Anti-pentosidine monoclonal antibody and pentosidine measurement reagent using the antibody

Abstract

This invention provides a novel anti-pentosidine monoclonal antibody, a hybridoma that produces the anti-pentosidine monoclonal antibody, a reagent for measuring pentosidine containing the anti-pentosidine monoclonal antibody, a pentosidine measurement kit containing the pentosidine measurement reagent, and a method for measuring pentosidine by immunoassay using the anti-pentosidine monoclonal antibody. [Solution] A monoclonal antibody against pentosidine or its antigen-binding fragment produced by a hybridoma with accession number NITE BP-04144 or NITE BP-04145.

Description

[Technical Field]

[0001] The present invention relates to a monoclonal antibody against pentosidine or its antigen-binding fragment, a hybridoma that produces a monoclonal antibody against pentosidine, an isolated polynucleotide encoding a monoclonal antibody against pentosidine or its antigen-binding fragment, an expression vector containing the polynucleotide, a host cell containing the expression vector, a method for producing a monoclonal antibody against pentosidine or its antigen-binding fragment, a reagent for measuring pentosidine containing a monoclonal antibody against pentosidine, a pentosidine measurement kit containing a pentosidine measurement reagent, and a method for immunoassaying pentosidine using a monoclonal antibody against pentosidine. [Background technology]

[0002] Advanced glycation end products (AGEs) are a general term for structures produced in the later stages of non-enzymatic glycation reactions (Maillard reactions) between proteins and sugars. The accumulation of AGEs has been reported to be involved in diabetic complications, vascular diseases, kidney diseases, and Alzheimer's disease (Non-Patent Literature 1).

[0003] Pentosidine is a type of advanced glycation end products (AGEs) with a structure in which lysine and arginine residues are cross-linked by a pentose sugar. Accumulation of pentosidine is known to be associated with various diseases, including diabetes, chronic renal failure, cataracts, osteoporosis, and Alzheimer's disease (Non-Patent Literature 2).

[0004] Furthermore, pentosidine is an abnormal crosslinking between collagen that weakens bone, and various clinical studies have reported that urinary pentosidine concentration is a useful indicator for evaluating bone quality (Non-Patent Literature 3 and Non-Patent Literature 4).

[0005] High-performance liquid chromatography (HPLC) is an established method for measuring pentosidine. While HPLC offers high separation ability and sensitivity, it has drawbacks such as complicated sample preparation, long measurement times, and unsuitability for processing multiple samples.

[0006] On the other hand, a method for measuring pentosidine using an anti-pentosidine polyclonal antibody by enzyme-linked immunosorbent assay (ELISA) has been reported (Non-Patent Literature 5). However, polyclonal antibodies have problems such as a high likelihood of cross-reactivity and the fact that lot-to-lot variability is likely to occur, making quality control complicated. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Bierhaus A, “AGEs and their interaction with AGE-receptors in vascular disease anddiabetes mellitus. I. The AGE concept”, Cardiovasc Res, Vol. 37(3), 586-600 (1998) [Non-Patent Document 2] Toriumi, Kazuya et al. “Glucuronic acid is a novel source of pentosidine, associated with schizophrenia.” Redox biology vol. 67 (2023): 102876. doi:10.1016 / j.redox.2023.102876 [Non-Patent Document 3] Shiraki, Masataka et al. “The association of urinary pentosidine levels with the prevalence of osteoporotic fractures in postmenopausal women.” Journal of bone and mineral metabolism vol. 37,6 (2019): 1067-1074. doi:10.1007 / s00774-019-01017-9 [Non-Patent Document 4] Hagino, Hiroshi et al. “Urinary pentosidine level is associated with the risk of fracture in community-dwelling older adults: a prospective observational study.” Osteoporosis international : a journal established as result of cooperation between the European Foundation for Osteoporosis and the National Osteoporosis Foundation of the USA vol. 34,10 (2023): 1703-1709. doi:10.1007 / s00198-023-06816-5 [Non-Patent Document 5] Kashiwabara, Shoji, et al. “Development and evaluation of novel ELISA for determination of urinary pentosidine.” Journal of Nutritional Science and Vitaminology 65.6 (2019): 526-533. [Overview of the project] [Problems that the invention aims to solve]

[0008] Given the current state of the technology, the present invention provides a novel anti-pentosidine monoclonal antibody, a hybridoma that produces the anti-pentosidine monoclonal antibody, a reagent for measuring pentosidine containing the anti-pentosidine monoclonal antibody, a pentosidine measurement kit containing the pentosidine measurement reagent, and a method for measuring pentosidine by immunoassay using the anti-pentosidine monoclonal antibody. [Means for solving the problem]

[0009] The inventors of this invention have succeeded in creating a novel monoclonal antibody against pentosidine, confirmed the suitability of this antibody for the measurement of pentosidine, and thus arrived at the present invention.

[0010] In other words, the present invention is as follows: [1] A monoclonal antibody against pentosidine or its antigen-binding fragment produced by the hybridoma of accession number NITE BP-04144. [2] A monoclonal antibody against pentosidine or its antigen-binding fragment produced by the hybridoma of accession number NITE BP-04145. [3] Hybridoma with accession number NITE BP-04144. [4] Hybridoma with accession number NITE BP-04145. A monoclonal antibody against pentosidine or an antigen-binding fragment thereof, comprising a light chain containing three CDRs (LCDR1, LCDR2, and LCDR3) of the light chain of the monoclonal antibody described in [5][1] or [2], and a heavy chain containing three CDRs (HCDR1, HCDR2, and HCDR3) of the heavy chain of the monoclonal antibody. The monoclonal antibody or antigen-binding fragment according to [5], comprising a light chain containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence of the light chain of the monoclonal antibody described in [6][1] or [2], and a heavy chain containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence of the heavy chain of the monoclonal antibody. [7] An antigen-binding fragment selected from the group consisting of Fab', (Fab')2, Fab, scF, Fv, dsFv, and dsscFv, as described in any of [1], [2], [5], and [6]. An isolated polynucleotide encoding a monoclonal antibody or its antigen-binding fragment as described in any of [8][1], [2], [5], and [6]. An expression vector containing the polynucleotides described in [9][8]. Host cells containing the expression vector described in

[10] [9]. A method for producing a monoclonal antibody or an antigen-binding fragment according to any one of [1], [2], [5], and [6], comprising culturing the host cells described in

[11] and

[10] under conditions capable of expressing the monoclonal antibody or an antigen-binding fragment thereof. A reagent for measuring pentosidine, comprising a monoclonal antibody or its antigen-binding fragment as described in any of

[12] [1], [2], [5], and [6]. A pentosidine measurement kit containing the pentosidine measurement reagents described in

[13]

[12] . A method for measuring pentosidine in a sample by an immunoassay using a monoclonal antibody or its antigen-binding fragment as described in any of

[14] [1], [2], [5], and [6]. The method according to

[14] , comprising the step of contacting a monoclonal antibody or an antigen-binding fragment thereof, as described in any of

[15] [1], [2], [5], and [6], with a sample.

[16] The method according to

[14] or

[15] , wherein the immunoassay is an enzyme-linked immunosorbent assay.

[17] The method according to any one of

[14] to

[16] , wherein the sample is serum, urine, or a dilution thereof. [Brief explanation of the drawing]

[0011] [Figure 1] The results of pentosidine measurement using competitive ELISA with Pentosidine 72-2-2-6 are shown. The vertical axis represents the absorbance at 450 nm, and the horizontal axis represents the concentration of pentosidine. [Figure 2] The results of pentosidine measurement by a competitive ELISA assay using Pentosidine 72-3-2-3 are shown. The vertical axis indicates the absorbance at 450 nm, and the horizontal axis indicates the concentration of pentosidine. [Figure 3] The results of the dilution linearity test of Pentosidine 72-2-2-6 are shown. The vertical axis indicates the concentration of pentosidine, and the horizontal axis indicates the dilution ratio of the sample. [Figure 4] The results of the dilution linearity test of Pentosidine 72-3-2-3 are shown. The vertical axis indicates the concentration of pentosidine, and the horizontal axis indicates the dilution ratio of the sample.

Mode for Carrying Out the Invention

[0012] Embodiments of the present invention will be described in detail. However, the present invention is not limited to the following embodiments.

[0013] In one embodiment, the anti-pentosidine monoclonal antibody is obtained by immunizing a mammal such as a mouse with pentosidine as an antigen. For example, it is produced by a hybridoma obtained by immunizing a mammal with pentosidine as an antigen and fusing the anti-pentosidine antibody-producing cells produced by the mammal with myeloma cells. Pentosidine is a compound represented by the following formula, and its molecular weight is

[0014] 378.43. Pentosidine has a structure that crosslinks lysine residues and arginine residues in proteins.

Chemical Formula

[0014] Antigens may be used directly for immunization, but to enhance antigenicity, it is preferable to use a complex of antigen with a carrier protein. Examples of carrier proteins include bovine serum albumin, thyroglobulin, hemocyanin, and keyhole limpet hemocyanin (KLH). Condensing agents such as glutaraldehyde, carbodiimide, and maleimide active esters can be used to prepare the complex of antigen and carrier protein. Preferably, pentosidine is used for immunization as a complex with KLH.

[0015] Mammals that can be immunized include mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, pigs, goats, horses, or cattle. Immunosensitization is performed by injecting or transplanting the immunogen subcutaneously, intramuscularly, intravenously, in the foot pad, or intraperitoneally into the above-mentioned mammals one to several times. Typically, immunization is performed one to four times at intervals of approximately 1 to 14 days from the initial immunization, and antibody-producing cells are obtained from the immunized mammals approximately 1 to 5 days after the final immunization. When administering the immunogen, it may be mixed with complete or incomplete Freund's adjuvant. Antibody-producing cells obtained from the spleen or lymph nodes of immunized animals are fused with myeloma cells to prepare hybridomas.

[0016] Hybridomas can be prepared according to the method of Köhler and Milstein (Nature. 256, 495. 1975) and similar methods.

[0017] Myeloma cells used in cell fusion are typically derived from mammals, such as mice, rats, or humans. For example, mouse-derived myeloma cells P3-X63-Ag8-U1 (P3U1), P3 / X63-AG8.653 (653), P3 / NSI / 1-Ag4-1 (NS-1), SP2 / 0-Ag14 (Sp2 / O, Sp2), PAI, F0, or BW5147; rat-derived myeloma 210RCY3-Ag.2.3; and human-derived myeloma cells U-266AR1, GM1500-6TG-A1-2, UC729-6, CEM-AGR, D1R11, or CEM-T15 can be used.

[0018] Screening for hybridoma clones that produce monoclonal antibodies can be performed by culturing the hybridomas, for example, in a microtiter plate, and measuring the reactivity of the culture supernatant from the wells to the immune antigen using an immunochemical method such as ELISA.

[0019] Clones can be obtained by further cloning using the limiting dilution method from wells containing hybridomas that produce the target antibody. Hybridoma selection and breeding are usually performed in animal cell medium containing 10-20% fetal bovine serum with the addition of HAT (hypoxanthine, aminopterin, thymidine).

[0020] Monoclonal antibodies from hybridomas can be produced by culturing hybridomas in vitro or by growing them in vivo in the ascites fluid of mammals such as mice and rats, and then isolating the resulting culture supernatant or the mammalian ascites fluid.

[0021] When culturing in vitro, it is possible to use a nutrient medium suitable for growing, maintaining, and preserving hybridomas and producing monoclonal antibodies in the culture supernatant, depending on the characteristics of the cell type being cultured and various conditions such as the culture method.

[0022] Monoclonal antibodies can be isolated and purified using methods known for purifying immunoglobulins. Examples include salting-out with ammonium sulfate or sodium sulfate, chromatography, ion-exchange chromatography, and affinity chromatography with protein A or protein G.

[0023] Anti-pentosidine monoclonal antibodies or their antigen-binding fragments can also be obtained by phage display. In phage display, phages selected from any phage antibody library are screened using the immunogen of interest to select phages with the desired binding affinity to the immunogen. Next, the antibody-corresponding sequence contained within the phage is isolated or sequenced, and an expression vector containing a nucleic acid molecule encoding the antibody or antigen-binding domain is constructed based on the isolated or sequenced information. Then, by culturing a cell line transfected with such an expression vector, monoclonal antibodies or antigen-binding fragments can be produced.

[0024] In one embodiment, the anti-pentosidine monoclonal antibody or its antigen-binding fragment is a monoclonal antibody against pentosidine or its antigen-binding fragment produced by the hybridoma (Pentosidine 72-2-2-6) of accession number NITE BP-04144 or the hybridoma (Pentosidine 72-3-2-3) of accession number NITE BP-04145.

[0025] Hybridomas Pentosidine 72-2-2-6 and Pentosidine 72-3-2-3 were received by the Patent Microorganism Depositary Center within the National Institute of Technology and Evaluation (NITE) on August 9, 2024, under receipt numbers NITE ABP-04144 (Pentosidine 72-2-2-6) and NITE ABP-04145 (Pentosidine 72-3-2-3). After confirmation of their viability on August 26, 2024, they were assigned accession numbers NITE BP-04144 (Pentosidine 72-2-2-6) and NITE BP-04145 (Pentosidine 72-3-2-3) (Certificate of Accession issued September 10, 2024). The following details identify the deposit. [1] Name of depositary institution and recipient Name: Patent Microbial Deposit Center, National Institute of Advanced Industrial Science and Technology (AIST), National Institute of Technology and Evaluation Address: 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan (Postal Code 292-0818) [2]Deposit date: August 9, 2024 [3] Accession number NITE BP-04144 (Hybridoma Pentosidine 72-2-2-6) NITE BP-04145 (Hybridoma Pentosidine 72-3-2-3)

[0026] In this invention, the "antigen-binding fragment" of an antibody refers to a part of an antibody that includes at least an antigen-binding region. Specific examples include Fab', (Fab')2, Fab, Fv, single-chain antibody (scFv), disulfide-stabilized antibody (dsFv), and disulfide-stabilized single-chain antibody (dsscFv).

[0027] In one embodiment, an anti-pentosidine monoclonal antibody or its antigen-binding fragment comprises a light chain containing three CDRs (LCDR1, LCDR2, and LCDR3) of the light chain of an anti-pentosidine antibody produced by hybridoma Pentosidine 72-2-2-6 and / or hybridoma Pentosidine 72-3-2-3, and a heavy chain containing three CDRs (HCDR1, HCDR2, and HCDR3) of the heavy chain of the monoclonal antibody, while also encompassing monoclonal antibodies or their antigen-binding fragments that are structurally identical or different from these produced antibodies except for the CDRs. Here, a complementarity-determining region (CDR) refers to the region of an immunoglobulin molecule's variable region that forms an antigen-binding site, also called a hypervariable region, and is a part of the immunoglobulin molecule where the amino acid sequence changes particularly large. There are three CDRs in both the light chain and the heavy chain.

[0028] Accordingly, in one embodiment, the anti-pentosidine monoclonal antibody or its antigen-binding fragment comprises a light chain containing the amino acid sequence of the light chain of the anti-pentosidine antibody produced by hybridoma Pentosidine 72-2-2-6 or hybridoma Pentosidine 72-3-2-3, and a heavy chain containing the amino acid sequence of the heavy chain of the anti-pentosidine antibody produced by hybridoma Pentosidine 72-2-2-6 or hybridoma Pentosidine 72-3-2-3. On the other hand, the amino acid sequences of the light and heavy chains contained in a monoclonal antibody or its antigen-binding fragment may have one or more amino acid substitutions, deletions, additions, or insertions (1 to 20, 1 to 10, or 1 to 5) in the amino acid sequence of an anti-pentosidine antibody produced by hybridoma Pentosidine 72-2-2-6 or hybridoma Pentosidine 72-3-2-3, as long as the ability to bind to pentosidine is maintained.

[0029] In one embodiment, the anti-pentosidine monoclonal antibody or its antigen-binding fragment comprises a light chain containing an amino acid sequence having 90% or more (preferably 95%, 96%, 97%, 98%, 99% or more) sequence identity with the amino acid sequence of the light chain of the anti-pentosidine antibody produced by hybridoma Pentosidine 72-2-2-6 or hybridoma Pentosidine 72-3-2-3, and a heavy chain containing an amino acid sequence having 90% or more (preferably 95%, 96%, 97%, 98%, 99% or more) sequence identity with the amino acid sequence of the heavy chain of the anti-pentosidine antibody produced by hybridoma Pentosidine 72-2-2-6 or hybridoma Pentosidine 72-3-2-3.

[0030] In this specification, amino acid sequence identity refers to the degree of amino acid agreement when two or more amino acid sequences being compared are optimally aligned. Amino acid sequence identity can be calculated using commercially available analysis tools or tools available via telecommunication lines (the Internet). For example, it can be calculated using the default parameters of the National Center for Biotechnology Information (NCBI) homology algorithm BLAST (Basic local alignment search tool) (http: / / www.ncbi.nlm.nih.gov / BLAST / ).

[0031] In one embodiment, the anti-pentosidine monoclonal antibody or its antigen-binding fragment may be labeled with a labeling substance.

[0032] Examples of labeling substances include enzymes, fluorescent substances, luminescent substances, radioactive substances, metal colloids, and colorants. Examples of enzymes include alkaline phosphatase, peroxidase, β-galactosidase, and luciferase. Examples of fluorescent substances include fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, and red fluorescent protein. Examples of luminescent substances include luciferin, aequorin, acridinium ester, tris(2,2'-bipyridyl)ruthenium, and luminol. Examples of radioactive substances include: 3 H, 14 C, 32 P, 35 S, 125 Examples include I. Examples of metal colloids include gold colloid, silver colloid, platinum colloid, iron oxide colloid, and aluminum hydroxide colloid. Examples of coloring substances include latex particles colored with dyes, pigments, etc. The labeling substance is not particularly limited. In the present invention, a preferred labeling substance is horseradish peroxidase (HRP). Labeling antibodies with labeling substances can be performed using a known method selected according to the type of labeling substance.

[0033] One embodiment of the present invention relates to an isolated polynucleotide encoding an anti-pentosidine monoclonal antibody or its antigen-binding fragment, an expression vector and host cells containing the same, and a method for producing an anti-pentosidine monoclonal antibody or its antigen-binding fragment using the same. Such polynucleotides can be obtained, for example, by the following method. First, total RNA is prepared from cells such as hybridomas that produce anti-pentosidine monoclonal antibodies using a commercially available RNA extraction kit, and cDNA is synthesized by reverse transcriptase using random primers, etc. Next, the cDNA encoding the antibody is amplified by PCR using oligonucleotides of sequences conserved in the variable regions of known antibody heavy chain genes and light chain genes, respectively, as primers. The sequence encoding the constant region can be obtained by amplifying a known sequence by PCR. The base sequence of the DNA can be determined by conventional methods, such as by incorporating it into a sequencing plasmid.

[0034] Expression vectors are appropriately selected depending on the intended use, the type of host cell, etc. For example, vectors that use E. coli as a host include M13 phage or its variants, λ phage or its variants, pBR322 or its variants (e.g., pB325, pAT153, pUC8), etc. Vectors that use yeast as a host include pYepSec1, pMFa, pYES2, pPIC3.5K, etc. Vectors that use insect cells as a host include pAc, pVL, etc. Vectors that use mammalian cells as a host include pcDNA, pCDM8, pMT2PC, etc.

[0035] Examples of host cells that can be used include mammalian, plant, insect, bacterial, and yeast host cells.

[0036] The obtained host cells can be cultured under conditions that allow expression of anti-pentosidine monoclonal antibodies or their antigen-binding fragments. The products can then be collected and, if necessary, purified to obtain anti-pentosidine monoclonal antibodies or their antigen-binding fragments. The culture medium composition and conditions can be those well known for antibody production.

[0037] In one embodiment, an anti-pentosidine monoclonal antibody or its antigen-binding fragment can be used in a method for measuring pentosidine in a sample by immunoassay. The method for measuring pentosidine includes the step of contacting the above-described anti-pentosidine antibody or its antigen-binding fragment with the sample.

[0038] Examples of immunoassay methods include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), chemiluminescent enzyme immunoassay (CLEIA), chemiluminescent immunoassay (CLIA), immunofluorescence assay (FAT), enzyme-fluorescence immunoassay (FEIA), electrochemiluminescence immunoassay (ECLIA), radioimmunoassay (RIA), immunoturbidimetry (TIA), immunochromatography, agglutination, and competitive assays. In the present invention, the preferred immunoassay method is the ELISA method. Furthermore, either a non-competitive method or a competitive method may be used for the immunoassay. Preferably, a competitive method is used. The detection method in the immunoassay may be either a direct method, in which a labeling substance is directly labeled onto the primary antibody, or an indirect method, in which the primary antibody is specifically detected using a secondary antibody labeled with a labeling substance. Preferably, the indirect method is used as the detection method in the immunoassay.

[0039] The ELISA method is a method for measuring a target antigen in a sample by capturing it with a specific antibody and using an enzymatic reaction. In the present invention, any of the following ELISA methods may be used: the direct method, the indirect method, the sandwich method, or the competitive method. The direct method is performed, for example, as follows: Pentosidine from the sample is immobilized on a microplate, an enzyme-labeled anti-pentosidine monoclonal antibody or its antigen-binding fragment is reacted with it, and after washing, the enzyme activity remaining on the microplate is detected. The indirect method is performed, for example, as follows: Pentosidine from the sample is immobilized on a microplate, an anti-pentosidine monoclonal antibody or its antigen-binding fragment is reacted with it, followed by a reaction with an enzyme-labeled secondary antibody against the antibody or its antigen-binding fragment, and after washing, the enzyme activity remaining on the microplate is detected. The sandwich method is performed, for example, as follows: An anti-pentosidine monoclonal antibody or its antigen-binding fragment is immobilized on a microplate, reacted with the sample, washed, then reacted with another enzyme-labeled anti-pentosidine monoclonal antibody or its antigen-binding fragment, washed, and the remaining enzyme activity on the microplate is detected. The competitive method can be performed, for example, as follows: Pentosidine is immobilized on a microplate, and the sample and an enzyme-labeled anti-pentosidine monoclonal antibody or its antigen-binding fragment are reacted simultaneously in the same microplate. After washing, the enzyme activity remaining in the microplate is detected. The anti-pentosidine monoclonal antibody or its antigen-binding fragment does not have to be enzyme-labeled; in this case, the sample and the anti-pentosidine monoclonal antibody or its antigen-binding fragment are reacted simultaneously in the same microplate, and after washing, an enzyme-labeled secondary antibody against that antibody or its antigen-binding fragment is reacted, and after washing, the enzyme activity remaining in the microplate is detected. Alternatively, the competitive method may be performed as follows: Anti-pentosidine monoclonal antibody or its antigen-binding fragment is immobilized on a microplate, and the sample and an enzyme-labeled pentosidine of known concentration are reacted simultaneously in the same microplate. After washing, the enzyme activity remaining in the microplate is detected. Enzymatic labeling of pentosidine can be performed using known methods. In the competitive method, if there is a large amount of antigen in the sample, the enzyme-labeled antibody that can bind to the antigen or the enzyme-labeled antigen that can bind to the antibody decreases, resulting in weaker color development. Conversely, if there is a small amount of antigen in the sample, the enzyme-labeled antibody that can bind to the antigen or the enzyme-labeled antigen that can bind to the antibody increases, resulting in stronger color development. In the present invention, a competitive ELISA method is preferably used. Furthermore, there are no particular restrictions on the method for detecting the labeled enzyme, and various detection systems can be used depending on the labeled enzyme used. For example, a sensitization method may be used in which the secondary antibody is biotinylated and a biotin-avidin (streptavidin) complex is formed for detection.

[0040] Chemiluminescent enzyme immunoassay (CLEIA) is performed, for example, in the case of a competitive assay, as follows: The sample is reacted with pentosidine immobilized on magnetic particles or beads, and an anti-pentosidine monoclonal antibody or its antigen-binding fragment. After washing (B / F separation), the sample is reacted with an enzyme-labeled secondary antibody, washed again (B / F separation), and then a chemiluminescent substrate is added for the enzymatic reaction, after which the luminescence intensity is measured. A sensitization method may also be used in which the secondary antibody is biotinylated to form a biotin-avidin (streptavidin) complex for detection.

[0041] Chemiluminescent immunoassay (CLIA) is performed, for example, in the case of a competitive assay, as follows: The sample is reacted with pentosidine immobilized on magnetic particles or the like, and with an anti-pentosidine monoclonal antibody or its antigen-binding fragment. After washing (B / F separation), a secondary antibody labeled with a chemiluminescent substance is reacted, and after washing (B / F separation), the luminescence intensity is measured. Acridinium and the like are used as labeling substances. A sensitization method may also be used in which the secondary antibody is biotinylated to form a biotin-avidin (streptavidin) complex for detection.

[0042] Fluorescent enzyme immunoassay (FEIA) is performed, for example, in the case of a competitive assay, as follows: The sample is reacted with immobilized pentosidine and an anti-pentosidine monoclonal antibody or its antigen-binding fragment. After washing (B / F separation), the sample is reacted with an enzyme-labeled secondary antibody, washed again (B / F separation), a fluorescent substrate is added, and after the enzymatic reaction, the fluorescence intensity is measured. HRP and ALP are used as labeling enzymes. A sensitization method may also be used in which the secondary antibody is biotinylated to form a biotin-avidin (streptavidin) complex for detection.

[0043] Electrochemiluminescence immunoassay (ECLIA) is performed, for example, in the case of a competitive assay, as follows: The sample is reacted with pentosidine immobilized on magnetic particles and an anti-pentosidine monoclonal antibody or its antigen-binding fragment. After washing (B / F separation), a secondary antibody labeled with an electrochemiluminescent substance is reacted, followed by washing (B / F separation), and the luminescence intensity is measured by electrical energy. Ruthenium is used as the labeling substance. Ru(bpy)3 is used as the labeling substance, and excitation and luminescence are repeatedly induced by oxidation by charging the electrode and reduction by tripropylamine (TPA), etc. A sensitization method may also be used in which the secondary antibody is biotinylated and a biotin-avidin (streptavidin) complex is formed for detection.

[0044] Radioimmunoassay (RIA) is performed, for example, in the case of a competitive assay, as follows: The sample is reacted with pentosidine immobilized on beads or the like, and with an anti-pentosidine monoclonal antibody or its antigen-binding fragment. After this, the mixture is washed (B / F separation) and the radioisotopes are removed. 125 A secondary antibody labeled with (I) is reacted, and after washing (B / F separation), the radiation dose of the radioactive isotope is measured.

[0045] Immunochromatography is an immunoassay method that utilizes capillary action, where the test sample moves along a test strip while dissolving reagents. Pentosidine in the sample forms an immunocomplex with the labeled antibody and capture antibody on the test strip, and the color of the labeled substance is observed. Antibody labeling can be done using gold colloid, enzymes, fluorescent substances, etc. When using enzyme-labeled antibodies, the enzyme substrate is placed on the test strip to induce color development.

[0046] Agglutination is a method of observing agglutination by reacting an antigen in a sample with an antibody in a reagent. Examples include methods that do not use a solid phase, particle agglutination (PA) which uses artificially produced particles as the solid phase, and latex agglutination (LA) which uses latex particles as a type of PA.

[0047] The competitive method, for example, involves binding pentosidine to a solid phase, simultaneously reacting the test sample with a certain amount of anti-pentosidine monoclonal antibody or its antigen-binding fragment, and measuring the amount of pentosidine in the sample from the amount of bound anti-pentosidine monoclonal antibody or its antigen-binding fragment.

[0048] Samples for measuring pentosidine include, for example, bodily fluids obtained from the subject, such as blood (whole blood, plasma, serum, etc.), urine, tissue fluid, lymph fluid, synovial fluid, breast milk, cerebrospinal fluid, pus, saliva, tears, mucus, nasal discharge, sputum, ascites, water, and semen; as well as washing solutions after washing the nasal cavity, bronchi, lungs, skin, abdominal cavity, various organs, joints, and bones; or cell culture supernatants; or column eluents. These samples are used for measurement either as is, or diluted with various buffers or concentrated after extraction.

[0049] The sample is preferably urine or a dilution thereof. In the case of a urine sample, pretreatment such as acid hydrolysis or enzymatic digestion at high temperatures is unnecessary, allowing for simpler and faster measurement of pentosidine.

[0050] One embodiment of the present invention relates to a reagent for measuring pentosidine. The reagent comprises the above-mentioned anti-pentosidine monoclonal antibody or its antigen-binding fragment, and can be used when measuring pentosidine by an immunoassay. The pentosidine measuring reagent may optionally contain additives. Examples of additives include protein stabilizers such as bovine serum albumin (BSA), preservatives such as sodium azide, and inorganic salts such as sodium chloride, which are known additives in immunoassay reagents.

[0051] The form of the reagent for measuring pentosidine is not particularly limited and may be solid (e.g., powder, crystals, lyophilized product, etc.) or liquid (e.g., solution, suspension, emulsion, etc.). If the reagent is liquid, the solvent is not particularly limited as long as it can dissolve and store the anti-pentosidine monoclonal antibody or its antigen-binding fragment. Examples of solvents include water, physiological saline, phosphate-buffered saline (PBS), Tris-buffered saline (TBS), and Good's buffer. Examples of Good's buffers include MES, Bis-Tris, ADA, PIPES, Bis-Tris-Propane, ACES, MOPS, MOPSO, BES, TES, HEPES, HEPPS, Tricin, Tris, Bicine, and TAPS.

[0052] The reagents for measuring pentosidine constitute a pentosidine measurement kit. The pentosidine measurement kit is used to measure pentosidine in a sample by immunoassay and may further include auxiliary reagents. Examples of auxiliary reagents include, but are not limited to, labeled substances that bind to the antibody or antigen-binding fragment of the present invention (e.g., secondary antibody, protein G, protein A, etc.), chromogenic substrates, fluorescent substrates, chemiluminescent substrates, specific binding substances such as biotin-streptavidin, insoluble carriers, blocking agents, diluents, washing solutions, reagents for stopping the detection reaction of labeled substances (reaction stop agents), standard substances, positive controls, negative controls, etc. The auxiliary reagents are used in appropriate combinations according to the method of measuring pentosidine. For example, when horseradish peroxidase (HRP) is used as the labeling substance, examples of chromogenic substrates include 3,3',5,5'-tetramethylbenzidine (TMB), 2,2'-azo-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS), and o-phenylenediamine (OPD). When alkaline phosphatase (ALP) is used as the labeling substance, examples of chromogenic substrates include p-nitrophenyl phosphate (pNPP). When β-galactosidase is used as the labeling substance, examples of chromogenic substrates include o-nitrophenyl-β-D-galactopyranoside (ONPD).

[0053] For example, a pentosidine assay kit for ELISA includes i) a pentosidine assay reagent containing an anti-pentosidine monoclonal antibody or its antigen-binding fragment (which may be labeled), and ii) a substrate necessary for detecting the labeled substance. If the anti-pentosidine monoclonal antibody or its antigen-binding fragment is not labeled, the pentosidine assay kit may also include iii) a labeled substance that binds to the anti-pentosidine monoclonal antibody or its antigen-binding fragment (e.g., a secondary antibody, protein G, protein A, etc.). [Examples]

[0054] The present invention will be described in more detail by the following examples, but the present invention is not limited to these examples.

[0055] <Example 1> Preparation of anti-pentosidine monoclonal antibody (1) Preparation of antigens for producing anti-pentosidine monoclonal antibodies Synthetic full-length pentosidine (manufactured by Peptide Laboratories, Inc., CAT No: 3242-v) was conjugated to keyhole limpet hemocyanin (KLH, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), a carrier protein, to give it antigenicity, thereby obtaining pentosidine-KLH.

[0056] (2) Immunity Pentosidine-KLH was diluted with 20 mM Tris-HCl (pH 7.2) to a concentration of 1 mg / ml. 50 μl of 1 mg / ml pentosidine-KLH solution (containing 50 μg of pentosidine-KLH) and 50 μl of Freund's complete adjuvant (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were thoroughly mixed until emulsified. The prepared suspension was administered intraperitoneally to Balb / c 6-week-old female mice (manufactured by CLEA Japan, Ltd.) under diethyl ether anesthesia. Two weeks later, 50 μl of 50 μg / ml pentosidine-KLH solution and 50 μl of Freund's incomplete adjuvant (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were thoroughly mixed until emulsified to obtain an emulsion suspension, and mice were sensitized with this emulsion suspension. The same procedure was performed two weeks later, and for the fourth and final immunization, 50 μl of 50 μg / ml pentosidine-KLH solution was administered by tail vein injection to the mice.

[0057] (3) Establishment of hybridomas Three days after final immunization, the spleen was surgically removed from pentosidine-sensitized mice under diethyl ether anesthesia. The removed spleen was aseptically dispersed to prepare spleen cells. Fusion was performed according to the method of Kohler and Milstein (Nature. 256, 495. 1975), using polyethylene glycol (PEG4000) (Merck) to fuse spleen cells with myeloma cells P3-X63-Ag8-U1 (P3U1). The fusion ratio was 8 × 10⁶ spleen cells. 7 Each cell contains 2 × 10 myeloma cells P3-X63-Ag8-U1 (P3U1). 7 The ratio was 4:1. The fused cells were dispersed in α-MEM medium (GIBCO) containing 10% FCS (INVITROGEN) and 50-fold diluted HAT (Cosmo Bio), dispensed into 48-well microtiter culture plates (Sumitomo Bakelite), and cultured at 37°C under 5% CO2 conditions.

[0058] (4) Screening After approximately two weeks, we checked the colony's growth and conducted screening. The screening method is described below. i) To prepare screening plates, synthetic full-length pentosidine (Peptide Laboratories, Inc., CAT No: 3242-v) was conjugated to BSA, the pentosidine-BSA conjugate was dissolved in 0.1 mol / L carbonate buffer (pH 9.6), and dispensed into 96-well plates (Thermo) at a concentration of 0.5 μg / 100 μL / well. The plates were left to stand at 4°C for two nights, then washed three times with Tris buffer containing 0.05% Tween-20 to obtain a pentosidine-BSA conjugate immobilized plate. 100 μL of the culture supernatant of fusion cells was added to the pentosidine-BSA conjugate immobilized plate and reacted. After washing, the secondary antibody, HRP-labeled anti-mouse immunoglobulin antibody (Jackson Laboratory), was added and reacted. After washing, 100 μL of 3,3'5,5'-tetramethylbenzidine (TMB) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), a chromogenic substrate for HRP, was added. After a certain period of color development, another 100 μL of 1N sulfuric acid was added as a stop solution, and the absorbance was measured at a measurement wavelength of 450 nm. Clones that tested positive in this measurement were re-cloned using the limiting dilution method, and the supernatant was screened again. ii) For rescreening, following the protocol in i) above, clones producing IgG were selectively selected using HRP-labeled anti-mouse IgG Fc antibody (Merck) as the secondary antibody. Recloning was then performed again using the limiting dilution method, and the supernatant was further screened and tested. iii) In the second re-screening, pentosidine-immobilized plates, pentosidine-BSA conjugate-immobilized plates, KLH-immobilized plates, and BSA-immobilized plates were prepared according to the protocol in i) above, and these plates were used for screening. Clones that were positive for the pentosidine-immobilized plate and pentosidine-BSA conjugate-immobilized plate, and negative for the BSA-immobilized plate and KLH-immobilized plate were re-cloned using the limiting dilution method to establish new clones.

[0059] (5) Confirmation of antibodies When the reactivity with pentosidine was confirmed by ELISA, clones Pentosidine 72-2-2-6 and Pentosidine 72-3-2-3 reacted sensitively with the pentosidine-immobilized plate. As a result, clones Pentosidine 72-2-2-6 and Pentosidine 72-3-2-3 were selected as those that recognized pentosidine. The obtained antibodies were assayed using a monoclonal antibody typing kit (manufactured by ROCHE), and the results were as shown in Table 1 below.

Table 1

[0060] (6) Preparation and purification of monoclonal antibodies 1×10 7 Hybrids Pentosidine 72-2-2-6 and Pentosidine 72-3-2-3 obtained in the above (5) per cell were each intraperitoneally administered to 10-week-old Balb / c female mice (manufactured by CLEA Japan Inc.) two weeks after administration of 0.5 ml of pristane (manufactured by Sigma-Aldrich). Approximately two weeks later, the ascites retained in the mouse peritoneal cavity was surgically collected under diethyl ether anesthesia. When the ascites was serially diluted and confirmed as a sample by the ELISA method performed in the screening of the above (4), a high concentration of monoclonal antibody was contained. This ascites was treated with 40% ammonium sulfate, dialyzed with PBS, purified by a protein G column (manufactured by GE Healthcare), and confirmed by SDS-PAGE. For both Pentosidine 72-2-2-6 and Pentosidine 72-3-2-3, a single band with a molecular weight of approximately 150,000 was observed under non-reducing conditions, and two bands with molecular weights of approximately 50,000 and 25,000 were observed under mercaptoethanol reduction conditions. The purified antibodies were approximately 10 mg or more per mouse for both Pentosidine 72-2-2-6 and Pentosidine 72-3-2-3, and were sufficient for industrial use.

[0061] <Example 2> Pentosidine measurement and specificity test by competitive ELISA measurement method (1) Pentosidine measurement by competitive ELISA assay Pentosidine was measured using a competitive ELISA assay with the monoclonal antibodies Pentosidine 72-2-2-6 and Pentosidine 72-3-2-3. Pentosidine solid-phase plates were prepared as follows: Synthetic pentosidine was dispensed onto solid-phase plates (Nunc) at a concentration of 10 nmol / well and allowed to stand at 4°C for 1 day. After washing three times with a washing solution of 10 mM disodium hydrogen phosphate dodecahydrate and 150 mM sodium chloride (pH 7.4) containing 0.05% Tween 20, 250 μL of 150 mM disodium hydrogen phosphate dodecahydrate and 150 mM sodium chloride (pH 7.4) containing 1.5% BSA was added, and the plates were blocked overnight at 4°C. The usefulness of pentosidine measurement using competitive ELISA was evaluated using the prepared pentosidine-immobilized plates. 100 μL of a mixed solution of 0-180 nmol of synthetic pentosidine and 0.01 μg of monoclonal antibody Pentosidine 72-2-2-6 or 0.04 μg of Pentosidine 72-3-2-3 was added to the pentosidine-immobilized plate. The reaction was allowed to proceed at room temperature for 2 hours. After the reaction was complete, the plate was washed three times with the aforementioned washing solution, and 100 μL of HRP-labeled anti-mouse immunoglobulin antibody (Agilent Technologies) was added. The reaction was further allowed to proceed at room temperature for 1 hour. After the reaction was complete, the mixture was washed three times with the aforementioned washing solution, and 100 μL of 3,3'5,5'-tetramethylbenzidine (TMB) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added. After a certain period of color development, another 100 μL of 1N sulfuric acid was added as a stop solution, and the absorbance was measured at a measurement wavelength of 450 nm. It was confirmed that the absorbance decreased in a concentration-dependent manner with pentosidine in the measurement system using the obtained monoclonal antibody (Figures 1 and 2).

[0062] (2) Testing of antibody specificity To investigate the specificity of the anti-pentosidine monoclonal antibodies Pentosidine 72-2-2-6 and Pentosidine 72-3-2-3 for pentosidine, a splice-recovery test and a dilution linearity test were performed using healthy human urine samples and synthetic pentosidine. In the add-and-recovery test, three samples of healthy human urine (A, B, and C) were used. One volume of physiological saline or one volume of pentosidine solution of known concentration (final concentration 40 pmol / mL) was added to nine volumes of healthy human urine samples, and the pentosidine concentration in the samples was measured using the standard curve tested in Example 2 (1). As shown in Tables 2 and 3, an increase in the amount of pentosidine measured was observed in proportion to the amount of pentosidine added, and the recovery rate was 95-117%. [Table 2] [Table 3]

[0063] In the dilution linearity test, human healthy urine samples were diluted 6 to 12 times with PBS Buffer, and the pentosidine concentration in the samples was measured. As shown in Figures 3 and 4, good dilution linearity was obtained. From these results, it was confirmed that the obtained anti-pentosidine monoclonal antibody can quantitatively and specifically measure pentosidine in the sample. [Industrial applicability]

[0064] As described in detail above, by using the antibody of the present invention, the target substance to be measured can be measured specifically and precisely.

Claims

1. A monoclonal antibody against pentosidine or its antigen-binding fragment produced by the hybridoma of accession number NITE BP-04144.

2. A monoclonal antibody against pentosidine or its antigen-binding fragment produced by the hybridoma of accession number NITE BP-04145.

3. Hybridoma with accession number NITE BP-04144.

4. Hybridoma with accession number NITE BP-04145.

5. A monoclonal antibody against pentosidine or an antigen-binding fragment thereof, comprising a light chain containing three CDRs (LCDR1, LCDR2, and LCDR3) of the light chain of the monoclonal antibody according to claim 1 or 2, and a heavy chain containing three CDRs (HCDR1, HCDR2, and HCDR3) of the heavy chain of the monoclonal antibody.

6. A monoclonal antibody or its antigen-binding fragment according to claim 5, comprising a light chain containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence of the light chain of the monoclonal antibody according to claim 1 or 2, and a heavy chain containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence of the heavy chain of the monoclonal antibody.

7. An antigen-binding fragment according to any one of claims 1, 2, 5, and 6, selected from the group consisting of Fab', (Fab')2, Fab, scF, Fv, dsFv, and dsscFv.

8. An isolated polynucleotide encoding a monoclonal antibody or its antigen-binding fragment according to any one of claims 1, 2, 5, and 6.

9. An expression vector comprising the polynucleotide described in claim 8.

10. A host cell comprising the expression vector described in claim 9.

11. A method for producing a monoclonal antibody or an antigen-binding fragment according to any one of claims 1, 2, 5, and 6, comprising culturing the host cells according to claim 10 under conditions capable of expressing the monoclonal antibody or an antigen-binding fragment thereof.

12. A reagent for measuring pentosidine, comprising a monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1, 2, 5, and 6.

13. A pentosidine measurement kit comprising the pentosidine measurement reagent described in claim 12.

14. A method for measuring pentosidine in a sample by an immunoassay using a monoclonal antibody or its antigen-binding fragment according to any one of claims 1, 2, 5, and 6.

15. The method according to claim 14, comprising the step of contacting a sample with a monoclonal antibody or an antigen-binding fragment according to any one of claims 1, 2, 5, and 6.

16. The method according to claim 14 or 15, wherein the immunoassay is an enzyme-linked immunosorbent assay.

17. The method according to any one of claims 14 to 16, wherein the sample is serum, urine, or a dilution thereof.

Images