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WO2020224189A1 - Kit de dosage immunologique de 24,25-dihydroxyvitamine d et son application - Google Patents

Kit de dosage immunologique de 24,25-dihydroxyvitamine d et son application Download PDF

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Publication number
WO2020224189A1
WO2020224189A1 PCT/CN2019/111359 CN2019111359W WO2020224189A1 WO 2020224189 A1 WO2020224189 A1 WO 2020224189A1 CN 2019111359 W CN2019111359 W CN 2019111359W WO 2020224189 A1 WO2020224189 A1 WO 2020224189A1
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Prior art keywords
vitd
working solution
antibody
solution
chemiluminescence
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Ceased
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PCT/CN2019/111359
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English (en)
Chinese (zh)
Inventor
欧赛英
涂策
李大军
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Suzhou Hybiome Biomedical Engineering Co Ltd
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Suzhou Hybiome Biomedical Engineering Co Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/82—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving vitamins or their receptors

Definitions

  • the invention relates to a 24,25-dihydroxyvitamin D immunoassay kit and its application, and belongs to the technical field of in vitro detection.
  • Vitamin D exists in the human body in two forms: cholecalciferol (D3) and ergocalciferol (D2). Among them, cholecalciferol (D3) is derived from sunlight and meat products, and ergocalciferol (D2) is derived from dietary plants. source. The vitamin D endocrine system plays an important biological function in the human body. Vitamin D enters the liver through the blood circulation, and is converted into 25-hydroxy vitamin D (25-OH-VitD, including 25-hydroxylase CYP2R1) under the action of 25-hydroxylase CYP2R1.
  • OH-VitD2 and 25-OH-VitD3 which are then catalyzed by 24-hydroxylase CYP24A1 in the kidney to produce 24,25-dihydroxyvitamin D (24,25-(OH)2-VitD), 25-OH-VitD It can also be metabolized in the body into biologically active 1,25-(OH)2-VitD and 3-epi-25OH-VitD.
  • 24,25-(OH)2-VitD is dependent on 25-OH-VitD, and the ratio of 25-OH-VitD to 24,25-(OH)2-VitD is in the serum 25-OH-VitD concentration It is significantly increased when the concentration is lower than 20ng/ml, and the ratio decreases when the serum 25-OH-VitD concentration is high. This may be due to the decrease of 25-OH-VitD in the blood circulation, which leads to the down-regulation of CYP24A1 to promote catabolism 1,25 The production of (OH)2-VitD; and individuals with high concentrations of 25-OH-VitD tend to produce 24,25-(OH)2-VitD to reduce the production of 1,25-(OH)2-VitD.
  • the determination of serum 24,25-(OH)2-VitD concentration is helpful to identify patients with CYP24A1 loss-of-function mutations, and the ratio of serum 25-OH-VitD to 24,25-(OH)2-VitD concentration can be As an indicator of vitamin D catabolic status, especially for patients with renal calcification or kidney stones, if the ratio of 25-OH-VitD to 24,25-(OH)2-VitD is detected to increase, it indicates that vitamin supplementation should be restricted or avoided D to prevent hypercalcemia and further stone formation.
  • the technical problem to be solved by the present invention is: to solve the technical problem that there is no immunological method in the prior art that can accurately detect 24,25-(OH)2-VitD, a 24,25-dihydroxyvitamin D immunoassay is provided Kit and its application.
  • a 24,25-dihydroxyvitamin D immunoassay kit including: perfluorooctanoic acid working solution, biotin working solution, antibody working solution, streptavidin-coated magnetic particle reagent, 24,25-(OH)2 -VitD calibrator solution and chemiluminescence substrate solution, the antibody working solution contains chemiluminescence marker-labeled anti-24,25-(OH)2-VitD immune monoclonal antibody, unlabeled anti-25-OH-VitD immune monoclonal antibody Anti-, unlabeled anti-1,25-(OH)2-VitD immune monoclonal antibody, unlabeled anti-3-epi-25OH-VitD immune monoclonal antibody.
  • the 25OH-VitD immune monoclonal antibodies are all sheep monoclonal antibodies.
  • the antibody working solution contains a heterophagic antibody blocker HBR
  • the concentration of the heterophagic antibody blocker HBR is preferably 0.5-3 mg/mL
  • the heterophagic antibody blocker HBR can consume the sample Heterophagic antibody, to avoid the reaction of heterophagic antibody with anti-24,25-(OH)2-VitD immunomab labeled with chemiluminescence marker, to further avoid false results
  • the antibody working solution is preferably based on PBS or CBS buffer solution is prepared, the concentration of PBS or CBS buffer solution is preferably 0.05-0.15M, and the pH is preferably 8.5-10.5.
  • the antibody working solution contains protein, polysaccharide and polyol;
  • the protein is albumin and/or casein, and the concentration is preferably 0.5-2 mg/mL;
  • the polysaccharide is fructose and/or sucrose, and the concentration is preferably 3-5mg/mL;
  • the polyol is glycerol and/or ethylene glycol, and the concentration is preferably 3-5mg/mL;
  • the polyol is glycerol and/or ethylene glycol, protein, polysaccharide and polyol
  • the addition of can effectively prevent the aggregation of antibodies and further avoid false positives.
  • the preparation method of the anti-24,25-(OH)2-VitD immunomab labeled with the chemiluminescence marker is: combining the anti-24,25-(OH)2-VitD immunomab with the chemiluminescence marker React in PBS or CBS buffer at a molar ratio of 1:10-20 for 0.5-1.5 hours. Dialysis removes uncross-linked small molecules to obtain chemiluminescent marker-labeled anti-24,25-(OH)2-VitD immunity Monoclonal antibody; the concentration of the PBS or CBS buffer is preferably 0.02-0.08M, and the pH is preferably 8.5-10.5.
  • the perfluorooctanoic acid working solution contains 5-15% by volume of ethylene glycol.
  • the perfluorooctanoic acid working solution is preferably prepared from PBS or CBS buffer.
  • the concentration of PBS or CBS buffer is preferably 0.05-0.15M, and the pH is preferably Is 8.5-10.5.
  • the 24,25-(OH)2-VitD calibrator solution is prepared from steroid-free serum, and the 24,25-(OH)2-VitD calibrator solution preferably contains 0.05wt%-0.2wt % Proclin300.
  • the chemiluminescent label is selected from luminol or its derivatives, isoluminol or its derivatives, acridine ester or acridine sulfonamide.
  • the chemiluminescence substrate liquid includes a chemiluminescence excitation liquid 1 and a chemiluminescence excitation liquid 2, the chemiluminescence excitation liquid 1 contains an inorganic acid and peroxide, and the chemiluminescence excitation liquid 2 contains a hydroxide.
  • the present invention also provides the application of the 24,25-(OH)2-VitD chemiluminescence immunoassay kit, which is used according to the following steps:
  • the present invention uses a chemiluminescence immunoassay kit to detect 24,25-(OH)2-VitD for the first time.
  • the detection method is simple and the sample amount is less. Compared with LC-MS/MS, it is more suitable for clinical promotion, especially the antibody work of the kit.
  • the solution contains chemiluminescent marker-labeled anti-24,25-(OH)2-VitD immunomab, unlabeled anti-25-OH-VitD immunomab, and unlabeled anti-1,25-(OH)2-VitD Immune monoclonal antibody, unlabeled anti-3-epi-25OH-VitD immunomab, can effectively avoid 24,25-(OH)2-VitD immunomab and 25-OH-VitD, 1,25-(OH)2 -VitD, 3-epi-25OH-VitD and other vitamin D metabolites cross-react, and the test results are accurate, which are expected to be used in clinical efficacy monitoring of vitamin D supplementation and screening of patients with CYP24A1 mutations; further, the antibody working solution
  • the added heterophagic antibody blocker HBR, protein, polysaccharide and polyol can further avoid false results.
  • Figure 1 is a comparison diagram of the content of 24,25-(OH)2-VitD in serum samples detected by the method of Comparative Example 1 and the method of Example 4 of the present invention.
  • This embodiment provides a 24,25-dihydroxyvitamin D immunoassay kit and a preparation method thereof.
  • the kit includes: perfluorooctanoic acid working solution, biotin working solution, antibody working solution, streptavidin-coated magnetic particles Reagents, 24,25-(OH)2-VitD calibrator solution and chemiluminescence substrate solution, the antibody working solution contains luminol-labeled anti-24,25-(OH)2-VitD sheep monoclonal antibody, unlabeled Anti-25-OH-VitD sheep monoclonal antibody, unlabeled anti-1,25-(OH)2-VitD sheep monoclonal antibody, unlabeled anti-3-epi-25OH-VitD sheep monoclonal antibody;
  • PBS buffer solution containing 5% by volume ethylene glycol was used to prepare a perfluorooctanoic acid working solution with a concentration of 100ng/mL, the PBS buffer solution had a concentration of 0.05M and a pH of 8.5;
  • Biotin working solution use PBS buffer with a concentration of 0.05M and pH of 8.5 to prepare a biotin working solution with a concentration of 100ng/mL;
  • the anti-24,25-(OH)2-VitD sheep monoclonal antibody solution was added to luminol for 0.5 hours, and the molar ratio of anti-24,25-(OH)2-VitD sheep monoclonal antibody to luminol was 1:10 Reaction, dialysis to remove uncross-linked small molecules, to obtain luminol-labeled anti-24,25-(OH)2-VitD immunomab;
  • the luminol-labeled anti-24,25-(OH)2-VitD sheep monoclonal antibody was diluted with PBS buffer at a concentration of 0.05M and pH 8.5, and unlabeled anti-25-OH-VitD sheep monoclonal antibody, Unlabeled anti-1,25-(OH)2-VitD sheep monoclonal antibody and unlabeled anti-3-epi-25OH-VitD sheep monoclonal antibody to obtain an antibody working solution.
  • concentration of epi-25OH-VitD sheep monoclonal antibody is 1mg/mL;
  • the PBS buffer contains 0.5mg/mL heterophagic antibody blocker, 0.5mg/mL albumin, 3mg/mL fructose and 5mg/mL mL glycerol;
  • chemiluminescence substrate solution includes chemiluminescence excitation solution 1 and chemiluminescence excitation solution 2, said chemiluminescence excitation solution 1 contains 0.1M nitric acid and 1wt% peroxide, said chemiluminescence excitation solution 2 Containing 0.1M sodium hydroxide.
  • This embodiment provides a 24,25-dihydroxyvitamin D immunoassay kit and a preparation method thereof.
  • the kit includes: perfluorooctanoic acid working solution, biotin working solution, antibody working solution, streptavidin-coated magnetic particles Reagents, 24,25-(OH)2-VitD calibrator solution and chemiluminescence substrate solution.
  • the antibody working solution contains isoluminol-labeled anti-24,25-(OH)2-VitD sheep monoclonal antibody, Labeled anti-25-OH-VitD sheep monoclonal antibody, unlabeled anti-1,25-(OH)2-VitD sheep monoclonal antibody, unlabeled anti-3-epi-25OH-VitD sheep monoclonal antibody;
  • Preparation of perfluorooctanoic acid working solution prepare perfluorooctanoic acid working solution with a CBS buffer solution containing 15% by volume of ethylene glycol, the concentration of the CBS buffer solution is 0.15M and the pH is 10.5;
  • Preparation of antibody working solution prepare anti-24,25-(OH)2-VitD sheep monoclonal antibody solution with PBS buffer with a concentration of 0.08M and a pH of 10.5;
  • the anti-24,25-(OH)2-VitD sheep monoclonal antibody solution was added to isoluminol for 1.5 hours, and the molar ratio of anti-24,25-(OH)2-VitD sheep monoclonal antibody to isoluminol was 1 : 20 reaction, dialysis to remove uncrosslinked small molecules, and isoluminol-labeled anti-24,25-(OH)2-VitD immunomab;
  • chemiluminescence substrate solution includes chemiluminescence excitation solution 1 and chemiluminescence excitation solution 2, said chemiluminescence excitation solution 1 contains 0.1M nitric acid and 1wt% peroxide, said chemiluminescence excitation solution 2 Containing 0.1M sodium hydroxide.
  • This embodiment provides a 24,25-dihydroxyvitamin D immunoassay kit and a preparation method thereof.
  • the kit includes: perfluorooctanoic acid working solution, biotin working solution, antibody working solution, streptavidin-coated magnetic particles Reagents, 24,25-(OH)2-VitD calibrator solution and chemiluminescence substrate solution, the antibody working solution contains acridinium ester-labeled anti-24,25-(OH)2-VitD sheep monoclonal antibody, unlabeled Anti-25-OH-VitD sheep monoclonal antibody, unlabeled anti-1,25-(OH)2-VitD sheep monoclonal antibody, unlabeled anti-3-epi-25OH-VitD sheep monoclonal antibody;
  • Preparation of perfluorooctanoic acid working solution prepare perfluorooctanoic acid working solution with a PBS buffer solution containing 10% by volume of ethylene glycol, the concentration of the PBS buffer solution being 0.1M and pH 9.5;
  • the acridinium ester-labeled antibody in the antibody working solution 24,25-(OH)2-VitD sheep monoclonal antibody, unlabeled anti-25-OH-VitD sheep monoclonal antibody, unlabeled anti-1,25-(OH)2-VitD sheep monoclonal antibody, unlabeled anti-3 -
  • concentration of epi-25OH-VitD sheep monoclonal antibody is 1 mg/mL;
  • the PBS buffer contains 2 mg/mL heterophagic antibody blocker, 1 mg/mL casein, 1 mg/mL albumin, and 5 mg/mL Sucrose sugar and 3mg/mL ethylene glycol;
  • chemiluminescence substrate solution includes chemiluminescence excitation solution 1 and chemiluminescence excitation solution 2, said chemiluminescence excitation solution 1 contains 0.1M nitric acid and 1wt% peroxide, said chemiluminescence excitation solution 2 Containing 0.1M sodium hydroxide.
  • This embodiment provides a method for detecting 148 clinical serum samples using the kit of embodiment 3, which includes the following steps:
  • Example 4 148 clinical serum samples of Example 4 were used to detect the content of 24,25-(OH)2-VitD in each clinical serum sample by LC-MS/MS, and the result of the LC-MS/MS detection was The abscissa, taking the detection result of Example 4 as the ordinate, draw a scatter diagram (as shown in Figure 1), and obtain a standard linear curve.
  • the LC-MS/MS method for detecting the content of 24,25-(OH)2-VitD in clinical serum samples has little difference from the method of Example 4, indicating that the detection method of the present invention can accurately detect 24,25-(OH)2-VitD in samples.
  • the content of 25-(OH)2-VitD The content of 25-(OH)2-VitD.
  • the kit of the present invention can accurately detect the concentration of 24,25-(OH)2-VitD in the sample, the vitamin D in the sample and other vitamins except 24,25-(OH)2-VitD D metabolites have little effect on the detection of 24,25-(OH)2-VitD.

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Abstract

La présente invention concerne un kit de dosage immunologique de 24,25-dihydroxyvitamine D et son application. Le kit comprend une solution de travail à base d'acide perfluorooctanoïque, une solution de travail à base de biotine, une solution de travail à base d'anticorps, un réactif sous forme de particules magnétiques revêtues de streptavidine, une solution d'étalonnage de 24,25-(OH)2-VitD, et une solution de substrat chimioluminescente. La solution de travail à base d'anticorps contient un anticorps monoclonal immun anti-24,25-(OH)2-VitD marqué par un marqueur chimioluminescent, un anticorps monoclonal immun anti-25-OH-VitD non marqué, un anticorps monoclonal immun anti-1,25-(OH)2-VitD non marqué, et un anticorps monoclonal immun anti-3-épi-25OH-VitD non marqué. L'invention concerne également un dosage immunologique chimioluminescent de 24,25-(OH)2-VitD à l'aide du kit. Le résultat de mesure est précis.
PCT/CN2019/111359 2019-05-05 2019-10-16 Kit de dosage immunologique de 24,25-dihydroxyvitamine d et son application Ceased WO2020224189A1 (fr)

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CN201910367609.5A CN110058028B (zh) 2019-05-05 2019-05-05 一种24,25-双羟基维生素d免疫检测试剂盒及其应用

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CN110058028B (zh) * 2019-05-05 2021-03-02 苏州长光华医生物医学工程有限公司 一种24,25-双羟基维生素d免疫检测试剂盒及其应用
CN110702928B (zh) * 2019-09-25 2023-12-12 迪瑞医疗科技股份有限公司 一种稳定的25-羟基维生素d化学发光免疫检测试剂盒

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US5064770A (en) * 1989-08-01 1991-11-12 Wisconsin Alumni Research Foundation Assay for 1,25-dihydroxy vitamin d receptor protein
CN101273272A (zh) * 2005-09-29 2008-09-24 霍夫曼-拉罗奇有限公司 维生素d化合物的释放试剂
CN102985826A (zh) * 2010-04-01 2013-03-20 未来诊断有限公司 游离的维生素d的免疫分析
CN104955841A (zh) * 2013-01-28 2015-09-30 索灵股份公司 用于检测1,25-二羟基维生素d的方法和试剂盒以及相关抗体
CN105556313A (zh) * 2013-09-17 2016-05-04 生物梅里埃公司 用于从维生素d结合蛋白解离维生素d的溶液、相关的检测方法及用途
CN107003305A (zh) * 2014-12-08 2017-08-01 豪夫迈·罗氏有限公司 用于测量维生素d的方法
CN107621499A (zh) * 2016-07-14 2018-01-23 上海可力梅塔生物医药科技有限公司 25‑羟基维生素d2和25‑羟基维生素d3高效液相色谱质谱联用法检测试剂盒
CN110058028A (zh) * 2019-05-05 2019-07-26 苏州长光华医生物医学工程有限公司 一种24,25-双羟基维生素d免疫检测试剂盒及其应用

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