 2.0 μg / ml, 150 μL was coated in each well, and the plate was incubated at 4°C overnight. The plate was washed twice with washing solution. 200 μL / well of blocking solution was added, and the plate was blocked at room temperature for 6 hours. The plate was washed 5 times with washing solution. The rabbit serum was diluted with PBS, and the dilution ratios were 1:2000, 1:8000, 1:32000, 1:128000, 1:512000, 1:1024000, 1:2048000 and 1:4096000, and 100 μL was added to each well. The plate was incubated at 37°C for 45 min. The plate was washed 5 times with 200 μL of washing solution per well. The HRP-labeled goat anti-rabbit secondary antibody was incubated at 37°C for 45 min. The plate was washed 5 times with 200 μL of washing solution per well. Freshly prepared substrate solution was added to each well, 100 μL per well, and the plate was incubated at 37°C for 10 min. 50 μL of 2 M H2SO4 was added to each well to terminate the reaction. The absorbance value of each well was determined by an enzyme-labeled instrument at a wavelength of 450 nm. The reading was taken within 10 min after termination. The results are shown in Figure 3 The pre-immune rabbit serum was used as a negative control. The titer of the polyclonal antibody prepared by immunizing the rabbit with the osteocalcin 25-43 segment polypeptide can reach 1:1024000.

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

[0048] The monoclonal antibody 3A421 which only recognizes the fully carboxylated osteocalcin is used as the capture antibody to specifically capture the fully carboxylated osteocalcin in the sample, and the polyclonal antibody of the rabbit anti-osteocalcin 25-43 segment polypeptide prepared in Example 5 is used as the detection antibody, to establish a specific detection method for the fully carboxylated osteocalcin (Gla-OC) based on the double antibody sandwich method. The method can detect all the fully carboxylated osteocalcin, including the fully carboxylated full-length osteocalcin and the fully carboxylated osteocalcin N-MID. The specific steps are as follows: the enzyme-linked plate is coated with the monoclonal antibody 3A421 which only recognizes the fully carboxylated osteocalcin, the concentration is 2.0 μg / mL, 100 μL per well, 4°C overnight, and the plate is washed with the washing solution for 2 times; 120 μL / well of blocking solution is added for blocking at room temperature for 6 hours, and the plate is washed with the washing solution for 5 times; the bone osteocalcin bOC extracted from the bovine bone and the recombinant osteocalcin rOC expressed in prokaryotes in Example 2 are respectively diluted with PBS in gradient, and the concentrations are 2000, 400, 80, 16, 3.2, 0.64, 0.32, 0.16 ng / mL respectively. 100 μL of each is added to the well, 37°C incubation for 60 min, and the liquid is discarded. The plate is washed with the washing solution for 5 times, 100 μL of the horseradish peroxidase-labeled rabbit anti-osteocalcin 25-43 segment polypeptide polyclonal antibody is added to each well, 37°C incubation for 60 min. The plate is washed for 5 times, dried, 50 μL of TMB color developing liquid A and B is added to each well, and the color is developed at room temperature in the dark for 15 min. 50 μL / well of 2 M H2SO4 stop solution is added to stop the reaction. The OD value of each well is determined by the enzyme label instrument at 450 nm, and the reading value is read within 10 min after the termination.

[0049] The results are shown in Table 1. Figure 4 As shown in Table 1, the double antibody sandwich detection method established only specifically detects the fully carboxylated osteocalcin contained in the bovine bone extracted osteocalcin bOC, and does not have cross reaction with the non-carboxylated prokaryotic expressed recombinant osteocalcin rOC, indicating that the method can be used for specific detection of the fully carboxylated osteocalcin. The critical value is three times the detection value of the negative control (Cut=0.207), and the minimum detection limit of the double antibody sandwich detection method established for the fully carboxylated osteocalcin is 0.32 ng / mL.

[0050] Example 7: Specific detection of fully carboxylated osteocalcin in blood samples

[0051] The 63 postmenopausal women and 63 premenopausal women were detected by the established double antibody sandwich method for detecting fully carboxylated osteocalcin, and the specific steps were as follows: the enzyme-linked plate was coated with the monoclonal antibody 3A421 which only recognized fully carboxylated osteocalcin, the concentration was 2.0 μg / mL, 100 μL per well, 4°C overnight, and the plate was washed twice with the washing solution; 120 μL / well of blocking solution was added and blocked at room temperature for 6 hours, and the plate was washed 5 times with the washing solution; 100 μL of serum sample was taken and added to the well, incubated at 37°C for 60 min, and the liquid was discarded. The plate was washed 5 times with the washing solution, 100 μL of horseradish peroxidase-labeled rabbit anti-osteocalcin 25-43 segment polyclonal antibody was added to each well, and incubated at 37°C for 60 min. The plate was washed 5 times, dried, and 50 μL of TMB color developing liquid A and B was added to each well, and the color was developed at room temperature for 15 min in the dark. 50 μL of 2 M H2SO4 stopping solution was added to each well to stop the reaction. The OD value of each well was detected by an enzyme-labeled instrument at 450 nm, and the reading was taken within 10 min after stopping.

[0052] The results are shown in Figure 5 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 0.479±0.021, and the difference between the two groups was significant (p<0.001), indicating that the method of the application can be used to prepare products for detecting fully carboxylated osteocalcin and assisting the diagnosis of osteoporosis patients.

[0053] Example 8: Subtype analysis of anti-fully carboxylated osteocalcin monoclonal antibody 3A421

[0054] The heavy chain and light chain subtypes of the mouse antibody were identified by the mouse antibody subtype rapid detection card (THJ-ISO-M8a-10 / 20) of Antegene (Beijing) Biotechnology Co., Ltd. First, the antibody was diluted to 1 μg / mL with PBS, then 100 μl of the diluted antibody was added to each well, and the results were observed and recorded after standing for 5-10 min. The results are shown in Figure 6 The anti-fully carboxylated osteocalcin monoclonal antibody 3A421 was of mouse IgG2a subtype, and the antibody light chain was of Igκ subtype.

[0055] Example 9: Determination of variable region sequence of anti-fully carboxylated osteocalcin monoclonal antibody 3A421

[0056] The mouse hybridoma cell line 3A421 was cultured, total RNA of the hybridoma cells was extracted by Trizol method, after reverse transcription of cDNA, PCR amplification was performed using the mouse monoclonal antibody Fab segment primer sequence synthesized by Beijing Genki Biotechnology Co., Ltd., 95°C preheating for 2 min, 95°C for 30 seconds, 58°C for 30 seconds, 72°C for 30 seconds for 30 cycles, and finally 72°C for 5 min, ligated with pMD18-T vector, transformed into E. coli JM109, and positive clones were selected for sequencing. The determination sequence was compared and analyzed in the IgBLAST (https: / / www.ncbi.nlm.nih.gov / igblast / ) module in the BLAST module of the NCBI website.

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

7.

2. The monoclonal antibody or its antigen-binding fragment 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 mouse hybridoma cell line 3A421 with accession number CGMCC No. 46547.

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

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

6. An expression carrier, characterized in that, It comprises the nucleic acid molecule as described in claim 5.

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

8. The recombinant cell according to claim 7, characterized in that, It is a recombinant of bacteria or yeast.

9. A hybridoma cell line that secretes a monoclonal antibody that specifically recognizes fully carboxylated osteocalcin, characterized in that, It is the mouse hybridoma cell line 3A421 with accession number CGMCC No. 46547.

10. The use of the monoclonal antibody or antigen-binding fragment thereof that specifically recognizes fully carboxylated osteocalcin as described in any one of claims 1 to 4 in the preparation of a product for detecting fully carboxylated osteocalcin.

11. A kit for detecting fully carboxylated osteocalcin, characterized in that, It comprises the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 4.

12. The kit according to claim 11, characterized in that, It is a detection kit using a double-antibody sandwich method, wherein the monoclonal antibody or its antigen-binding fragment serves as the capture antibody.

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