Osteoprotectin recombinant protein, monoclonal antibody and application thereof

Abstract

The invention provides an osteoprotegerin recombinant protein, a monoclonal antibody and application of the osteoprotegerin recombinant protein and the monoclonal antibody, and relates to the technical field of biological detection, and the amino acid sequence of the recombinant protein is as shown in SEQ ID No.1. The invention provides the osteoprotegerin recombinant protein, a monoclonal antibody pair with high specificity is screened on the basis of the osteoprotegerin recombinant protein, an osteoprotegerin double-antibody sandwich method is established, the osteoprotegerin protein can be specifically detected, the sensitivity is high, a simple and convenient biological detection method is provided, and the application prospect is relatively good.

Description

Technical Field

[0001] This invention relates to the field of biodetection technology, and in particular to a recombinant osteoprotein protein, a monoclonal antibody, and their applications. Background Technology

[0002] Osteoprotegerin (OPG), also known as osteoprotective protein and osteoclastogenesis inhibitor, is a new member of the tumor necrosis factor (TNF) receptor family. It was initially discovered simultaneously by two independent laboratories in 1997 and is also called osteoclastogenesis inhibitory factor (OCIF). The main function of osteoprotegerin is to influence bone metabolism, regulating it through the OPG / RANK / RANK ligand system. Specifically, OPG competitively binds to RANKL, preventing the mutual binding of RANK and RANKL, thereby inhibiting osteoclast differentiation and promoting apoptosis of mature osteoclasts.

[0003] In recent years, with extensive and in-depth collaboration among medical researchers, molecular biologists, and pharmaceutical researchers, the mechanism of action of osteoprotegerin in bone metabolism has been discovered. In addition, osteoprotegerin plays a crucial role in the treatment of rheumatoid arthritis, osteoporosis, osteosclerosis, atherosclerosis, diabetes, immune system diseases, and tumors, and its clinical applications are widespread and increasingly attracting attention. Osteoporosis is considered a regulator of vascular calcification in atherosclerotic plaques, related to vascular smooth muscle cell proliferation and collagen production. Therefore, OPG serves as a potential biomarker for calcified carotid artery plaques and carotid artery stenosis. Clinically, elevated OPG levels are commonly seen in patients with rheumatoid arthritis, ankylosing spondylitis, lung cancer, diabetes, and prostate cancer; serum OPG levels in postmenopausal women are positively correlated with age. Decreased OPG levels are mainly seen in osteolytic destruction caused by tumor metastasis.

[0004] Osteoprotectin not only participates in bone metabolism but also in vascular calcification, making it significant for the detection of cardiovascular and cerebrovascular diseases. In recent years, with the increase in diseases such as atherosclerosis and coronary heart disease, the development of osteoprotectin reagent kits has become crucial. Summary of the Invention

[0005] In view of this, the present invention proposes a recombinant osteoprotegerin protein, a monoclonal antibody, and their applications. The present invention utilizes bioinformatics software to select a segment of osteoprotegerin sequence, which, after predictive analysis, can be expressed in *E. coli* to obtain the recombinant protein. Following immune screening, specific monoclonal antibody pairs for osteoprotegerin can be obtained. Based on this, a double-antibody sandwich ELISA system is established, which can be applied to detect osteoprotegerin for purposes other than disease diagnosis and treatment.

[0006] The technical solution of this invention is implemented as follows: In a first aspect, the present invention provides a recombinant protein for preparing a monoclonal antibody targeting osteoprotegerin, the amino acid sequence of which is shown in SEQ ID No. 1: MNNLLCQVTVFLDISIKWTTQETFPPKYLHYDKETSHQLLCDKCPPGTYLKQHCTAKWKTVCAPCPDHYYTDSWHTSDECLYCSPVCKELQYVKQECNRTHNRVCECKEGRYLEIEFCLKHRNLGPGFGVVQAGTPERNTVCKRCPDGFFSNETSSKAPCRKHTNSSQFGLLLTQKGNATHDNICSGNSESTQKCGIDVTL CEEAFFRFAVPTKFTVCKLSVLVDNLPGTKVNAESVERIKRQHSSQEQTFQLLKLWKHQNKDQDIVKKIIQDIDLCENSVQRHIGHANLTFEQLRSLMES LPGKKVGAEDIEKTIKACKPWKTILKLLSLWRIKNGDQDTLKGLMHALKHSKTYHFGNTVTQSLKKTIRFLHSFTMYKLPKKLFLEMIGNQVQSVKISCL.

[0007] Based on the above technical solutions, the recombinant protein further includes an amino acid sequence with the same function obtained by substituting, deleting and / or adding one or more amino acids to the amino acid sequence of the recombinant protein.

[0008] Based on the above technical solution, the nucleotide sequence of the recombinant protein is further shown in SEQ ID No. 2:

[0009] Secondly, the present invention also provides the use of the recombinant protein in the preparation of monoclonal antibodies targeting osteoprotegerin.

[0010] Thirdly, the present invention provides a biomaterial for preparing a monoclonal antibody targeting osteoprotegerin, wherein the biomaterial contains the recombinant protein.

[0011] Based on the above technical solutions, the biomaterials further include recombinant bacteria, expression vectors, and / or cells.

[0012] Fourthly, the present invention provides a monoclonal antibody targeting osteoprotein, wherein the monoclonal antibody is obtained by immunizing animals with the recombinant protein as an immunogen.

[0013] Based on the above technical solutions, the monoclonal antibody further includes the coating antibody OPG-5G9 and the detection antibody OPG-7D9; The amino acid sequences of the complementarity-determining region of the heavy chain variable region of the coated antibody OPG-5G9 are GYGGNSDRA, ITGSGSN, and VRGQNFAT, respectively. The amino acid sequences of the complementarity-determining region of the light chain variable region of the coated antibody OPG-5G9 are EQDDSRTNST, YKRSN and SEQ ID No. 3: QQKGNDPGGTGRG; The amino acid sequences of the complementarity-determining region of the heavy chain variable region of the detection antibody OPG-7D9 are GRTFDSYA, RGTNSVTF and SEQ ID No. 4: VQPQASTSFTN, respectively. The amino acid sequences of the complementarity-determining region of the light chain variable region of the detection antibody OPG-7D9 are QGRDNTL, YYTVT, and QTGDDRGWT, respectively.

[0014] Based on the above technical solution, the amino acid sequence of the heavy chain variable region of the coated antibody OPG-5G9 is shown in SEQ ID No. 5: LELQQSGPGLVKPSQSLSLTCTVTGYGGNSDRAWNWIRQFPGNRLEWMGYITGSGSNSYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCVRGQNFATWGKGTLVTVSA; The amino acid sequence of the light chain variable region of the coated antibody OPG-5G9 is shown in SEQ ID No. 6: MTQSPASLAVSLGQRAPYPCRASEQDDSRTNSTLHWYQQKPGQPPKLLIYKRSNLQSGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQKGNDPGGTGRGTKLEIK; The amino acid sequence of the heavy chain variable region of the detection antibody OPG-7D9 is shown in SEQ ID No. 7: EVQLVESGGGLMQPKGSLKLSCAASGRTFDSYALNWVRQAPGTGLEWVARIRGTSNSVTFYYADSVKDRFTISRDDSQNMLYLQMNNLKTEDTAMYYCVQPQASTSFTNWGQGTLVTVSA; The amino acid sequence of the light chain variable region of the detection antibody OPG-7D9 is shown in SEQ ID No. 8: MTQTPSSLSASLGDRVTISCRASQGRDNTLNWYQQGPGGTVKVLIYYTVTLHSGVPSRFSGSGSGTDYSLTISNLEQEDFATYFCQTGDDRGWTFGGGTKLEIK.

[0015] Fifthly, the present invention provides a nucleic acid molecule encoding the monoclonal antibody, the nucleic acid molecule comprising a heavy chain variable region and a light chain variable region encoding the coated antibody OPG-5G9 and the detection antibody OPG-7D9; The nucleotide sequence encoding the heavy chain variable region of the coated antibody OPG-5G9 is shown in SEQ ID No. 9: ctggaactgcagcagagcggcccgggcctggtgaaaccgagccagagcctgagcctgacctgcaccgtgaccggctatggcggcaacagcgatcgcgcgtggaactggattcgccagtttccgggcaaccgcctggaatggatgggctatattaccggcagcggcagcaacagctataacccgagcctgaaaagccgcattagcattacccgcgataccagcaaaaaccagttttttctgcagctgaacagcgtgaccaccgaagataccgcgacctattattgcgtgcggccagaactttgcgcg; The nucleotide sequence encoding the light chain variable region of the coated antibody OPG-5G9 is shown in SEQ ID No. 10: atgacccagagcccggcgagcctggcggtgagcctgggccagcgcgcgccgtatccgtgccgcgcgagcgaacaggatgatagccgcaccaacagcaccctgcattggtatcagcagaaaccgggccagccgccgaaactgctgatttataaacgcagcaacctgcagagcggcattccggcgcgctttagcggcagcggcagccgcaccgattttaccctgaccattaacccggtggaagcggatgatgtggcgacctattattgccagcagaaaggcaacgatccgggcggcgctttagcggcagcggcagccgcaccgattttaccctgaccattaacccggtggaagcggatgatgtggcgacctattattgccagcagaaaggcaacgatccgggcggcaccggccgcggcaccaaactggaaattaaa; The nucleotide sequence encoding the heavy chain variable region of the detection antibody OPG-7D9 is shown in SEQ ID No. 11: gaagtgcagctggtggaaagcggcggcggcctgatgcagccgaaaggcagcctgaaactgagctgcgcggcgagcggccgcacctttgatagctatgcgctgaactgggtgcgccaggcgccgggcaccggcctggaatgggtggcgcgcattcgcggcaccagcaacagcgtgaccttttattatgcggatagcgtgaaagatcgctttaccattagccgcgatgatagccagaacatgctgtatctgcagatgaacaacctgaaaaccgaagataccgcgatgtattattgcgtgcagccgcaggcgagcaccagctttaccaactggggccagggcaccctggtgaccgtgagcgcg; The nucleotide sequence encoding the light chain variable region of the detection antibody OPG-7D9 is shown in SEQ ID No. 12: atgacccagaccccgagcagcctgagcgcgagcctgggcgatcgcgtgaccattagctgccgcgcgagccagggccgcgataacaccctgaactggtatcagcagggcccgggcggcaccgtgaaagtgctgatttattataccgtgaccctgcatagcggcgtgccgagccgctttagcggcagcggcagcggcaccgattatagcctgcaacctggaacaggaagagattttgcgacctatttttgccagaccggcgatgatcgcggctggacctttggcggcggcaccaaactggaaattaaa.

[0016] Sixthly, the present invention provides an application of the monoclonal antibody, used in the preparation of products for detecting osteoprotegerin, or for detecting osteoprotegerin for purposes other than disease diagnosis and treatment.

[0017] In a seventh aspect, the present invention provides a detection product comprising the monoclonal antibody, the detection product being used to detect osteoprotegerin not for the purpose of disease diagnosis and treatment.

[0018] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a recombinant osteoprotein protein, and based on this, screens out highly specific monoclonal antibody pairs, and establishes a double-antibody sandwich method for osteoprotein, which can specifically detect osteoprotein protein with high sensitivity. It provides a simple and convenient biological detection method with good application prospects. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 Electrophoresis diagram of osteoprotegerin OPG recombinant protein; Figure 2 The graph shows the plot of the osteoprotegerin ELISA kit. Detailed Implementation

[0021] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0022] In the following specific implementation methods, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0023] Example 1: A recombinant osteoprotegerin protein and its preparation method This embodiment provides a recombinant osteoprotein protein and its preparation method. The amino acid sequence of the recombinant protein is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2.

[0024] The preparation method includes the following steps: (1) Constructing plasmids Based on the NCBI database and bioinformatics analysis software, major antigenic epitopes containing osteoprotegerin (OPG) were selected for expression. The amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2. The PCR program was as follows: 98°C pre-denaturation for 3 min, 98°C denaturation for 10 s, 52°C annealing for 15 s, 72°C extension for 1 min, and 72°C incubation for 10 min, repeated for 30 cycles.

[0025] After PCR, the cells were centrifuged, and the PCR products were electrophoresed on a 1% agarose gel at 80 V for 1 h. The gel was then excised and the products were recovered. The recovered products and the pET-28a vector were digested with EcoRI and NotI, respectively. Homologous recombination of the purified OPG gene and the pET-28a fragment was performed, and the recombinant product was transformed into E. coil DH5α competent cells. The transformed product was plated on LB agar plates containing kanamycin and incubated at 37°C for 10 h.

[0026] A single colony was inoculated into a 50 mL Erlenmeyer flask containing 10 mL of LB medium and incubated at 37°C and 200 r / min for approximately 12 h in a shaker. 4 mL of the bacterial culture was transferred to a 2 mL centrifuge tube (repeated twice), centrifuged at 12000 r / min for 1 min, and the supernatant was discarded, retaining the bacterial cells. Plasmids were extracted using a kit (AxyPrep), following the instructions. Plasmid DNA was eluted with 70 μL of preheated Elution Buffer (65°C), and the concentration was determined. The extracted plasmid was then identified by PCR and sequencing. The correctly identified plasmid was named pET-28a-HOPG.

[0027] (2) Induced expression 50 ng of pET-28a-HOPG recombinant plasmid was transformed into E. coli BL21(DE3) competent cells and plated on LB agar plates containing kanamycin.

[0028] Single colonies were picked and inoculated into LB liquid medium containing kanamycin and cultured at 37°C with shaking. The bacterial suspension was then inoculated into 10 mL of LB liquid medium containing kanamycin at a ratio of 1:100 and cultured at 37°C and 200 r / min in a shaking incubator for about 3 h until the OD600 nm value was between 0.6 and 0.8. IPTG was then added to a final concentration of 0.1 mmol / L and induced at 37°C and 200 r / min for 6 h.

[0029] After induction, the bacterial culture was collected by centrifugation at 8000 r / min for 10 min at 4℃. The bacteria were resuspended in 1 mL of sterile PBS and transferred to a 1.5 mL centrifuge tube. 200 μL was taken as the bacterial culture sample, and the remaining bacterial culture was sonicated. The 1.5 mL centrifuge tube containing the bacterial culture was placed in an ice-water mixture and then placed in an ultrasonic homogenizer. The homogenization conditions were: 20% power, 4 s operation, 6 s pause, and 2 min homogenization.

[0030] After disruption, centrifuge at 12000 r / min for 15 min at 4℃. Take 200 μL of supernatant and add 50 μL of 5×Loading Buffer. Discard the remaining supernatant. Resuspend the precipitate in 200 μL of 1×Loading Buffer. Add 50 μL of 5×Loading Buffer to the previously prepared whole bacterial samples as well.

[0031] Three samples—whole bacteria, supernatant, and precipitate—were boiled in a 95°C water bath for 10 min, centrifuged at 12000 r / min for 1 min at 4°C, and then subjected to SDS-PAGE to determine whether the target protein was expressed in the form of inclusion bodies.

[0032] (3) Protein purification The pET-28a-HOPG expression strain was expanded and induced to express protein in large quantities. After cell disruption, the protein was purified using affinity chromatography with a Protein A column. The purified protein sample was identified by SDS-PAGE. The purified protein was confirmed to be recombinant osteoprotegerin OPG protein, as shown in the electrophoresis image below. Figure 1 As shown.

[0033] Example 2 Screening of monoclonal antibodies This embodiment provides a method for obtaining monoclonal antibodies based on the recombinant protein osteoprotegerin OPG screening, including the following steps: (1) Mouse immunization The purified OPG recombinant protein expressed in Example 1 was mixed with an adjuvant in equal volume and emulsified. Two 6-week-old female Balb / c mice (100 μg / mouse) were immunized by subcutaneous injection at multiple points on the back of the neck. Freund's complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for subsequent booster immunizations. Each immunization was 3 weeks apart, for a total of 4 immunizations. Blood was collected from the tail on day 21 after the 4th immunization.

[0034] The serum titer of immunized mice was detected by coating ELISA plates with purified recombinant OPG protein (10 μg / mL) and using indirect enzyme-linked immunosorbent assay (iELISA). The results are shown in Table 1. As can be seen from Table 1, mouse No. 2 had a relatively high titer, and mouse No. 2 was selected for subsequent experiments.

[0035] Table 1 Serum iELISA titers of immunized mice

[0036] The results showed that mouse No. 2 had the highest titer. Mice were given booster immunization by intraperitoneal injection of antigen at twice the immunization dose without adjuvant. Seven days later, the spleens of the mice were aseptically harvested for cell fusion.

[0037] The specific operating procedure is as follows: Sacrifice the mice and dissect the spleens. Grind the spleen cells to prepare a cell suspension. Mix the immunized mouse spleen cell suspension ( ) with the Sp2 / 0 myeloma cell suspension ( ), and centrifuge at 1000 r / min for 10 min. Discard the supernatant, gently shake the centrifuge tube to loosen the cells. In a 37℃ water bath, add 1 mL of preheated fusion promoter PEG while stirring. Add preheated RPMI-1640 / HAT / 10% FBS culture medium, and seed the cells into 100 μL per well of a 96-well plate containing feeder cells. Incubate at 37℃ in a 5% CO2 cell culture incubator.

[0038] (2) Screening of hybridoma cell lines On day 10 of culture, the hybridoma cell supernatant was tested using an indirect ELISA method. Hybridoma cells with positive results were transferred to 24-well cell plates for further culture for 4 days. Wells with strong positive results were selected for limiting dilution three times. Finally, healthy monoclonal hybridoma cells were selected, expanded, centrifuged, collected, resuspended in serum-free cell cryopreservation medium, and frozen in liquid nitrogen for later use.

[0039] (3) Production of monoclonal antibodies using in vivo ascites induction method Ten-week-old female Balb / c mice were pre-sensitized by intraperitoneal injection of incomplete Freund's adjuvant (0.5 mL / mouse); three days later, the expanded hybridoma cell lines were collected, washed twice with sterile PBS, and finally resuspended in 0.5 mL of sterile PBS. The cells were then injected intraperitoneally into the mice. After approximately 14 days of observation, when the mice's abdomens were noticeably distended, ascites fluid was aseptically collected, centrifuged at 5000 rpm for 15 minutes, and the supernatant was collected. Monoclonal antibodies were purified using Protein A affinity chromatography and stored at -20°C for later use.

[0040] (4) Identification of monoclonal antibody activity The osteoprotein OPG recombinant protein was coated using an indirect ELISA method. The purified antibody was then subjected to ELISA to determine its affinity. The antibody was diluted to 1 μg / ml. The results are shown in Table 2. Table 2 shows that different antibodies exhibited different binding activities, with antibodies 1 and 6 showing relatively stronger binding activities.

[0041] Table 2. Osteoporosis monoclonal antibody binding activity

[0042] Example 3: Establishment of an ELISA detection method based on the monoclonal antibody This embodiment provides an ELISA detection method for obtaining monoclonal antibodies based on the aforementioned screening, comprising the following steps: (1) Coating of ELISA plate The purified monoclonal antibodies were diluted to 2 μg / mL with CBS buffer (1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L of pure water, pH 9.6), and 100 μL / well was used for coating. The solutions were incubated overnight at 4 °C.

[0043] The next day, remove the coated microplate, equilibrate to room temperature, discard the coating solution, wash the plate twice with PBST, and pat dry.

[0044] Use PBS solution containing 3% BSA and 3% sucrose as the blocking solution, 200 μL / well for blocking, at 37°C for 1 h. Discard the blocking solution, pat dry, and vacuum seal for storage at -20°C.

[0045] (2) HRP-labeled antibody The monoclonal antibody was HRP-labeled using the sodium periodate method. After labeling, the antibody was dialyzed with PBS buffer, collected, and PBS buffer was added. Glycerol was then added to bring the final concentration to 0.5 mg / mL.

[0046] (3) Antibody screening The obtained antibody pairs were cross-paired to screen for the optimal reaction conditions, which were then used to establish the ELISA detection method. Some cross-pairing results are shown in Table 3. Table 3 shows that OPG-5G9 as the coating antibody and OPG-7D9 as the detection antibody resulted in good protein and sample recognition. The other pairing combinations did not show ideal sample recognition.

[0047] Table 3 Results of cross-detection with different monoclonal antibodies

[0048] The optimal pairing combination was selected through cross-pairing: coating antibody OPG-5G9 and detection antibody OPG-7D9.

[0049] The full-length amino acid sequence of the heavy chain (H chain) variable region of the monoclonal antibody OPG-5G9 is shown in SEQ ID No. 5, and the nucleotide sequence is shown in SEQ ID No. 9; the full-length amino acid sequence of the light chain (L chain) variable region is shown in SEQ ID No. 6, and the nucleotide sequence is shown in SEQ ID No. 10.

[0050] The full-length amino acid sequence of the heavy chain (H chain) variable region of the monoclonal antibody OPG-7D9 is shown in SEQ ID No. 7, and the nucleotide sequence is shown in SEQ ID No. 11; the full-length amino acid sequence of the light chain (L chain) variable region is shown in SEQ ID No. 8, and the nucleotide sequence is shown in SEQ ID No. 12.

[0051] (4) Preparation of double-antibody sandwich reagent kit The optimal reaction conditions were determined by adjusting the coating concentration and the HRP-labeled antibody concentration.

[0052] The specific operating steps are as follows: Dilute with CBS buffer to 0.5, 1, or 2 μg / mL, coat 100 μL / well, and incubate overnight at 4°C.

[0053] The next day, remove the coated microplate, equilibrate to room temperature, discard the coating solution, wash twice with PBST, and pat dry. Block with 200 μL / well of PBS solution containing 3% BSA and 3% sucrose, at 37°C for 1 hour. Discard the blocking solution and pat dry.

[0054] The recombinant osteoprotein protein was diluted to 1000 pg / mL with PBS, and then serially diluted to concentrations of 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL, 31.25 pg / mL, and 15.63 pg / mL. The diluted protein was added to an ELISA plate coated with monoclonal antibody at a rate of 50 μL / well, and parallel replicates were set. Dilute the HRP-labeled antibody proportionally and add 50 μL / well to the microplate. Gently shake the plate to mix the protein and antibody thoroughly. Incubate at 37°C for 1 hour. After the reaction time, shake off the liquid from the plate, wash the plate 5 times with PBST, pat dry, and add 50 μL / well of TMB chromogenic buffer. Incubate at 37°C for 15 minutes. After the incubation period, add 50 μL / well of stop buffer and take an OD reading at 450 nm.

[0055] The optimal coating conditions were selected: 2 μg / mL and HRP-labeled antibody concentration of 0.5 μg / mL. Specific linearity data are shown in Table 4, and the standard curve is shown below. Figure 2 As shown, the linearity was 0.9998 when fitted using Origin software.

[0056] Table 4. Curve data

[0057] (5) Validation of performance parameters of double-antibody sandwich reagent kit Based on the calibrated kit parameters, sensitivity, recovery rate, intra- and inter-batch precision, and specificity were tested. The specific validation results are shown in Table 5-7.

[0058] As shown in Table 5, this double-antibody sandwich method kit has high sensitivity, strong specificity, and good reproducibility.

[0059] As shown in Table 6, the double-antibody sandwich method reagent kit has a good recovery rate, ensuring the accuracy of the test.

[0060] As shown in Table 7, the intra-plate and inter-plate precision of this double-antibody sandwich method reagent kit is within 10%, indicating good repeatability.

[0061] Table 5. Reagent linearity and sensitivity

[0062] Table 6. Reagent kit dilution recovery rate detection

[0063]

[0064] Table 7. Intra- and Inter-assay Precision Detection Range of the Reagent Kit

[0065] In summary, this invention provides a recombinant osteoprotein protein, a monoclonal antibody, and their applications. Based on the recombinant osteoprotein protein, highly specific monoclonal antibody pairs were screened, and a double-antibody sandwich method for osteoprotein was established. This method can specifically detect osteoprotein protein with high sensitivity, providing a simple and convenient biological detection method with good application prospects.

[0066] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A recombinant protein for preparing monoclonal antibodies targeting osteoprotegerin, characterized in that, The amino acid sequence of the recombinant protein is shown in SEQ ID No.

1.

2. The recombinant protein for preparing a monoclonal antibody targeting osteoprotegerin as described in claim 1, characterized in that, The nucleotide sequence of the recombinant protein is shown in SEQ ID No.

2.

3. The use of the recombinant protein as described in any one of claims 1 to 2 in the preparation of monoclonal antibodies targeting osteoprotegerin.

4. A biomaterial for preparing monoclonal antibodies targeting osteoprotegerin, characterized in that, The biomaterial contains the recombinant protein as described in any one of claims 1 to 2.

5. A monoclonal antibody targeting osteoprotegerin, characterized in that, The monoclonal antibody is obtained by immunizing animals with the recombinant protein as described in any one of claims 1 to 2.

6. The monoclonal antibody targeting osteoprotegerin as described in claim 5, characterized in that, The monoclonal antibody includes the coating antibody OPG-5G9 and the detection antibody OPG-7D9; The amino acid sequences of the complementarity-determining region of the heavy chain variable region of the coated antibody OPG-5G9 are GYGGNSDRA, ITGSGSN, and VRGQNFAT, respectively. The amino acid sequences of the complementarity-determining region of the light chain variable region of the coated antibody OPG-5G9 are EQDDSRTNST, YKRSN and SEQ ID No. 3, respectively; The amino acid sequences of the complementarity-determining region of the heavy chain variable region of the detection antibody OPG-7D9 are GRTFDSYA, RGTNSVTF and SEQ ID No. 4, respectively. The amino acid sequences of the complementarity-determining region of the light chain variable region of the detection antibody OPG-7D9 are QGRDNTL, YYTVT, and QTGDDRGWT, respectively.

7. The monoclonal antibody targeting osteoprotegerin as described in claim 6, characterized in that, The amino acid sequence of the heavy chain variable region of the coated antibody OPG-5G9 is shown in SEQ ID No. 5, and the amino acid sequence of the light chain variable region of the coated antibody OPG-5G9 is shown in SEQ ID No.

6. The amino acid sequence of the heavy chain variable region of the detection antibody OPG-7D9 is shown in SEQ ID No. 7; The amino acid sequence of the light chain variable region of the detection antibody OPG-7D9 is shown in SEQ ID No.

8.

8. A nucleic acid molecule encoding a monoclonal antibody as described in any one of claims 5 to 7, characterized in that, The nucleic acid molecule includes a nucleic acid molecule encoding the heavy chain variable region and the light chain variable region of the coating antibody OPG-5G9 and the detection antibody OPG-7D9; The nucleotide sequence encoding the heavy chain variable region of the coated antibody OPG-5G9 is shown in SEQ ID No. 9, and the nucleotide sequence encoding the light chain variable region of the coated antibody OPG-5G9 is shown in SEQ ID No. 10; the nucleotide sequence encoding the heavy chain variable region of the detection antibody OPG-7D9 is shown in SEQ ID No. 11, and the nucleotide sequence encoding the light chain variable region of the detection antibody OPG-7D9 is shown in SEQ ID No.

12.

9. The application of the monoclonal antibody according to any one of claims 5 to 7, characterized in that, It can be used to prepare products for detecting osteoprotegerin, or for detecting osteoprotegerin when the purpose is not for disease diagnosis or treatment.

10. A testing product, characterized in that, The detection product includes the monoclonal antibody according to any one of claims 5 to 7, and the detection product is used to detect osteoprotegerin for purposes other than disease diagnosis and treatment.

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