Alpha kl protein antibody and use thereof

Abstract

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

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of antibodies, in particular to an alpha KL protein antibody and its application. BACKGROUND

[0002] The KL gene, also known as the klotho gene, is located at position 13q13.1 on human chromosome. The protein encoded by this gene belongs to the Glycoside hydrolase family 1 family, which is a class of proteins related to glycoside hydrolase. It includes alpha KL protein and beta KL protein. The protein product of the KL gene plays an important role in cells, especially in extracellular matrix and calcification regulation. KL protein can inhibit the calcification process, thereby protecting cells and tissues from calcification damage. This is not only crucial for bone health, but also has an important impact on the normal function of the cardiovascular system. In cells, KL protein is also involved in various signaling pathways, such as the insulin-like growth factor (IGF) signaling pathway. Through these signaling pathways, KL protein can regulate cell growth, differentiation, and metabolic processes.

[0003] Currently, there are some KL antibodies and diagnostic kits available on the market, but the existing KL antibodies lack specificity and cannot effectively immunoprecipitate KL from human serum. The current immunology-based KL detection method is costly and lacks sensitivity and specificity. Overcoming the lack of sensitivity and specificity of KL protein antibodies is the primary task of solving the clinical application of KL protein detection.

[0004] Prior art US10228374B2 provides a specific antibody sb106 with a dissociation constant of about 2 nM or less, which binds to alpha Klotho polypeptide, introduces sequence diversity into the complementarity determining region (CDR) using synthetic antibody technology, and can be used for accurate immunoprecipitation and detection in human serum and urine, and combined with phage display to obtain high affinity and specificity. However, the antibody sb106 can only recognize the native conformation of KL and cannot recognize denatured KL protein, which limits the detection. SUMMARY

[0005] The purpose of the present application is to provide an alpha KL protein antibody with significantly enhanced sensitivity and specificity and its application in detecting KL protein.

[0006] To achieve the above purpose, the technical scheme adopted by the present application is:

[0007] An aKlotho antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a heavy chain CDR1 (CDR-H1), a heavy chain CDR2 (CDR-H2), and a heavy chain CDR3 (CDR-H3), and the light chain variable region comprising a light chain CDR1 (CDR-L1), a light chain CDR2 (CDR-L2), and a light chain CDR3 (CDR-L3), wherein:

[0008] the sequence of the heavy chain CDR1 is set forth in SEQ ID NO. 1, the sequence of the heavy chain CDR2 is set forth in SEQ ID NO. 2, the sequence of the heavy chain CDR3 is set forth in SEQ ID NO. 3, the sequence of the light chain CDR1 is set forth in SEQ ID NO. 4, the sequence of the light chain CDR2 is set forth in SEQ ID NO. 5, and the sequence of the light chain CDR3 is set forth in SEQ ID NO. 6.

[0009] SEQ ID NO. 1: SGFTFSNF.

[0010] SEQ ID NO. 2: LEWVAYITETGGRTYYP.

[0011] SEQ ID NO. 3: GFDYT.

[0012] SEQ ID NO. 4: QSLLHNSGDT.

[0013] SEQ ID NO. 5: PKLLIYKVSNRF.

[0014] SEQ ID NO. 6: STHYPW.

[0015] wherein the sequences of the heavy chain CDRs and the light chain CDRs are according to the definition of Kabat.

[0016] According to the embodiments of the present application, the present application can be further optimized, and the following is the technical solution formed after optimization:

[0017] In one preferred embodiment, the aKlotho antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region has an amino acid sequence set forth in SEQ ID NO. 7; and / or the light chain variable region has an amino acid sequence set forth in SEQ ID NO. 8.

[0018] SEQ ID NO. 7:

[0019] EVQL ES GGGLQQPGGSLKLSCAASGFTFSNFYMAWVRQTPEKRLEWVAYITETGGRTYYPDTVK GRFTISRDDAKNTLYLEMSSLRSEDTATYYCSRGFDYTATLLDYWGQGVTVTVSSAKTTAPS VYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSV TVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKI KDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALP IQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCM VTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVV HEGLHNHHTTKSFSRTPGK.

[0020] SEQ ID NO. 8:

[0021] DVVMTQTPLSLPVSLGDQASLSCRSSQSLLHNSGDTYLHWYLQRPGQSPKLLIYKVSNRFSG VPDRFSGSGSGTDFTLKISRVEPEDLGVYFCSQSTHYPWTFGAGTKLEIKRADAAPTVSIFP PSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTL TLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.

[0022] Based on the same inventive concept, the present application also claims a nucleic acid molecule encoding the αKlotho antibody.

[0023] Based on the same inventive concept, the present application also claims a vector containing the nucleic acid molecule.

[0024] Based on the same inventive concept, the present application also claims an αKlotho protein detection kit containing the αKlotho antibody or the nucleic acid molecule.

[0025] Based on the same inventive concept, the present application also claims the use of the aKlotho antibody, the nucleic acid molecule, the vector or the aKlotho protein detection kit, which is to evaluate the aKlotho protein level in the serum sample of an individual.

[0026] Based on the same inventive concept, the present application also claims a reagent for detecting aKlotho protein, which comprises the aKlotho antibody, the nucleic acid molecule, the vector or the aKlotho protein detection kit.

[0027] In a preferred embodiment of the present application, the evaluation is based on detecting the content of aKlotho protein in the serum sample of an individual.

[0028] Based on the same inventive concept, the present application also claims the use of the aKlotho antibody, the nucleic acid molecule, the vector or the aKlotho protein detection kit, which is as a detection reagent for chronic kidney disease.

[0029] Based on the same inventive concept, the present application also claims a reagent for diagnosing chronic kidney disease, which comprises the aKlotho antibody, the nucleic acid molecule, the vector or the aKlotho protein detection kit.

[0030] Compared with the prior art, the present application has the following beneficial effects:

[0031] The present application immunizes mice with KL full-length protein, combines flow screening and hybridoma technology. The specific antibodies with high affinity to KL screened are sequenced, the CDR region binding to KL is identified, and the light chain and heavy chain expression vectors are constructed to transform them into full-length IgG from mammalian cells, thereby obtaining aKlotho antibody with high sensitivity and specificity, which can significantly outperform the existing KL antibody performance and can be applied to the clinical detection of chronic kidney disease. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 is the result of ELISA screening of hybridoma cells; wherein, Figure 1 a in is the ELISA graph of 96 hybridoma cells, Figure 1 b in is the column chart of the absorbance value at 450 nm of 5 KL antibody-producing clones, Figure 1 c in is the ELISA graph of 5 KL antibody-producing clones after gradient dilution, Figure 1 d in is the column chart of the absorbance value at 450 nm of 5 KL antibody-producing clones after gradient dilution.

[0033] Figure 2WB detection of KL antibody specific recognition of endogenous (KL) and exogenous (KL-GFP) KL protein chemiluminescence imaging.

[0034] Figure 3 WB detection of KL antibody specific recognition of endogenous (KL) and exogenous (KL-GFP) KL protein chemiluminescence imaging.

[0035] Figure 4 ELISA detection of KL antibody specific recognition of KL protein sensitivity results, wherein Figure 4 a in the above is the color developing photo of the ELISA plate of each antibody, Figure 4 b in the above is the column chart of the ELISA detection results of each antibody.

[0036] Figure 5 CKD mouse serum KL protein level detection results column chart, wherein, Figure 5 a in the above is the column chart of the level of KL protein in the serum of UUO model mice, Figure 5 b in the above is the column chart of the level of KL protein in the serum of 5 / 6 nephrectomy model mice. DETAILED DESCRIPTION

[0037] The present application is not limited to the following detailed description, and those skilled in the art can use other various embodiments to implement the present application according to the disclosure of the present application, or any simple changes or modifications made by using the design structure and ideas of the present application, all fall within the scope of the present application. It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict.

[0038] Noun explanation of related technical terms

[0039] The term "antibody" broadly refers to any immunoglobulin (Ig) molecule comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains. In a full-length antibody, each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is comprised of one domain, CL. The VHand VLregions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), and framework regions (FR). Each VHand VLis composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The first, second, and third CDRs of a VHdomain are typically referred to as CDR-H1, CDR-H2, and CDR-H3; likewise, the first, second, and third CDRs of a VLdomain are typically referred to 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. Murine and human Ig heavy and light chain constant domain amino acid sequences are known in the art.

[0040] The term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that can be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic determinant (epitope). Furthermore, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

[0041] The term "CDR" refers to a complementarity determining region within the variable domain sequence of an antibody. There are three CDRs in each of the variable regions of the heavy and light chains, which are referred to as CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3. The term "CDR set" as used herein refers to the set of three CDRs found in a single variable region that is capable of binding an antigen. The exact boundaries of these CDRs have been defined differently by different systems. 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 an unambiguous residue numbering system applicable to any variable region of an antibody, but also precise residue boundaries defining the three CDRs.

[0042] The term "plasmid" refers to a circular double-stranded DNA molecule that can have additional DNA segments attached. The vector is capable of bringing the DNA sequence of interest into a host cell for autonomous replication and expression in the host cell.

[0043] The term "transformation" refers to any process by which exogenous DNA enters a host cell. The transformation can be carried out under native or artificial conditions using various methods known in the art. The transformation can rely on the formation of cationic liposomes, neutral liposomes, dummy liposomes, immunoliposomes, or other lipid-based transporters which can fuse with cell membranes and allow the entry of the DNA into the cell. The transformation can rely on the use of polybrene, protamine, or other agents to facilitate the entry of the DNA into the cell. The transformation can rely on electroporation, lipofection, or particle bombardment.

[0044] The term "UUO" (unilateral ureteral obstruction) is a classical experimental method to establish an animal model of renal fibrosis by surgically ligating one side of the ureter. The main 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 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 clear pathological process, the UUO model is widely used to study the pathogenesis of renal fibrosis and as a gold standard tool for screening and evaluating the efficacy of anti-fibrosis drugs.

[0045] The term "5 / 6 nephrectomy" is a classical method to establish an animal model of chronic renal insufficiency by surgically removing most of the kidney tissue in rats. The main modeling method is performed in two steps: first, 2 / 3 of the kidney on one side (upper and lower poles) is removed by surgery, and about a week later, the opposite kidney is completely removed. Finally, only about 1 / 6 of the total amount of the original kidney (1 / 3 of one kidney) functional tissue is left. The core use of this model is to simulate the progression of chronic kidney disease (CKD) in humans caused by various reasons (such as glomerulosclerosis, hypertension, diabetes, etc.). Its characteristic is to stably reproduce the pathological process of adaptive hyperfiltration, systemic hypertension, proteinuria, and progressive glomerulosclerosis and interstitial fibrosis, so it is widely used to study the pathogenesis of CKD and evaluate drugs that delay the progression of kidney disease.

[0046] Example 1

[0047] Construction and purification of full-length KL protein vector

[0048] The DNA sequence of human full-length KL protein was connected to the expression vector pT7-6xHis-Twinstrep vector by PCR to construct the pT7-6xHis-KL-Twinstrep expression vector. The first step of protein purification was carried out by Ni-NTA binding His-tag, and the second round of purification was carried out by Strep-tag to obtain purified full-length KL protein. The specific steps are as follows:

[0049] 1. Construction of expression vector:

[0050] The cDNA sequence of KL (NM_004795.4) was amplified by primer F and primer R: primer F: TGCCGCGCGGCAGCCATATGATGCCCGCCAGCGCCCCGCC (SEQ ID NO. 9); primer R: CTCCAAGCACTGTAAGCTTGTTTGTAACTTCTTCTGCCTTTC (SEQ ID NO. 10).

[0051] The cDNA sequence of KL is:

[0052]

[0053] Amplification with Phanta UniFi Super-Fidelity DNA Polymerase Kit (Novozyme, item number P506), 2x buffer was added according to the instructions in a 50 μL PCR system, and 10 ng KL template plasmid pCDH-CMV-KL(human)-EGFP (Mingling, item number P56339) was added. The PCR conditions for amplifying KL are as follows: 98°C, 10 seconds; 60°C, 10 seconds; 72°C, 165 seconds; cycle 30 times, to obtain the KL fragment.

[0054] At the same time, the vector pT7-6xHis-Twinstrep (Mingling, item number P57424) was treated with NdeI and HindIII to linearize the vector. The enzyme digestion system is as follows: in a 50 μL system, according to the product instructions of NdeI and HindIII (both enzymes are purchased from NEB company), 5 μL 10x buffer was added, 2 μg plasmid was added, Nde1, Hind III was 1 μL, 37°C enzyme digestion for 2 hours.

[0055] Then the KL fragment and the linearized vector were recombined under the action of the recombination enzyme. The recombination was carried out using the full-size pEASY®-Basic Seamless Cloning and Assembly Kit reagent kit, and the linearized vector and KL fragment were added according to the instructions, incubated at 50°C for 30 minutes, and then transformed DH5α competent cells. The process of transforming DH5α competent cells is as follows: 5 μL of recombination product was added to 100 μL of DH5α competent cells, and incubated on ice for 30 minutes; after 42°C heat shock for 90 seconds, 900 μL of SOC medium was added; after 37°C shaking table recovery for 45 minutes, the bacteria were spread on 60 mm LB plate; 37°C incubator culture overnight. Two colonies were picked from the overnight plate, and each was added to a plastic shake flask containing 2 mL of LB medium. After 37°C shaking table culture for 4 hours, 0.5 mL of bacteria was taken to a sequencing company (Huada Gene) for T7 primer sequencing. It was proved that the positive clone pT7-6xHis-KL-Twinstrep selected was sequenced correctly.

[0056] 2. Purification of KL protein:

[0057] 2.1 The positive clone pT7-6xHis-KL-Twinstrep plasmid was transformed into Rosetta competent cells (Biyun Tian, item number D1065S, the transformation process was the same as that of DH5α competent cells), and cultured in a 37°C incubator overnight.

[0058] 2.2 The next day, a clone was picked and inoculated into 5 mL of LB medium containing kanamycin, and cultured at 200 rpm and 37°C overnight.

[0059] 2.3 On the third day, inoculate the overnight culture into 500 mL LB medium containing kanamycin, 200 rpm, 37°C, until OD 0.5-0.6, add 0.5 mM of isopropyl-β-D-thiogalactopyranoside (IPTG, Aladdin, Cat# I104812), induce at 18°C for 16 hours, centrifuge (3000 g, 5 min) to collect the bacteria, resuspend the bacteria with 20 mL of equilibration buffer Buffer B (0.15 M NaCl, 20 mM Na2HPO4, pH 7.2), and perform ultrasonic disruption.

[0060] 2.4 After disruption, centrifuge at 10000 g, 4°C, for 20 min, and collect the supernatant, which is the bacterial lysate.

[0061] 2.5 Prepare the gel, centrifuge 500 µL Strep-Tactin XT Sepharose gel (Biolinkedin, Cat# L-2302) at 1000 g for 1 min, and discard the supernatant; wash the gel with 2.5 mL of washing buffer Buffer W (0.15 M NaCl, 20 mM Na2HPO4, 0.1% Tween-20, pH 7.2), centrifuge at 1000 g for 1 min, discard the supernatant, and repeat twice.

[0062] 2.6 Mix the equilibrated Strep-Tactin XT Sepharose gel with the bacterial lysate, and combine at 4°C for 2 hours; collect the Sepharose gel; wash the gel with 5 mL of washing buffer Buffer W, wash 3 times, and remove the combined impurities; elute the protein (target protein, i.e., KL protein) with elution buffer (2.5 mM D-desulfitobiotin, 0.15 M NaCl, 20 mM Na2HPO4, 0.1% Tween-20, pH 7.2), collect 1 mL in a 1.5 mL Ep tube, repeat the operation, and collect 10 tubes of elution buffer.

[0063] Verify the collected protein elution sample by SDS-PAGE, combine tubes 1-5 containing the KL protein, centrifuge (3000 g, 45 min) in a 30 KD ultrafiltration tube, and replace into Phosphate-Buffered Saline solution (PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4).

[0064] 2.7 Preparation of Ni-NTA gel column, 500 μL of Ni-NTA agarose gel (Qiagen, 30210) was equilibrated with lysis buffer (20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 10 mM imidazole, 10% glycerol); the KL protein solution obtained in the previous step was added to the equilibrated Ni-NTA agarose gel, and combined at 4°C for 2 hours; the agarose gel was collected; the bound impurities were removed with 50 mL of washing buffer (20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 20 mM imidazole, 10% glycerol, adjusted to pH = 8.0); the protein (target protein, KL protein) was eluted with elution buffer (20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 250 mM imidazole, 10% glycerol), 1 mL was collected in a 1.5 mL Ep tube, and the operation was repeated, and 5 tubes of elution buffer were collected.

[0065] 2.8 Protein concentration determination and aliquoting: 5 μL of the elution buffer was mixed with 100 μL of Bradford (Bi Yun Tian, P0006C) per tube, and the presence or absence of protein in the 5 tubes of elution buffer collected was determined according to the color change. 10 μL of each of the 5 tubes of elution buffer was taken and 10 μL of 2x SDS loading buffer (Bi Yun Tian, P0015B) was added, and boiled in a boiling water bath for 10 minutes. Protein electrophoresis was performed by SDS-PAGE (5 μL of each of the above samples was subjected to protein electrophoresis), and the protein gel was stained with Coomassie Brilliant Blue staining solution (Bi Yun Tian, P0018B) for 30 minutes, and then decolorized with decolorizing solution (Bi Yun Tian, P0017C) for 1 hour. The presence or absence of target protein KL protein concentration and purity in the elution buffer was determined. Tubes 1-5 containing KL protein were combined, and centrifuged in a 30 KD ultrafiltration tube (3000g, 45 minutes), and replaced in PBS solution. The replacement solution was concentrated to a final volume of 0.5-1 mL. The concentrated protein was determined for protein concentration by Bradford (Bi Yun Tian, P0006C), and the result showed that the protein concentration was 5 μg / μL. 100 μL / tube was aliquoted and stored at -80°C.

[0066] The sequence of the KL protein is as follows (SEQ ID NO. 12):

[0067]

[0068] Example 2

[0069] Immunization of mice with KL protein to obtain monoclonal antibody

[0070] 1. Immunization of mice with human KL full-length protein, the specific steps are as follows:

[0071] Antigen coupling: KL protein was coupled with ovalbumin (OVA) to obtain OVA-KL antigen. The coupling reagent was cross-linking reagent SMCC (succinimidyl 4-[N-maleimidomethyl] cyclohexane-1-carboxylate, manufacturer ThermoFisher Scientific, item number A35394). According to the reagent instruction, the coupling steps are as follows: ① 100 µL of SMCC with a concentration of 1.5 mg / mL was added to 1 mL of PBS solution containing 1 mg / mL KL protein, and incubated at room temperature for 30 minutes; ② Then, according to the instruction of Zeba™ dye and biotin removal centrifugal column and filter plate (5 mL centrifugal column, ThermoFisher Scientific, item number A44300), remove the un-crosslinked SMCC; the obtained protein is KL-SMCC, and the protein concentration is detected; ③ Mix KL-SMCC and ovalbumin (OVA) at a molar ratio of 1:1, and incubate at room temperature for 30 minutes; ④ After the reaction is completed, the system is added to Amicon Ultra-15 centrifugal filter with Ultracel-50 filter membrane, and centrifuged at 2000 rpm (revolutions per minute) for 10 minutes. The protein filtered by Ultracel-50 filter membrane is OVA-KL, and the protein concentration is detected and the protein is stored.

[0072] Immunization of mice with OVA-KL protein, the steps are as follows:

[0073] 1. Primary immunization: OVA-KL (100 µg / 200 µL / each) was mixed with complete Freund's adjuvant, and subcutaneously injected into female Balb / c mice (Jiangsu Huacheng Xinnuo Pharmaceutical Technology Co., Ltd.).

[0074] 2. Reinforced immunization on day 7: OVA-KL (100 µg / 200 µL / each) was mixed with complete Freund's adjuvant, and subcutaneously injected into mice.

[0075] 3. Day 35 ELISA detection of antibody titer: Take the tail tip blood to detect the serum antibody titer by ELISA, and the ELISA detection process refers to the prior art (Kim, HY., Stojadinovic, A., Izadjoo, M.J. (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).

[0076] 4. Day 38 impact immunization: Immunize the mice by intraperitoneal injection of OVA-KL (35 μg / 300 μL / one mouse) (no need for adjuvant), and obtain the immunized mice.

[0077] 2. Generation of hybridoma

[0078] According to the method described in the prior art (Kim, HY., Stojadinovic, A., Izadjoo, M.J. (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), the spleen cells of the prepared immunized mice are fused with SP2 / 0 cells (Wuhan Punsai) at a ratio of 5:1 to generate hybridoma. The process of fusion refers to the prior art (Antibodies a Laboratory Manual, Second Edition, p274-278), and the specific process is as follows: SP2 / 0 cells are cultured in culture medium, and the culture medium is DMEM high glucose medium (Gibco, item number 11965) containing 10% FBS (fetal bovine serum, Gibco, item number 10100147C) and 1× penicillin-streptomycin (Gibco, item number 15140148).

[0079] Then 1×108 Splenocytes were mixed with 2 x 10 7 mL of 50% (w / v) polyethylene glycol solution (Sigma-Aldrich, Cat. No. P7181) was added slowly, followed by 10 mL of DMEM high glucose medium (Gibco, Cat. No. 11965). After incubation in a 37°C water bath for 10 minutes, the cells were centrifuged at 1000 rpm. After centrifugation, the supernatant was removed and the precipitated cells were resuspended in 200 mL of DMEM high glucose medium containing 20% FBS (fetal bovine serum, Gibco, Cat. No. 10100147C), 1 x Hybri-Max™ HAT medium supplement (Sigma-Aldrich, Cat. No. H0262), and 1 x penicillin-streptomycin (Gibco, Cat. No. 15140148). The cells were then seeded at 200 µL per well in a 96-well cell culture dish and incubated at 37°C for 7 to 10 days to obtain the fusion product.

[0080] The fusion product was seeded in a 96-well plate at a density of 1 x 10 5 Splenocytes per well in a selection medium containing hypoxanthine-aminopterin-thymidine (HAT) (Sigma-Aldrich, Cat. No. H0262). After 7 to 10 days of incubation, visible hybridoma colonies were observed. The supernatant from each well containing hybridoma colonies was tested for the presence of KL antibodies by ELISA. The KL enzyme-linked immunosorbent assay (ELISA) detection process was as follows: To determine whether the anti-KL mAb (mouse monoclonal antibody) binds to human KL, an ELISA plate with high protein affinity was coated with KL protein at 37°C for 2 hours. After washing 3 times with washing buffer (PBS containing 0.05% Tween 20), a solution of PBST (PBS containing 0.05% Tween 20) containing 5% (w / v) skim milk powder was added to the wells and incubated at room temperature for 1 hour. After washing 3 times with washing buffer (PBS containing 0.05% Tween 20), the KL antibody was added and incubated at 4°C overnight. The wells were washed 4 times with washing buffer, and HRP-conjugated anti-mouse IgG antibody (Sigma) was diluted 1:5000 and added to the wells at 100 µL per well. The plate was incubated at 37°C for 1 hour and washed 4 times with washing buffer. 100 µL of tetramethylbenzidine (TMB) color developing solution was added to each well. After color development, the reaction was stopped with 1 M HCl, and the absorbance was measured at 450 nm. The data were processed using GraphPad software.

[0081] The results are shown in Figure 1 , where Figure 1 a is the ELISA graph of 96 hybridoma cells, Figure 1 b is the absorbance value at 450 nm of 5 KL antibody-producing clones,Figure 1 c is the ELISA graph of the gradient dilution of the 5 KL antibody producing clones in b, Figure 1 d is the absorbance value at 450 nm of the gradient dilution of the 5 KL antibody producing clones in b. The results show that, Figure 1 a and Figure 1 b show that, among the 96 hybridoma cells selected, only 5 clones (A1, B1, C1, F3, and D5) can recognize KL protein, i.e., only 5 positive clones. Most of the others cannot recognize KL protein. In the experiments of the present application, about 10 8 cells can be obtained from each mouse, and after fusion, the cell survival rate is about 10 -5 i.e., only about 1000 fused cells can survive. However, the antibodies secreted by the hybridoma have great randomness.

[0082] 3. Purification of KL antibody

[0083] Subsequently, the hybridoma cells capable of producing antibodies that specifically bind to human KL were expanded and subcloned by limiting dilution, as follows: the monoclonal hybridoma cells were expanded in hybridoma serum-free medium (Gibco, item 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 monoclonal antibodies. The purified mAbs (monoclonal antibodies) were tested for their ability to bind to KL using the ELISA described above.

[0084] The ability of the supernatant of the 5 positive clones to recognize KL protein after gradient dilution was tested, and the results are shown in c and d of Figure 1 Figure 2 The results show that, after dilution 3200 times, the sensitivity of the antibody produced by clone A1 to recognize KL protein is still the highest, and although the other clones can also recognize KL protein, i.e., can produce KL antibodies, the sensitivity of the KL antibodies produced by them to recognize KL protein is significantly lower than that of the antibody produced by clone A1. Therefore, the antibody from clone A1 was selected for subsequent sequencing (the antibody from clone A1 is described below as KL antibody).

[0085] Example 3

[0086] Sequencing of KL antibody

[0087] ​The purified mAb (monoclonal antibody) specifically binding to KL was sent to a company (Kurso) for sequencing of the protein light chain and heavy chain, and the complementarity determining regions (CDRs) in the variable domain were identified based on the Kabat numbering system. The determined purified mAb (monoclonal antibody) of the present application, i.e. the aKlotho antibody, includes a heavy chain variable region including a heavy chain CDR1 (CDR-H1), a heavy chain CDR2 (CDR-H2) and a heavy chain CDR3 (CDR-H3), and a light chain variable region including a light chain CDR1 (CDR-L1), a light chain CDR2 (CDR-L2) and a light chain CDR3 (CDR-L3), wherein: the sequence of the heavy chain CDR1 is SEQ ID NO. 1, the sequence of the heavy chain CDR2 is SEQ ID NO. 2, the sequence of the heavy chain CDR3 is SEQ ID NO. 3, the sequence of the light chain CDR1 is SEQ ID NO. 4, the sequence of the light chain CDR2 is SEQ ID NO. 5, and the sequence of the light chain CDR3 is SEQ ID NO. 6.

[0088] SEQ ID NO. 1: SGFTFSNF.

[0089] SEQ ID NO. 2: LEWVAYITETGGRTYYP.

[0090] SEQ ID NO. 3: GFDYT.

[0091] SEQ ID NO. 4: QSLLHNSGDT.

[0092] SEQ ID NO. 5: PKLLIYKVSNRF.

[0093] SEQ ID NO. 6: STHYPW.

[0094] The heavy chain CDR and light chain CDR sequences are defined according to Kabat.

[0095] The heavy chain variable region has an amino acid sequence shown in SEQ ID NO. 7; and the light chain variable region has an amino acid sequence shown in SEQ ID NO. 8.

[0096] SEQ ID NO. 7:

[0097] EVQL ES GGGLQQPGGSLKLSCAASGFTFSNFYMAWVRQTPEKRLEWVAYITETGGRTYYPDTVK GRFTISRDDAKNTLYLEMSSLRSEDTATYYCSRGFDYTATLLDYWGQGVTVTVSSAKTTAPS VYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSV TVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKI KDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALP IQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTC MVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSV VHEGLHNHHTTKSFSRTPGK.

[0098] SEQ ID NO. 8:

[0099] DVVMTQTPLSLPVSLGDQASLSCRSSQSLLHNSGDTYLHWYLQRPGQSPKLLIYKVSNRF SGVPDRFSGSGSGTDFTLKISRVEPEDLGVYFCSQSTHYPWTFGAGTKLEIKRADAAPTVS IFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMS STLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.

[0100] Example 4

[0101] WB detection of antibody specificity and sensitivity

[0102] 1. Detection of antibody specificity:

[0103] In 6-well cell culture plates, 293T cells were transfected according to the instructions of the Transporter™ 5 Transfection Reagent kit (Polysciences, Cat. No. 26008). 48 hours after transfection, the proteins of the control group 293T cells and the experimental group 293T cells transfected with KL template plasmid pCDH-CMV-KL(human)-EGFP were extracted using 500 μL of RIPA lysis solution (strong, Biyun Tian, Cat. No. P0013B), and the BCA protein concentration determination kit (enhanced, Biyun Tian, P0010S) was used for accurate quantification. 40 μg of protein sample was loaded, and according to the molecular weight of KL protein (135 kDa), a 8% concentration of separation gel was prepared for SDS-PAGE electrophoresis, and the process was as follows:

[0104] Transferring: Transferring conditions 300 mA, 120 minutes, ice bath. The proteins were transferred from the gel to the PVDF membrane. After transferring, the transfer effect was observed quickly by staining with ponceau red, and then washed with water.

[0105] Immunoblotting and antibody incubation

[0106] Blocking: The PVDF membrane was blocked with a PBST solution (0.05% Tween 20 in PBS) containing 5% (w / v) skimmed milk powder on a shaker at room temperature for 1 hour.

[0107] Primary antibody incubation: The KL antibody of the present application and various brand antibodies were diluted 1:1000 in PBST containing 5% milk, and the various brand antibodies were: Ab203576, sc-315939, KM2076. Each antibody was incubated with the membrane on a shaker at 4°C overnight.

[0108] Washing the membrane: The next day, wash quickly with TBST on a shaker for 3 times, 5 minutes each time.

[0109] Secondary antibody incubation: Place all membrane strips in the corresponding HRP-labeled secondary antibody solution (HRP-labeled goat anti-mouse antibody: Jackson ImmunoResearch Laboratories, Cat. No. 115-035-146), and incubate on a shaker at room temperature for 1 hour.

[0110] Washing the membrane: Wash with TBST on a shaker for 3 times, 5 minutes each time, to remove unbound secondary antibodies and reduce background.

[0111] Chemiluminescence detection and imaging: Add ECL luminescent working solution (SuperSignal™ West Pico PLUS chemiluminescent substrate, ThermoFisher Scientific, Cat. No. 34580) evenly to the membrane, and react for 1-2 minutes.

[0112] The same exposure time was used to acquire images in the chemiluminescence imaging system and recorded. The results are shown in Figure 2 Figure 3 The results show that the KL antibody can specifically recognize endogenous (KL) and exogenous (KL-GFP) KL protein.

[0113] 2. Antibody sensitivity detection:

[0114] The experiment first extracted the lysate of KL-GFP transfected 293T cells. The extraction process was as follows: 2 mL of pre-cooled PBS was added to the 293T cells (60 mm cell culture dish) transfected with pCDH-CMV-KL (human)-EGFP plasmid to wash the cells once, then 1 mL of RIPA lysis buffer (QIAGEN, Catalog No. P0013B) was added, and the lysis buffer was incubated on ice for 20 minutes. The lysis buffer was transferred to a centrifuge tube, centrifuged at 4°C and 12000 rpm for 15 minutes, and the supernatant was collected, which was the 293T cell lysate containing KL-GFP.

[0115] After the 293T cell lysate containing KL-GFP was quantified by BCA method, 40 µg to 5 µg concentration gradient samples (40 µg, 20 µg, 10 µg, 5 µg) were prepared, treated with loading buffer (SDS-PAGE protein loading buffer (6X), QIAGEN, Catalog No. P0015F) and incubated at 60°C for 10 minutes. Then SDS-PAGE electrophoresis (8% separation gel) was performed, and Marker and gradient protein samples were loaded in turn. The membrane was transferred to PVDF membrane at 300 mA for 120 minutes, and the transfer effect was verified by ponceau staining. After blocking the membrane with 5% skim milk for 1 hour, the primary antibody (including self-made KL antibody and multiple brand antibodies KM2076, SC-315939, Ab203576) diluted 1:1000 was incubated at 4°C overnight. After washing with PBST, the HRP-labeled secondary antibody (HRP-labeled goat anti-mouse antibody, purchased from Jackson Immuno Research Laboratories, Catalog No. 115-035-146) was incubated at room temperature for 1 hour. After washing again, ECL luminescence solution was used for reaction, and finally the images were acquired by uniform exposure in the chemiluminescence imaging system. The results are shown in Figure 3 Figure 4 The results show that the KL antibody prepared by the present application has a sensitivity significantly higher than that of the other three brands of antibodies. The KL antibody prepared by the present application can still sensitively detect KL protein at a concentration of 5 µg.

[0116] Using the same method, the specific antibody sb106 in the prior art US10228374B2 was detected, and the results showed that the specific antibody sb106 could not detect KL protein at a concentration of 40 µg. ​​

[0117] Example 5

[0118] ELISA comparison of sensitivity of different brands of antibodies

[0119] 1. Coat high protein affinity ELISA plate with KL protein at 37°C for 2 hours. After washing 3 times with washing buffer (PBS containing 0.05% Tween 20), block the plate with PBST solution (PBS containing 0.05% Tween 20) containing 5% (w / v) skimmed milk powder at room temperature for 1 hour.

[0120] 2. Dilute the KL antibodies prepared according to the present application and antibodies of different brands at different gradients: 1 / 1600, 1 / 3200, 1 / 6400, 1 / 12800, 1 / 25600, 1 / 51200, 1 / 102400.

[0121] 3. After washing the blocked ELISA plate 3 times with washing buffer, add the diluted antibodies and incubate overnight at 4°C. Wash the wells 4 times with washing buffer, and add 100 μL per well of HRP-conjugated corresponding anti-mouse IgG antibody (Jackson ImmunoResearch Laboratories Inc. 115-035-146), anti-rat IgG antibody (Servicebio, GB23302) or anti-rabbit IgG antibody (Jackson ImmunoResearch Laboratories Inc. 111-005-144) diluted 1:5000 with PBST solution containing 5% (w / v) skimmed milk powder.

[0122] The method of HRP-conjugated antibody is similar to the method of KL-OVA conjugation, and is as follows:

[0123] ① To 1 mL of solution containing 1 mg / mL KL antibody, 100 μL of SMCC with a concentration of 1.5 mg / mL was added, and incubated at room temperature for 30 minutes; ② Then, the un-crosslinked SMCC was removed by Zeba™ Dye and Biotin Desalting Spin Columns and Filter Plates (5 mL spin columns, Thermo Fisher Scientific, Cat. No. A44300) according to the instructions; the obtained protein was KL antibody-SMCC, and the concentration of KL antibody-SMCC was tested; ③ KL-SMCC and horseradish peroxidase (HRP) protein (Aldrich, Cat. No. P105528) were mixed at a ratio of 1:1, and incubated at room temperature for 30 minutes; ④ After the reaction was completed, the solution was added to an Amicon Ultra-15 centrifugal filter with an Ultracel-100 filter membrane, and centrifuged at 2000 rpm (revolutions per minute) for 10 minutes; the protein cut off was HRP-conjugated KL antibody, and the concentration was measured by a BCA kit, and the protein was stored.

[0124] The plate was incubated at 37°C for 1 hour and washed 4 times in washing buffer. 100 μL of tetramethylbenzidine (TMB) chromogenic solution was added to each well. After color development, the reaction was terminated with 1M HCl, and the absorbance was measured at 450 nm. The data were processed by GraphPad software. The results are shown in FIG. 1, wherein Figure 4 a in FIG. 1 is a color development photograph of the ELISA plate of each antibody, Figure 4 b in FIG. 1 is a column chart of the ELISA detection results of each antibody. Figure 5 Figure 5 It is shown that the sensitivity of the KL antibody prepared by the present application is significantly better than that of other brands of antibodies at all dilutions.

[0125] Example 6

[0126] Detection of KL protein levels in serum of CKD mice

[0127] 1. Sample preparation

[0128] Mouse serum samples:

[0129] ​Experimental group: CKD model mice (5 / 6 nephrectomy mice after 8 weeks, UUO model mice after 2 weeks). The preparation method of UUO model refers to the prior art (Elena Martínez-Klimova, Aparicio-Trejo O E, 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 prior art (Tan R Z, Zhong X, Li J C, 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.).

[0130] Control group: healthy control of the same strain (sham group, i.e. Sham group). The sham group only opened the abdominal cavity during the operation, without kidney resection or unilateral ureter ligation (UUO) operation, and then sutured the wound. That is, except for not doing kidney resection or unilateral ureter ligation (UUO) operation, the sham group is completely the same as the model group.

[0131] Sample collection: whole blood was collected by orbital blood sampling, and the blood was allowed to stand at room temperature for 30 minutes, then centrifuged at 4°C and 3000 rpm for 15 minutes, and the supernatant (i.e. serum) was aspirated, aliquoted and stored at -80°C to avoid repeated freezing and thawing.

[0132] Dissolution reagent: all reagents and serum samples were taken out from -80°C and slowly thawed on ice.

[0133] 2. Antibody incubation and color development

[0134] ELISA plate coating: Dilute KL antibody (Cosmo Bio, KAL-KO603) to 2 pg / mL with PBS and add to the wells of the ELISA plate. Then add 50 pL of 2 pg / mL KL antibody (antibody prepared by the present application) to each well of the ELISA plate and incubate at room temperature for 2 hours. Wash each well with 200 pL of PBST (PBS containing 0.05% Tween 20) for 3 times.

[0135] Blocking: Add 100 pL of PBST containing 5% skimmed milk to each well of the ELISA plate and incubate at room temperature for 1 hour. Then wash each well with 200 pL of PBST for 3 times.

[0136] Sample addition: Take 100 pL of standard or diluted serum sample and add to the corresponding microwells. Set up blank wells (add diluent only). Gently shake the reaction plate and cover with sealing film, and incubate at 37°C for 2 hours.

[0137] Plate washing: Discard the liquid in the wells, add 200 pL of PBST containing 5% skimmed milk to each well, and discard after standing for 30 seconds. Repeat this process 4 times. Finally, pat dry on absorbent paper.

[0138] Add detection antibody: Add 100 pL of HRP-conjugated KL antibody (antibody prepared by the present application) to each well, cover with a new sealing film, and incubate at 37°C for 1 hour.

[0139] Plate washing: Same as above, wash the plate 4 times and pat dry.

[0140] Color development: Add 90 pL of TMB color development substrate to each well and develop color at 37°C for 15-30 minutes in the dark. Observe closely during this period, and when the high concentration wells of the standard show a clear blue gradient, proceed to the next step.

[0141] Reaction termination: Add 50 pL of termination solution (2M H2SO4) to each well, and the solution color immediately changes from blue to yellow. Gently shake the reaction plate to ensure uniform mixing.

[0142] Plate reading: Within 15 minutes after adding the termination solution, measure the absorbance (OD value) of each well at 450 nm wavelength using a microplate reader. Use 630 nm as the reference wavelength for correction.

[0143] Statistical analysis: Use GraphPad Prism software to make a column chart of the data values of each group of 6. Use unpaired t-test (compare two groups), P<0.05 is considered to have statistical significance. The results are shown in FIG. 1, where, Figure 5 a is a column chart of the level of KL protein in the serum of UUO model mice, ​ ​ ​The b in the above table is a column chart of the KL protein level in the serum of the 5 / 6 kidney cut model mice. The results shown in the chart are the results of 450 nm wavelength. The results show that the KL protein level in the serum of the chronic kidney disease model animals (5 / 6 kidney cut and UUO) mice is significantly lower than that of the sham operation group (Sham). This is consistent with the results of the gold standard detection, and the KL level in the fibrosis model mice is significantly lower than that in the control sham operation group. It is shown that the antibody prepared by the application can be used for the detection of the KL protein in the serum, and the result has high accuracy and high sensitivity.

[0144] It should be noted that the above examples are only examples for clearly illustrating the present application, and are not intended to limit the embodiments of the present application. Based on the above description, other different forms of changes or variations can also be made by those of ordinary skill in the art. Here, all the embodiments cannot be exhausted. Any obvious changes or variations derived from the technical scheme of the present application are still within the scope of protection of the present application.

Claims

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

6.

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

8.

3. A nucleic acid molecule, characterized in that, which encodes the alpha Klotho antibody of claim 1 or 2.

4. A vector, characterized by, which contains the nucleic acid molecule of claim 3.

5. An αKlotho protein detection kit, characterized by, which contains the alpha Klotho antibody of claim 1 or 2 or the nucleic acid molecule of claim 3.

6. Use of the αKlotho antibody of claim 1 or 2, the nucleic acid molecule of claim 3, the vector of claim 4, or the αKlotho protein detection kit of claim 5, characterized in that, the use as a reagent for the preparation of an assay for assessing the level of alpha Klotho protein in a serum sample.

7. Use according to claim 6, characterized in that, the assessment is based on detecting the amount of alpha Klotho protein in a serum sample from the individual.

8. A reagent for detecting αKlotho protein, characterized by, which comprises the alpha Klotho antibody of claim 1 or 2, the nucleic acid molecule of claim 3, or the vector of claim 4.

9. Use of the αKlotho antibody of claim 1 or 2, the nucleic acid molecule of claim 3, the vector of claim 4, or the αKlotho protein detection kit of claim 5, characterized in that, the use as a detection reagent for chronic kidney disease.

10. A reagent for diagnosing chronic kidney disease, characterized by comprising an antibody that binds to a polypeptide of claim 1. which comprises the alpha Klotho antibody of claim 1 or 2, the nucleic acid molecule of claim 3, or the vector of claim 4.

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