Alpha KL protein antibody and application thereof

Abstract

The invention relates to the technical field of antibodies, in particular to an alpha KL protein antibody and application thereof. According to the alpha Klotho antibody, the sequence of a heavy chain CDR1 is as shown in SEQ ID NO.1 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence as shown in SEQ ID NO.1, the sequence of a heavy chain CDR2 is as shown in SEQ ID NO.2 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence as shown in SEQ ID NO.2, and the sequence of a heavy chain CDR3 is as shown in SEQ ID NO.3 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence as shown in SEQ ID NO.3. The alpha Klotho antibody with high sensitivity and specificity is obtained, and the performance of the alpha Klotho antibody is obviously superior to that of an existing KL antibody.

Description

Technical Field

[0001] This invention relates to the field of antibody technology, specifically to an αKL protein antibody and its applications. Background Technology

[0002] The KL gene, also known as the klotho gene, is located on human chromosome 13q13.1. The protein encoded by this gene belongs to the Glycoside hydrolase family 1, a class of proteins associated with glycoside hydrolases, including αKL and βKL proteins. The protein products of the KL gene play important roles in cells, particularly in the regulation of the extracellular matrix and calcification. KL proteins can inhibit the calcification process, thereby protecting cells and tissues from calcification damage. This is crucial not only for bone health but also for the normal function of the cardiovascular system. Intracellularly, KL proteins also participate in various signaling pathways, such as the insulin-like growth factor (IGF) signaling pathway. Through these pathways, KL proteins regulate cellular growth, differentiation, and metabolic processes.

[0003] Currently, some KL antibodies and diagnostic kits are available on the market, but existing KL antibodies lack specificity and cannot effectively immunoprecipitate KL from human serum. Current immunoassay-based KL detection methods are costly and lack sufficient sensitivity and specificity. Overcoming the insufficient sensitivity and specificity of KL protein antibodies is a primary task in solving the clinical application of KL protein detection.

[0004] Existing technology US10228374B2 provides a specific antibody, sb106, with a dissociation constant of approximately 2 nM or less. This antibody binds to the α-Klotho peptide and utilizes synthetic antibody technology to introduce sequence diversity into the complementarity-determining region (CDR), enabling accurate immunoprecipitation and detection (CDR) in human serum and urine. It can also bind to phage display to achieve high affinity and specificity. However, this antibody, sb106, can only recognize the native conformation of KL and cannot recognize denatured KL protein, thus limiting its detection capabilities. Summary of the Invention

[0005] The purpose of this invention is to provide an αKL protein antibody with significantly enhanced sensitivity and specificity and its application in the detection of KL protein.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: An αKlotho antibody includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes heavy chain CDR1 (CDR-H1), heavy chain CDR2 (CDR-H2), and heavy chain CDR3 (CDR-H3), and the light chain variable region includes light chain CDR1 (CDR-L1), light chain CDR2 (CDR-L2), and light chain CDR3 (CDR-L3), wherein: The sequence of heavy chain CDR1 is shown in SEQ ID NO.1, the sequence of heavy chain CDR2 is shown in SEQ ID NO.2, the sequence of heavy chain CDR3 is shown in SEQ ID NO.3, the sequence of light chain CDR1 is shown in SEQ ID NO.4, the sequence of light chain CDR2 is shown in SEQ ID NO.5, and the sequence of light chain CDR3 is shown in SEQ ID NO.6.

[0007] SEQ ID NO.1: SGFTFSNF.

[0008] SEQ ID NO. 2: LEWVAYITETGGRTYYP.

[0009] SEQ ID NO.3: GFDYT.

[0010] SEQ ID NO.4: QSLLHNSGDT.

[0011] SEQ ID NO.5: PKLLIYKVSNRF.

[0012] SEQ ID NO.6: STHYPW.

[0013] The heavy chain CDR and light chain CDR sequences are defined according to Kabat.

[0014] According to embodiments of the present invention, the present invention can be further optimized, and the optimized technical solution is as follows:

[0015] In one preferred embodiment, the αKlotho antibody includes a heavy chain variable region and a light chain variable region, the heavy chain variable region having the amino acid sequence shown in SEQ ID NO. 7; and / or the light chain variable region having the amino acid sequence shown in SEQ ID NO. 8.

[0016] SEQ ID NO.7: EVQLAESGGGLQQPGGSLKLSCAASGFTFSNFYMAWVRQTPEKRLEWVAYITETGGRTYYPDTVKGRFTISRDDAKNTLYLEMSSLRSEDTATYYCSRGFDYTATLLDYWGQ GVTVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIK PCPPCKCPAPNLLGGPSVFIFPPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERT ISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK.

[0017] SEQ ID NO.8: DVVMTQTPLSLPVSLGDQASLSCRSSQSLLHNSGDTYLHWYLQRPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEPEDLGVYFCSQSTHYPWTFGAGTKLE IKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.

[0018] Based on the same inventive concept, the present invention also claims protection for a nucleic acid molecule that encodes the αKlotho antibody.

[0019] Based on the same inventive concept, the present invention also claims protection for a carrier containing the aforementioned nucleic acid molecule.

[0020] Based on the same inventive concept, the present invention also claims an αKlotho protein detection kit containing the αKlotho antibody or the nucleic acid molecule described herein.

[0021] Based on the same inventive concept, the present invention also claims protection for the application of the αKlotho antibody, the nucleic acid molecule, the vector, or the αKlotho protein detection kit for assessing the αKlotho protein level in an individual's serum sample.

[0022] Based on the same inventive concept, the present invention also claims a reagent for detecting αKlotho protein, comprising the αKlotho antibody, the nucleic acid molecule, the vector, or the αKlotho protein detection kit.

[0023] In a preferred embodiment of the invention, the assessment is based on the detection of αKlotho protein content in the serum sample of the individual being tested.

[0024] Based on the same inventive concept, the present invention also claims protection for the use of the αKlotho antibody, the nucleic acid molecule, the vector, or the αKlotho protein detection kit as a reagent for the preparation of a detection reagent for chronic kidney disease.

[0025] Based on the same inventive concept, the present invention also claims a reagent for diagnosing chronic kidney disease, comprising the αKlotho antibody, the nucleic acid molecule, the carrier, or the αKlotho protein detection kit.

[0026] Compared with the prior art, the beneficial effects of the present invention are: This invention involves immunizing mice with the full-length KL protein, combined with flow cytometry screening and hybridoma technology. The selected high-affinity KL-binding specific antibodies are sequenced to identify the CDR region binding to KL. By constructing expression vectors for the light and heavy chains, these antibodies are converted into full-length IgG from mammalian cells, resulting in a highly sensitive and specific αKlotho antibody. This antibody significantly outperforms existing KL antibodies and can be applied to the clinical detection of chronic kidney disease. Attached Figure Description

[0027] Figure 1 This is the result of ELISA screening for hybridoma cells; among them, Figure 1 In the image, 'a' represents an ELISA image of 96 hybridoma cells. Figure 1 In the graph, b is a bar chart showing the absorbance values ​​of five clones capable of producing KL antibodies at 450 nm. Figure 1 In the image, c represents the ELISA results after serial dilution of five clones capable of producing KL antibodies. Figure 1 The d in the figure is a bar graph showing the absorbance values ​​of five clones that can produce KL antibodies after gradient dilution at 450 nm.

[0028] Figure 2This is a chemiluminescent imaging image obtained by Western blotting of KL antibody specific recognition of endogenous (KL) and exogenous (KL-GFP) KL protein.

[0029] Figure 3 This is a chemiluminescent imaging image showing the sensitivity of each KL antibody to recognize the KL protein as detected by Western blotting.

[0030] Figure 4 This is the result of ELISA testing of the sensitivity of each KL antibody to recognize the KL protein, among which... Figure 4 In the image, 'a' represents the chromogenic image of each antibody on the ELISA plate. Figure 4 In the graph, b represents the bar chart of the ELISA detection results for each antibody.

[0031] Figure 5 This is a bar chart showing the results of KL protein level detection in the serum of CKD mice. Figure 5 In the figure, 'a' represents a bar chart showing the level of KL protein in the serum of UUO model mice. Figure 5 In the figure, b is a bar chart showing the level of KL protein in the serum of 5 / 6 nephrectomy model mice. Detailed Implementation

[0032] This invention is not limited to the specific embodiments listed below. Those skilled in the art can implement this invention using various other specific embodiments based on the content disclosed herein. Any modifications or alterations made to the design structure and concept of this invention fall within the protection scope of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0033] Glossary of relevant technical terms

[0034] The term "antibody" broadly refers to any immunoglobulin (Ig) molecule composed of four polypeptide chains (two heavy (H) chains and two light (L) chains). In a full-length antibody, each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain: CL. The VH and VL regions can be further subdivided into highly variable regions called complementarity-determining regions (CDRs) and framework regions (FRs). Each VH and VL consists of three CDRs and four FRs in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, arranged from the amino terminus to the carboxyl terminus. The first, second, and third CDRs of the VH domain are typically denoted as CDR-H1, CDR-H2, and CDR-H3; similarly, the first, second, and third CDRs of the VL domain are typically denoted as CDR-L1, CDR-L2, and CDR-L3. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. The amino acid sequences of the constant domains of the mouse and human Ig heavy and light chains are known in the art.

[0035] The term "monoclonal antibody" refers to antibodies derived from a substantially homogeneous group of antibodies, meaning that the individual antibodies constituting the group are identical, except for a small number of potentially naturally occurring mutations. Monoclonal antibodies are highly specific, targeting a single antigenic determinant (epitope). Furthermore, unlike polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen.

[0036] The term “CDR” refers to the complementarity-determining region within the variable domain sequence of an antibody. Three CDRs exist in each variable domain of the heavy and light chains, designated CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3. The term “CDR group” as used herein refers to a group of three CDRs present in a single variable domain capable of binding the antigen. The exact boundaries of these CDRs have been defined differently depending on the system. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Maryland (1987) and (1991)) provides not only a definitive residue numbering system applicable to any variable domain of an antibody but also precise residue boundaries defining the three CDRs.

[0037] The term "plasmid" refers to a circular double-stranded DNA molecule that can be linked to another DNA segment. Vectors can carry the target DNA sequence into host cells, replicate autonomously, and be expressed in the host cells.

[0038] The term "transformation" refers to any process by which exogenous DNA enters a host cell. Transformation can be performed under natural or artificial conditions using a variety of methods known in the art. Transformation can rely on any known method to introduce a exogenous nucleic acid sequence into a prokaryotic or eukaryotic host cell. The method is selected based on the host cell to be transformed and may include, but is not limited to, viral infection, electroporation, lipid transfection, and particle bombardment.

[0039] The term "UUO" (unilateral ureteral obstruction) is a classic experimental method for establishing an animal model of renal fibrosis by surgically ligating one ureter. Its primary application is to simulate the progression of chronic kidney disease (CKD) in humans caused by urinary tract obstruction. This model can highly reproduce key pathological changes such as renal tubular atrophy, interstitial inflammation, myofibroblast activation, and extracellular matrix deposition, ultimately leading to irreversible renal interstitial fibrosis. Due to its relatively simple operation, good reproducibility, and clearly defined pathological process, the UUO model is widely used to study the mechanisms of renal fibrosis and serves as the gold standard tool for screening and evaluating the efficacy of antifibrotic drugs.

[0040] The term "5 / 6 nephrectomy" refers to a classic method for establishing a chronic renal failure animal model by surgically removing most of the kidney tissue in rats. The modeling process involves two steps: first, two-thirds (upper and lower poles) of one kidney is surgically removed; approximately one week later, the contralateral kidney is completely removed. Ultimately, only about one-sixth (one-third of a kidney's total volume) of functional tissue remains. The core purpose of this model is to simulate the progression of chronic kidney disease (CKD) in humans caused by various factors such as glomerulosclerosis, hypertension, and diabetes. Its key feature is its ability to stably reproduce the pathological processes of adaptive hyperfiltration, systemic hypertension, proteinuria, and progressive glomerulosclerosis and interstitial fibrosis; therefore, it is widely used to study the pathogenesis of CKD and to evaluate drugs that slow the progression of kidney disease.

[0041] Example 1

[0042] Construction and purification of full-length KL protein vector

[0043] The full-length human KL protein DNA sequence was ligated into the expression vector pT7-6×His-Twinstrep via PCR to construct the pT7-6×His-KL-Twinstrep expression vector. Protein purification was performed firstly using Ni-NTA binding to a His-tag, followed by a second round of purification using a Strep-tag to obtain the purified full-length KL protein. The specific steps are as follows: 1. Construction of the expression carrier: The cDNA sequence of KL was amplified using primers F and R (NM_004795.4): Primer F was: TGCCGCGCGGCAGCCATATGATGCCCGCCAGCGCCCCGCC (SEQ ID NO.9); Primer R was: CTCCAAGCACTGTAAGCTTGTTTGTAACTTCTTCTGCCTTTC (SEQ ID NO.10).

[0044] The cDNA sequence of KL is:

[0045] Amplification was performed using the Phanta UniFi Ultra-Fidelity DNA Polymerase Kit (Novozymes, catalog number P506). In a 50µL PCR system, 2x buffer and 10ng of KL template plasmid pCDH-CMV-KL(human)-EGFP (Miaoling, catalog number P56339) were added according to the manufacturer's instructions. The PCR conditions for amplifying KL were as follows: 98℃ for 10 seconds; 60℃ for 10 seconds; 72℃ for 165 seconds; 30 cycles to obtain the KL fragment.

[0046] Simultaneously, the vector pT7-6×His-Twinstrep (Miaoling, catalog number P57424) was linearized by digestion with NdeI and HindIII. The digestion system was as follows: in a 50µL system, according to the product instructions of NdeI and HindIII (both enzymes purchased from NEB), 5µL of 10x buffer was added, along with 2µg of plasmid, 1µL each of NdeI and HindIII, and digestion was carried out at 37℃ for 2 hours.

[0047] The KL fragment and the linearized vector were then recombinantly ligated using recombinase. The pEASY®-Basic Seamless Cloning and Assembly Kit was used for recombination ligation. Following the instructions, the linearized vector and KL fragment were added, and the mixture was incubated at 50°C for 30 minutes before transformation into DH5α competent cells. The transformation process for DH5α competent cells was as follows: 5 µL of the recombinant product was added to 100 µL of DH5α competent cells and incubated on ice for 30 minutes; after heat shock at 42°C for 90 seconds, 900 µL of SOC medium was added; after recovery on a shaker at 37°C for 45 minutes, the bacteria were plated onto 60 mm LB plates and incubated overnight at 37°C. Two colonies were picked from the overnight plate and added to a plastic shaker tube containing 2 mL of LB medium. After incubation on a shaker at 37°C for 4 hours, 0.5 mL of the colony was sent to a sequencing company (BGI Genomics) for sequencing using T7 primers. The selected positive clone pT7-6×His-KL-Twinstrep was confirmed to be correctly sequenced.

[0048] 2. Purification of KL protein: 2.1 The positive clone pT7-6×His-KL-Twinstrep plasmid was transformed into Rosetta competent cells (Beyotime, catalog number D1065S, transformation process is the same as DH5α competent cells) and cultured overnight at 37℃.

[0049] 2.2 On the second day, select one clone, inoculate it into 5 mL of LB medium containing kanamycin, and incubate overnight at 37°C and 200 rpm.

[0050] 2.3 On the third day, the bacteria that had been cultured overnight were inoculated into 500 mL of LB medium containing kanamycin and cultured at 37 °C at 200 rpm until the OD reached 0.5-0.6. 0.5 mM isopropyl-β-D-thiogalactoside (IPTG, Aladdin, catalog number I104812) was added and the bacteria were induced at 18 °C for 16 hours. The bacteria were collected by centrifugation (3000 g, 5 min) and resuspended in 20 mL of equilibration buffer B (0.15 M NaCl, 20 mM Na2HPO4, pH 7.2). The bacteria were then ruptured by superpressure.

[0051] 2.4 After lysis, centrifuge at 10000g, 4℃ for 20 minutes, and collect the supernatant, which is the bacterial lysate.

[0052] 2.5 Prepare the gel: Centrifuge 500µL of Strep-Tactin XT agarose gel (Biolinkedin, cat# L-2302) at 1000 g for 1 minute and discard the supernatant; add 2.5mL of washing buffer W (0.15M NaCl, 20mM Na2HPO4, 0.1% Tween-20, pH 7.2) to wash the gel, centrifuge at 1000 g for 1 minute and discard the supernatant, repeat the operation twice.

[0053] 2.6 Mix the equilibrated Strep-Tactin XT agarose gel with bacterial lysis buffer and incubate at 4°C for 2 hours; collect the agarose gel; wash the gel three times with 5 mL of washing buffer W to remove bound impurities; elute the protein (target protein, i.e., KL protein) with elution buffer (2.5 mM D-dethiobiotin, 0.15 M NaCl, 20 mM Na2HPO4, 0.1% Tween-20, pH 7.2), collecting 1 mL of elution buffer in a 1.5 mL Eppendorf tube. Repeat the operation to collect 10 tubes of elution buffer.

[0054] The collected protein eluent samples were validated by SDS-PAGE. Tubes 1-5 containing KL protein were combined, centrifuged in 30KD ultrafiltration tubes (3000g, 45 minutes), and then replaced with Phosphate-Buffered Saline solution (PBS: 137mM NaCl, 2.7mM KCl, 10mM Na2HPO4, 1.8mM KH2PO4, pH 7.4).

[0055] 2.7 Prepare a Ni-NTA gel column. Equilibrate 500 µL of Ni-NTA agarose gel (Qiagen, 30210) with lysis buffer (20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 10 mM imidazole, 10% glycerol). Add the equilibrated Ni-NTA agarose gel to the KL protein solution obtained in the previous step and incubate at 4°C for 2 hours. Collect the agarose gel. Remove bound impurities with 50 mL of washing buffer (20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 20 mM imidazole, 10% glycerol). Elute with elution buffer (20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 250 mM imidazole, 10% glycerol). Elute the protein (target protein, i.e., KL protein) with glycerol, collect 1 mL in a 1.5 mL Eppendorf tube, repeat the operation, and collect 5 tubes of eluent.

[0056] 2.8 Protein Concentration Determination and Aliquoting: 5 μL of eluent from each tube was mixed with 100 μL of Bradford (Beyotime, P0006C). The presence of protein in the collected 5 tubes of eluent was determined based on the color change. 10 μL of each of the 5 tubes of eluent was then added to 10 μL of 2×SDS loading buffer (Beyotime, P0015B), and the mixture was boiled in a water bath for 10 minutes. Protein electrophoresis was then performed using SDS-PAGE (5 μL of each sample was used for electrophoresis). The resulting protein gel was stained with Coomassie Brilliant Blue (Beyotime, P0018B) for 30 minutes and then destained with destaining solution (Beyotime, P0017C) for 1 hour to determine the concentration and purity of the target protein KL in the eluent. Combine tubes 1-5 containing KL protein, centrifuge in 30KD ultrafiltration tubes (3000g, 45 min), displace with PBS solution, and concentrate to a final volume of 0.5-1 mL. The concentrated protein concentration was determined by Bradford (Beyotime, P0006C), showing a concentration of 5 μg / μL. Aliquots were then stored at -80°C in 100 μL tubes.

[0057] The sequence of the KL protein is as follows (SEQ ID NO.12):

[0058] Example 2

[0059] Mice were immunized with KL protein to obtain monoclonal antibodies.

[0060] 1. Immunize mice with full-length human KL protein, the specific steps are as follows: Antigen conjugation: KL protein was conjugated with ovalbumin (OVA) to obtain OVA-KL antigen. The conjugation reagent was the cross-linking reagent SMCC (succinimide-4-[N-maleimide-methyl]cyclohexane-1-carboxylate, manufacturer: Thermo Fisher Scientific, catalog number A35394). The coupling steps are as follows, following the reagent instructions: ① Add 100µL of 1.5mg / mL SMCC to 1mL of PBS solution containing 1mg / mL KL protein, and incubate at room temperature for 30 minutes; ② Then remove uncrosslinked SMCC according to the instructions for Zeba™ dye and biotin removal centrifuge column and filter plate (5mL centrifuge column, ThermoFisher Scientific, catalog number A44300); the resulting protein is KL-SMCC, and the protein concentration is measured; ③ Mix KL-SMCC and ovalbumin (OVA) in a 1:1 molar ratio, and incubate at room temperature for 30 minutes; ④ After the reaction is complete, add the system to an Amicon Ultra-15 centrifuge filter equipped with an Ultracel-50 filter membrane, centrifuge at 2000rpm for 10 minutes, and the protein filtered through the Ultracel-50 filter membrane is OVA-KL, the protein concentration is measured, and the protein is stored.

[0061] Mice were immunized with OVA-KL protein, following these steps: 1. Primary immunization: OVA-KL (100µg / 200µL / mouse) was mixed with complete Freund's adjuvant and injected subcutaneously into female Balb / c mice (Jiangsu Huachuang Xinno Pharmaceutical Technology Co., Ltd.).

[0062] 2. On day 7, a booster immunization was performed: OVA-KL (100µg / 200µL / mouse) was mixed with complete Freund's adjuvant and injected subcutaneously into mice.

[0063] 3. Antibody titer was measured by ELISA on day 35: serum antibody titer was measured by ELISA of tail blood. The ELISA procedure was performed in accordance with existing techniques (Kim, HY., Stojadinovic, A., Izadjoo, MJ (2014). Immunization, Hybridoma Generation, and Selection for Monoclonal Antibody Production. In: Ossipow, V., Fischer, N. (eds) Monoclonal Antibodies. Methods in Molecular Biology, vol 1131. Humana Press, Totowa, NJ. p33-45).

[0064] 4. Day 38 immunization: Mice were immunized by intraperitoneal injection of OVA-KL (35µg / 300µL / mouse) (no adjuvant required) to obtain immunized mice.

[0065] 2. The formation of hybridomas

[0066] According to the method described in the existing technology (Kim, HY., Stojadinovic, A., Izadjoo, MJ (2014). Immunization, Hybridoma Generation, and Selection for Monoclonal Antibody Production. In: Ossipow, V., Fischer, N. (eds) Monoclonal Antibodies. Methods in Molecular Biology, vol 1131. Humana Press, Totowa, NJ. p33-45), spleen cells from prepared immunized mice were fused with SP2 / 0 cells (Wuhan Pronosai) at a ratio of 5:1 to generate hybridomas. The fusion process was performed in accordance with existing techniques (Antibodies a Laboratory Manual, Second Edition, pp. 274-278), as follows: SP2 / 0 cells were cultured in a high-glucose DMEM medium (Gibco, catalog number 11965) containing 10% FBS (fetal bovine serum, Gibco, catalog number 10100147C) and 1× penicillin-streptomycin (Gibco, catalog number 15140148).

[0067] Then 1×108 Spleen cells and 2×10 7 Mix SP2 / 0 cells, remove the culture medium, and slowly add 1 mL of 50% (w / v) polyethylene glycol solution (Sigma-Aldrich, catalog number P7181), followed by slowly adding 10 mL of DMEM high-glucose medium (Gibco, catalog number 11965). Incubate at 37°C for 10 minutes, then centrifuge at 1000 rpm. After centrifugation, remove the supernatant, and resuspend the precipitated cells in 200 mL of DMEM high-glucose medium containing 20% ​​FBS (fetal bovine serum, Gibco, catalog number 10100147C), 1×Hybri-Max™ HAT supplement (Sigma-Aldrich, catalog number H0262), and 1×penicillin-streptomycin (Gibco, catalog number 15140148). Seed 200 µL per well in 96-well cell culture dishes and incubate at 37°C for 7 to 10 days to obtain the fusion product.

[0068] The fusion product was encapsulated in a 96-well plate at a rate of 1 × 10⁻⁶ ppm per well. 5 Spleen cells were seeded at a density of 100 cells / well in selective medium (Sigma-Aldrich, catalog number H0262) containing hypoxanthine-aminopterin-thymidine (HAT). After 7 to 10 days of culture, visible hybridoma colonies were observed. The presence of KL antibody in the supernatant from each well containing hybridoma colonies was detected by ELISA. The KL enzyme-linked immunosorbent assay (ELISA) procedure was as follows: To determine whether anti-KL mAb (mouse monoclonal antibody) binds to human KL, a high protein affinity ELISA plate was first coated with KL protein and incubated at 37°C for 2 hours. After washing three times with washing buffer (PBS containing 0.05% Tween 20), the plate was blocked at room temperature for 1 hour with PBST solution containing 5% (w / v) skim milk powder (PBST containing 0.05% Tween 20). After washing three times with washing buffer (PBS containing 0.05% Tween 20), KL antibody was added and incubated overnight at 4°C. Wash the wells four times with washing buffer, and add 100 μL of HRP-conjugated anti-mouse IgG antibody (Sigma) diluted 1:5000 to each well. Incubate the plate at 37°C for 1 hour and wash four times with washing buffer. Add 100 μL of tetramethylbenzidine (TMB) chromogenic solution to each well. After chromogenic development, terminate the reaction with 1M HCl and measure the absorbance at 450 nm. Process the data using GraphPad software.

[0069] The results are as follows Figure 1 As shown, where, Figure 1 In the image, 'a' represents an ELISA image of 96 hybridoma cells. Figure 1 In the figure, b represents the absorbance values ​​of five clones capable of producing KL antibodies at 450 nm. Figure 1 In the image, c represents the ELISA results after serial dilution of five clones capable of producing KL antibodies. Figure 1 In the figure, d represents the absorbance values ​​at 450 nm of five clones capable of producing KL antibodies after serial dilution. The results show that... Figure 1 a and Figure 1 Figure b shows that of the 96 selected hybridoma cells, only 5 clones were able to recognize the KL protein (A1, B1, C1, F3, and D5), meaning only 5 were positive clones. The majority of the others did not recognize the KL protein. In the experiments of this invention, approximately 10 [cells / clones] could be obtained from each mouse. 8 After fusion, the cell viability rate is approximately 10%. -5 That is, only about 1,000 fused cells can survive. Furthermore, the antibodies secreted by the hybridoma are highly random.

[0070] 3. Purification of KL antibody

[0071] Subsequently, hybridoma cells capable of producing antibodies specifically binding to human KL were amplified and subcloned through limiting dilution. The specific procedure was as follows: Monoclonal hybridoma cells were amplified in serum-free hybridoma medium (Gibco, catalog number 12300067) containing 2.5% low-IgG fetal bovine serum. An average of 200 mL of culture supernatant was harvested from each hybridoma, concentrated, and purified by protein A / G affinity chromatography (Antibodies: a Laboratory Manual, Second Edition, Chapter 10, Antibody Purification and Storage, p393-396) to obtain the monoclonal antibody. The ability of the purified mAb (monoclonal antibody) to bind to KL was tested using the ELISA method described above.

[0072] The ability to identify KL protein was detected by serially diluting the supernatant of these 5 positive clones. The results are as follows: Figure 1 c and Figure 1 As shown in d in the figure. The results showed that after a 3200-fold dilution, the antibody produced by clone A1 still exhibited the highest sensitivity in recognizing the KL protein. While other clones could also recognize the KL protein and produce KL antibodies, the sensitivity of their KL antibodies in recognizing the KL protein was significantly lower than that of the antibody produced by clone A1. Therefore, the antibody derived from clone A1 was selected for subsequent sequencing (the antibody derived from clone A1 will be described using KL antibodies later).

[0073] Example 3

[0074] KL antibody sequencing

[0075] The purified mAb (monoclonal antibody) that specifically binds to KL was sent to the company (rapid sequencing) for sequencing of the protein light and heavy chains, and the complementarity-determining regions (CDRs) in the variable domains were identified based on the Kabat numbering system. The mAb (monoclonal antibody) purified by this invention, namely αKlotho antibody, comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region includes heavy chain CDR1 (CDR-H1), heavy chain CDR2 (CDR-H2), and heavy chain CDR3 (CDR-H3), and the light chain variable region includes light chain CDR1 (CDR-L1), light chain CDR2 (CDR-L2), and light chain CDR3 (CDR-L3). The sequence of heavy chain CDR1 is SEQ ID NO.1, the sequence of heavy chain CDR2 is SEQ ID NO.2, the sequence of heavy chain CDR3 is SEQ ID NO.3, the sequence of light chain CDR1 is SEQ ID NO.4, the sequence of light chain CDR2 is SEQ ID NO.5, and the sequence of light chain CDR3 is SEQ ID NO.6.

[0076] SEQ ID NO.1: SGFTFSNF.

[0077] SEQ ID NO. 2: LEWVAYITETGGRTYYP.

[0078] SEQ ID NO.3: GFDYT.

[0079] SEQ ID NO.4: QSLLHNSGDT.

[0080] SEQ ID NO.5: PKLLIYKVSNRF.

[0081] SEQ ID NO.6: STHYPW.

[0082] The heavy chain CDR and light chain CDR sequences are defined according to Kabat.

[0083] The heavy chain variable region has the amino acid sequence shown in SEQ ID NO.7; the light chain variable region has the amino acid sequence shown in SEQ ID NO.8.

[0084] SEQ ID NO.7: EVQLAESGGGLQQPGGSLKLSCAASGFTFSNFYMAWVRQTPEKRLEWVAYITETGGRTYYPDTVKGRFTISRDDAKNTLYLEMSSLRSEDTATYYCSRGFDYTATLLDYWGQGVTVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK。

[0085] SEQ ID NO.8: DVVMTQTPLSLPVSLGDQASLSCRSSQSLLHNSGDTYLHWYLQRPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEPEDLGVYFCSQSTHYPWTFGAGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC。

[0086] Example 4

[0087] WB detection of antibody specificity and sensitivity

[0088] 1. Antibody specificity detection: 293T cells were transfected into 6-well cell culture plates according to the Transporter™ 5 Transfection Reagent Kit (Polysciences, catalog number 26008). Forty-eight hours post-transfection, proteins were extracted from control 293T cells and experimental 293T cells transfected with the KL template plasmid pCDH-CMV-KL(human)-EGFP using 500 µL RIPA lysis buffer (Strong, Beyotime, catalog number P0013B). Precise quantification was performed using the BCA protein assay kit (Enhanced, Beyotime, catalog number P0010S). 40 μg of protein sample was loaded, and based on the molecular weight of KL protein (135 kDa), an 8% separating gel was prepared for SDS-PAGE electrophoresis, as follows:

[0089] Transfer: Transfer conditions: 300 mA, 120 minutes, ice bath. Transfer the protein from the gel to a PVDF membrane. After transfer, quickly observe the uniformity of the transfer by staining with Ponceau S, then wash with water.

[0090] Immunoblotting and antibody incubation

[0091] Blocking: Block the PVDF membrane with a PBST solution containing 5% (by weight / volume) skim milk powder (PBST containing 0.05% Tween 20 is PBST) on a shaker at room temperature for 1 hour.

[0092] Primary antibody incubation: The KL antibody of this invention and antibodies from various brands were diluted 1:1000 in PBST containing 5% milk. The antibodies from various brands were: Ab203576, sc-315939, and KM2076. Each antibody and membrane were incubated overnight on a shaker at 4°C.

[0093] Washing: The next day, wash the membrane quickly three times on a shaker with TBST for 5 minutes each time.

[0094] Secondary antibody incubation: Place all membrane strips into the corresponding HRP-labeled secondary antibody solution (HRP-labeled goat anti-mouse antibody: Jackson ImmunoResearch Laboratories, catalog number 115-035-146) and incubate on a shaker at room temperature for 1 hour.

[0095] Wash the membrane: Wash three times with TBST on a shaker for five minutes each time to remove unbound secondary antibody and reduce background.

[0096] Chemiluminescence detection and imaging: The ECL working solution (SuperSignal™ West Pico PLUS chemiluminescent substrate, ThermoFisher Scientific, catalog number 34580) was uniformly dropped onto the membrane and reacted for 1-2 minutes.

[0097] Images were acquired and recorded using the same exposure time in the chemiluminescence imaging system. The results are as follows: Figure 2 As shown. Figure 2 The results showed that the KL antibody could specifically recognize both endogenous (KL) and exogenous (KL-GFP) KL proteins.

[0098] 2. Antibody sensitivity detection: The experiment first extracted the lysate of 293T cells transfected with KL-GFP. The extraction process was as follows: 2 mL of pre-chilled PBS was added to 293T cells (in a 60 mm cell culture dish) transfected with pCDH-CMV-KL(human)-EGFP plasmid and washed once. Then, 1 mL of RIPA lysis buffer (Strong, Beyotime, catalog number P0013B) was added, and the cells were incubated on ice for 20 minutes. The lysis buffer was then transferred to a centrifuge tube and centrifuged at 12,000 rpm for 15 minutes at 4°C. The supernatant was collected, which was the lysate of 293T cells containing KL-GFP.

[0099] After quantification of 293T cell lysates containing KL-GFP using the BCA method, concentration gradient samples (40µg, 20µg, 10µg, 5µg) were prepared from 40µg to 5µg. These samples were treated with loading buffer (SDS-PAGE protein loading buffer (6X), Beyotime, catalog number P0015F) and incubated at 60℃ for 10 minutes. Subsequently, SDS-PAGE electrophoresis (8% separating gel) was performed, with markers and gradient protein samples loaded sequentially. The samples were then transferred to a PVDF membrane at 300mA for 120 minutes, and the transfer efficiency was verified by Ponceau S staining. After blocking the membrane with 5% skim milk for 1 hour, it was incubated overnight at 4°C with a 1:1000 diluted primary antibody (including our own KL antibody and multiple brand antibodies KM2076, SC-315939, and Ab203576). After washing with PBST, it was incubated at room temperature for 1 hour with HRP-labeled secondary antibody (HRP-labeled goat anti-mouse antibody, purchased from Jackson Immuno Research Laboratories, catalog number 115-035-146). After washing again, it was reacted with ECL chemiluminescence solution, and finally, images were acquired by uniform exposure in a chemiluminescence imaging system. Results are as follows: Figure 3 As shown. Figure 3 The results showed that the KL antibody prepared in this invention had significantly higher sensitivity in recognizing KL protein than antibodies from the other three brands. The KL antibody prepared in this invention could still sensitively detect KL protein at a concentration of 5 µg.

[0100] Using the same method, the specific antibody sb106 in the existing technology US10228374B2 was detected. The results showed that the specific antibody sb106 could not detect KL protein even at a concentration of 40 µg.

[0101] Example 5

[0102] Comparison of the sensitivity of different brands of antibodies using ELISA

[0103] 1. Coat high protein affinity ELISA plates with KL protein at 37°C for 2 hours. Wash three times with washing buffer (PBS containing 0.05% Tween 20), then block at room temperature for 1 hour with PBST solution containing 5% (w / v) skim milk powder (PBST containing 0.05% Tween 20).

[0104] 2. The KL antibody prepared in this invention and antibodies from different brands were diluted in different gradients: 1 / 1600, 1 / 3200, 1 / 6400, 1 / 12800, 1 / 25600, 1 / 51200, and 1 / 102400.

[0105] 3. After washing the sealed ELISA plate three times with washing buffer, add the diluted antibody and incubate overnight at 4°C. Wash the wells four times with washing buffer, and add 100 μL of the corresponding HRP-conjugated anti-mouse IgG antibody (Jackson Immuno Research Laboratories Inc. 115-035-146), anti-rat IgG antibody (Servicebio, GB23302), or anti-rabbit IgG antibody (Jackson Immuno Research Laboratories Inc. 111-005-144) diluted 1:5000 with PBST solution containing 5% (w / v) skim milk powder to each well.

[0106] The method for HRP-conjugated antibodies is similar to the KL-OVA conjugation method, as detailed below: ① Add 100 μL of 1.5 mg / mL SMCC to 1 mL of a solution containing 1 mg / mL KL antibody and incubate at room temperature for 30 minutes; ② Then remove the centrifuge column and filter plate (5 mL centrifuge column, Thermo Fisher Scientific, catalog number A44300) with Zeba™ dye and biotin, and remove uncrosslinked SMCC according to the instructions; the resulting protein is KL antibody-SMCC, and test the concentration of KL antibody-SMCC; ③ Mix KL-SMCC and horseradish peroxidase (HRP) protein (Aladdin, catalog number P105528) at a 1:1 ratio and incubate at room temperature for 30 minutes; ④ After the reaction is complete, add the solution to an Amicon Ultra-15 centrifuge filter equipped with an Ultracel-100 filter membrane, centrifuge at 2000 rpm for 10 minutes, and the intercepted protein is the HRP-conjugated KL antibody. Measure the concentration with a BCA kit and store the protein.

[0107] The plate was incubated at 37°C for 1 hour and washed four times with washing buffer. 100 μL of tetramethylbenzidine (TMB) chromogenic solution was added to each well. After chromogenic development, the reaction was terminated with 1M HCl, and the absorbance was measured at 450 nm. Data were processed using GraphPad software. Results are as follows: Figure 4 As shown, where Figure 4 In the image, 'a' represents the chromogenic image of each antibody on the ELISA plate. Figure 4 In the graph, b represents the bar chart of the ELISA detection results for each antibody. Figure 4 The results show that the KL antibody prepared by this invention has significantly better sensitivity than other brands of antibodies at all dilutions.

[0108] Example 6

[0109] Detection of KL protein levels in serum of CKD mice

[0110] 1. Sample preparation

[0111] Mouse serum samples: Experimental group: CKD model mice (mice 8 weeks after 5 / 6 nephrectomy, mice 2 weeks after UUO model). The preparation method of the UUO model refers to the existing technology (Elena Martínez-Klimova, Aparicio-Trejo OE, Tapia E, et al. Unilateral Ureteral Obstruction as a Model to Investigate Fibrosis-Attenuating Treatments[J].Biomolecules, 2019, 9(4):141.DOI:10.3390 / biom9040141.), and the method of 5 / 6 nephrectomy refers to the existing technology (Tan RZ, Zhong X, Li JC, et al.An optimized 5 / 6 nephrectomy mouse model based on unilateral kidney ligation and its application in renal fibrosis research[J].Renal Failure, 2019, 41(1):555-566.DOI:10.1080 / 0886022X.2019.1627220.).

[0112] Control group: The same strain as healthy controls (sham group). In the sham group, only the abdominal cavity was opened during surgery, without nephrectomy or unilateral ureteral ligation (UUO), and the wound was subsequently sutured. That is, except for the absence of nephrectomy or unilateral ureteral ligation (UUO), the sham group was treated exactly the same as the model group.

[0113] Sample collection: Whole blood was collected by snipping the eye socket, allowed to stand at room temperature for 30 minutes, centrifuged at 3000 rpm for 15 minutes at 4°C, and the supernatant (i.e., serum) was collected, aliquoted and stored at -80°C to avoid repeated freeze-thaw cycles.

[0114] Dissolve reagents: Remove all reagents and serum samples from -80°C and place them on ice to thaw slowly.

[0115] 2. Antibody incubation and color development

[0116] ELISA plate coating: Dilute KL antibody (Cosmo Bio, KAL-KO603) to 2 µg / mL with PBS and add it to the wells of the ELISA plate. Then add 50 µL of 2 µg / mL KL antibody (the antibody prepared in this invention) to each well of the ELISA plate and incubate at room temperature for 2 hours. Wash each well with 200 µL of PBST (PBS containing 0.05% Tween 20), repeating 3 times.

[0117] Blocking: Add 100 µL of PBST containing 5% skim milk powder to each well of the ELISA plate and incubate at room temperature for 1 hour. Then wash with 200 µL of PBST to each well, repeating 3 times.

[0118] Sample addition: Add 100 µL of standard or diluted serum sample to the corresponding well. Set up blank wells (add diluent only). Gently shake the reaction plate, cover with sealing film, and incubate at 37°C for 2 hours.

[0119] Wash the plate: Discard the liquid in the wells, add 200µL of PBST containing 5% skim milk powder to each well, let stand for 30 seconds, then discard. Repeat this process 4 times. Finally, pat dry on absorbent paper.

[0120] Add detection antibody: Add 100µL of HRP-conjugated KL antibody (the antibody prepared in this invention) to each well to cover the new sealing membrane, and incubate at 37°C for 1 hour.

[0121] Washing the plate: Same as above, wash the plate 4 times and pat it dry.

[0122] Color development: Add 90µL of TMB substrate to each well and incubate at 37°C in the dark for 15-30 minutes. Observe closely during this period; proceed to the next step when a clear blue gradient appears in the high-concentration wells of the standard.

[0123] To terminate the reaction: Add 50 µL of stop solution (2 M H₂SO₄) to each well. The solution color will immediately change from blue to yellow. Gently shake the reaction plate to ensure thorough mixing.

[0124] Plate reading: Within 15 minutes of adding the stop solution, measure the absorbance (OD value) of each well using a microplate reader at a wavelength of 450 nm. Use 630 nm as a reference wavelength for calibration.

[0125] Statistical analysis: GraphPad Prism software was used to create bar charts with 6 data points per group. An unpaired t-test was used to compare two groups, with P < 0.05 considered statistically significant. Results are as follows: Figure 5 As shown, where, Figure 5 In the figure, 'a' represents a bar chart showing the level of KL protein in the serum of UUO model mice. Figure 5 Figure b is a bar graph showing the KL protein level in the serum of 5 / 6 nephrectomy model mice. The results are shown at a wavelength of 450 nm. The results show that the KL protein level in the serum of chronic kidney disease model mice (5 / 6 nephrectomy and UUO) was significantly lower than that in the sham-operated group. This is consistent with the results of gold standard detection, where the KL level in fibrosis model mice was significantly lower than that in the control sham-operated group. This indicates that the antibody prepared in this invention can be used for the detection of KL protein in serum with high accuracy and sensitivity.

[0126] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of this invention are still within the scope of protection of this invention.

Claims

1. An αKlotho antibody, 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 sequence of heavy chain CDR1 is shown in SEQ ID NO.1, the sequence of heavy chain CDR2 is shown in SEQ ID NO.2, the sequence of heavy chain CDR3 is shown in SEQ ID NO.3, the sequence of light chain CDR1 is shown in SEQ ID NO.4, the sequence of light chain CDR2 is shown in SEQ ID NO.5, and the sequence of light chain CDR3 is shown in SEQ ID NO.

6.

2. The αKlotho antibody according to claim 1, characterized in that, The heavy chain variable region has the amino acid sequence shown in SEQ ID NO.7; and / or the light chain variable region has the amino acid sequence shown in SEQ ID NO.

8.

3. A nucleic acid molecule, characterized in that, It encodes the αKlotho antibody as described in claim 1 or 2.

4. A carrier, characterized in that, It contains the nucleic acid molecule as described in claim 3.

5. An αKlotho protein detection kit, characterized in that, It contains the αKlotho antibody as described in claim 1 or 2 or the nucleic acid molecule as described in claim 3.

6. The application of the αKlotho antibody according to claim 1 or 2, the nucleic acid molecule according to claim 3, the vector according to claim 4, or the αKlotho protein detection kit according to claim 5, characterized in that, The application is to assess the level of αKlotho protein in serum samples.

7. The application according to claim 6, characterized in that, The assessment is based on the detection of αKlotho protein levels in the serum samples of the tested individuals.

8. A reagent for detecting αKlotho protein, characterized in that, It includes the αKlotho antibody as described in claim 1 or 2, the nucleic acid molecule as described in claim 3, the vector as described in claim 4, or the αKlotho protein detection kit as described in claim 5.

9. The application of the αKlotho antibody according to claim 1 or 2, the nucleic acid molecule according to claim 3, the vector according to claim 4, or the αKlotho protein detection kit according to claim 5, characterized in that, The application is as a diagnostic reagent for chronic kidney disease.

10. A reagent for diagnosing chronic kidney disease, characterized in that, It includes the αKlotho antibody as described in claim 1 or 2, the nucleic acid molecule as described in claim 3, the vector as described in claim 4, or the αKlotho protein detection kit as described in claim 5.

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