WO2022233106A1 - Procédé de préparation de thyroxine marquée par une phosphatase alcaline - Google Patents

Procédé de préparation de thyroxine marquée par une phosphatase alcaline Download PDF

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Publication number
WO2022233106A1
WO2022233106A1 PCT/CN2021/117137 CN2021117137W WO2022233106A1 WO 2022233106 A1 WO2022233106 A1 WO 2022233106A1 CN 2021117137 W CN2021117137 W CN 2021117137W WO 2022233106 A1 WO2022233106 A1 WO 2022233106A1
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Prior art keywords
buffer
thyroxine
preparation
alkaline phosphatase
reagent
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PCT/CN2021/117137
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English (en)
Chinese (zh)
Inventor
李宗祥
罗继全
徐�明
贾亮
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三诺生物传感股份有限公司
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Publication of WO2022233106A1 publication Critical patent/WO2022233106A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
    • 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
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
    • 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
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/78—Thyroid gland hormones, e.g. T3, T4, TBH, TBG or their receptors

Definitions

  • the invention relates to the technical field of biochemical detection, in particular to a preparation method of alkaline phosphatase-labeled thyroxine.
  • the detection of thyroxine includes the detection of free thyroxine (FT4) and total thyroxine (TT4), the results of which can directly reflect thyroid function. Because the radioimmunoassay method pollutes the environment and there are many factors that affect the detection results, chemiluminescence method is often used to detect thyroxine.
  • the components to be included in the free thyroxine (FT4) and total thyroxine (TT4) chemiluminescence kits include alkaline phosphatase (ALP)-labeled thyroxine (ALP-T4).
  • ALP alkaline phosphatase
  • ALP-T4 alkaline phosphatase-labeled thyroxine
  • most of the methods for labeling T4 are to conjugate macromolecules (such as BSA) through T4 and then label ALP enzyme or coat magnetic beads; or to conjugate T4 to NHS and then label ALP enzyme; there are also some methods
  • the coupling is carried out by activating ALPase and T4 respectively.
  • the technical problem to be solved by the present invention is to provide a preparation method of thyroxine labeled with alkaline phosphatase, and the thyroxine-labeled alkaline phosphatase can be used for more accurate detection of thyroxine.
  • the alkaline phosphatase is activated in an acidic solution
  • the free activator is removed, and then it is coupled with thyroxine in an alkaline solution, and the alkaline phosphatase-labeled thyroxine is obtained after the reaction is terminated.
  • the acidic solution in the present invention is MES buffer with pH ⁇ 7, PBS buffer, Tris-HCl buffer, phosphate buffer, citrate buffer or glycine buffer.
  • the activator is selected from EDC/NHS, glutaraldehyde or Sulfo-SMCC (4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfosuccinimide at least one of ester sodium salt).
  • the method for removing the activator includes dialysis or ultrafiltration.
  • the alkaline solution is MES buffer with pH>7, PBS buffer, Tris-HCl buffer, phosphate buffer, citrate buffer or glycine buffer.
  • the carboxyl group of alkaline phosphatase is coupled with the amino group of thyroxine.
  • an amino group-containing buffer is used for the termination reaction.
  • the method further includes: mixing the product with the preservation solution and removing by-products.
  • the preservation solution is MES buffer, PBS buffer, Tris-HCl buffer, phosphate buffer, citrate buffer or glycine buffer with a pH of 7.0-7.5;
  • the by-products include free thyroxine
  • Methods of such removal include dialysis or ultrafiltration.
  • the present invention also provides a thyroxine detection reagent, which includes R1 reagent, R2 reagent and R3 reagent;
  • the R1 reagent is a magnetic bead suspension coated with T4 antibody
  • the R2 reagent is an alkaline phosphatase-labeled thyroxine solution
  • the R3 reagent is a magnetic bead buffer
  • the R2 reagent includes alkaline phosphatase-labeled thyroxine, enzyme-labeled buffer C and glycerol prepared by the preparation method of the present invention.
  • the preparation method provided by the present invention directly connects the alkaline phosphatase with thyroxine after activation, and does not need to activate T4, and does not need to couple macromolecules such as BSA.
  • the prepared ALP-T4 complex has good activity and does not contain by-products. And the preparation method has simple process and low cost. It has been verified that the ALP-T4 prepared by this method can detect free thyroxine or total thyroxine with good sensitivity, accuracy, precision and specificity.
  • the present invention provides a method for preparing alkaline phosphatase-labeled thyroxine, which can be achieved by those skilled in the art by appropriately improving the process parameters for reference. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present invention.
  • the method and application of the present invention have been described through the preferred embodiments, and it is obvious that relevant persons can make changes or appropriate changes and combinations of the methods and applications herein without departing from the content, spirit and scope of the present invention, so as to realize and apply the present invention. Invention technology.
  • alkaline phosphatase-labeled thyroxine In the preparation method of alkaline phosphatase-labeled thyroxine provided by the present invention, firstly, under acidic conditions, an activating group is connected to the carboxyl group of ALP enzyme to form a stable intermediate product, and then the free activating agent is removed. Then in alkaline conditions, without further addition of activating agent, the activating group on ALP enzyme is removed, and the carboxyl group of ALP enzyme is coupled with the amino group of T4. After that, the uncoupled carboxyl groups on ALP were blocked with buffer containing amino groups, and then free small molecules such as T4 were removed to obtain ALP-T4. In the obtained ALP-T4, the amino group of T4 is directly coupled with the carboxyl group of ALP without connecting other groups or molecules in the middle.
  • the acidic solution is MES buffer with pH ⁇ 7, PBS buffer, Tris-HCl buffer, phosphate buffer, citrate buffer or glycine buffer.
  • the acidic solution is denoted as enzyme labeling buffer A.
  • the enzyme labeling buffer A includes water and: MES and NaCl.
  • the pH of the enzyme labeling buffer A was 4.5.
  • the activator of the present invention is used to activate the carboxyl group of ALP. After activation, the carboxyl group of ALP is connected with an activating group. The selection of the activator is based on its ability to activate ALP, and the activator with better activation performance is preferred. Commonly used activators include EDC/NHS, or glutaraldehyde, or Sulfo-SMCC. In the embodiment of the present invention, the activator is EDC/NHS, and EDC and NHS are used as the activator of ALP. Specifically, the NHS is sulfo-NHS. The mass ratio of the EDC to sulfo-NHS was 500:50.
  • the mass ratio of alkaline phosphatase, EDC and sulfo-NHS was 250:500:50.
  • the concentration of alkaline phosphatase in the activated system was 1 ⁇ g/ ⁇ L, the concentration of EDC was 2 ⁇ g/ ⁇ L, and the concentration of sulfo-NHS was 0.1 ⁇ g/ ⁇ L; the activation conditions included 25° C., 1100 rpm shaking for 1 h.
  • the free activator is removed before coupling, and other possible small molecule impurities are also removed, so as to ensure the accurate coupling of ALP-T4.
  • the method of removal may include dialysis or ultrafiltration.
  • the method of ultrafiltration is adopted.
  • the molecular weight cut-off of the filter membrane of the ultrafiltration tube is 30 kDa.
  • the conditions of the ultrafiltration were centrifugation at 9000 rpm/min for 25 min.
  • ultrafiltration is carried out under low temperature conditions.
  • the low temperature condition is 0-4°C.
  • the alkaline solution is MES buffer with pH>7, PBS buffer, Tris-HCl buffer, phosphate buffer, citrate buffer or glycine buffer.
  • the acidic solution is denoted as enzyme labeling buffer B.
  • the enzyme labeling buffer B includes water and : Na2HPO4.12H2O , NaH2PO4.2H2O , and NaCl .
  • the pH of the enzyme labeling buffer B was 9.0.
  • the coupling step no activator or other components such as coupling agent are added, and only the activated ALP and T4 are subjected to a coupling reaction in an alkaline solution.
  • the reaction conditions described in the present invention are determined to be the optimum conditions. Coupling under this condition not only avoids the self-linking of T4 molecules, but also avoids the generation of other by-products, thereby improving the accuracy of coupling, which has positive significance for improving the accuracy of detection.
  • the coupling step specifically includes: mixing the activated ALP solution with thyroxine, and incubating with enzyme labeling buffer B to a constant volume. The volume ratio of the activated ALP solution to the enzyme labeling buffer B was 250:150.
  • the mass ratio of the alkaline phosphatase to thyroxine was 250:20.
  • the enzyme labeling buffer B was used to make the volume to a concentration of 0.2 ⁇ g/ ⁇ L of thyroxine.
  • the incubation conditions were 25°C for 3h.
  • the buffer solution containing amino group is recorded as blocking solution in the present invention.
  • the blocking solution is Tris buffer.
  • the blocking solution is Tris buffer with a pH value of 8.0.
  • the volume ratio of the blocking solution and the enzyme labeling buffer B after constant volume was 50:100.
  • the blocking condition is room temperature blocking for 0.5 h.
  • the room temperature is preferably 18-30°C.
  • the step further includes: mixing the product with the preservation solution, and then removing by-products.
  • the by-products include free T4 unconjugated ALP and possibly other small molecule impurities.
  • the method of removing by-products includes dialysis or ultrafiltration.
  • the ultrafiltration step the molecular weight cut-off of the filter membrane of the ultrafiltration tube is 30 kDa.
  • the conditions of the ultrafiltration were centrifugation at 9000 rpm/min for 25 min.
  • ultrafiltration is carried out under low temperature conditions.
  • the low temperature condition is 0-4°C. After ultrafiltration, collect the liquid in the ultrafiltration tube after ultrafiltration.
  • the preservation solution of the product is denoted as enzyme labeling buffer C.
  • the enzyme labeling buffer C included water Na 2 HPO 4 ⁇ 12H 2 O, NaH 2 PO 4 ⁇ 2H 2 O and NaCl.
  • the pH value of the enzyme labeling buffer C is 7.4.
  • the liquid in the ultrafiltration tube can be directly used for the detection of T4. But it is usually diluted and made up to volume before use.
  • the enzyme labeling buffer C is used to make constant volume, and glycerol is added as a cryoprotectant.
  • the preparation method of alkaline phosphatase-labeled thyroxine provided by the present invention comprises:
  • Step 1 Mix alkaline phosphatase, EDC, sulfo-NHS and enzyme labeling buffer A, after activation, mix with enzyme labeling buffer B, and perform ultrafiltration to obtain an activated alkaline phosphatase solution;
  • Step 2 Mix the activated alkaline phosphatase solution with thyroxine, make up the volume with enzyme labeling buffer B, block after incubation, mix with enzyme labeling buffer C, and obtain alkaline phosphatase after ultrafiltration labeled thyroxine;
  • the present invention also provides a thyroxine detection reagent, which includes R1 reagent, R2 reagent and R3 reagent;
  • the R1 reagent is a magnetic bead suspension coated with T4 antibody
  • the R2 reagent is an alkaline phosphatase-labeled thyroxine solution
  • the R3 reagent is a magnetic bead buffer.
  • the R2 reagent includes alkaline phosphatase-labeled thyroxine, enzyme-labeled buffer C and glycerol prepared by the preparation method of the present invention.
  • a dissociating agent is also included, and the dissociating agent is 0.5 mg/mL ANS solution.
  • the present invention also provides a method for detecting thyroxine, which is detected by using the detecting reagent of the present invention.
  • the thyroxine is free thyroxine or total thyroxine.
  • the preparation method provided by the present invention directly connects the alkaline phosphatase with thyroxine after activation, and does not need to activate T4, and does not need to couple macromolecules such as BSA.
  • the prepared ALP-T4 complex has good activity and does not contain by-products.
  • Preliminary experiments show that, compared with the scheme using other activators, the technical scheme provided by the present invention has reasonable selection of process parameters, which not only greatly shortens the preparation process, but also improves the coupling effect and reduces the content of by-products, so that the prepared Reagents allow for more accurate detection of thyroxine.
  • test materials used in the present invention are all common commercial products and can be purchased in the market. Below in conjunction with embodiment, the present invention is further elaborated:
  • R2 Dilute the ALP-T4 stock solution 16,000 times with enzyme labeling buffer to prepare R2.
  • R3 Magnetic Bead Buffer.
  • Minimum detection limit test 20 zero-concentration calibrators, and calculate the average of the luminescence values and the standard deviation SD, will Substitute into the dose-response curve equation, the obtained concentration value is the minimum detection limit, which should not be higher than 2.00pmol/L.
  • the test results are shown in Table 1
  • T3 with a concentration of not less than 200ng/mL
  • rT3 with 100ng/mL
  • 3,3'-diiodothyronine with a concentration of 200ng/mL
  • the results are shown in Table 4:
  • R1 Dilute the magnetic bead-coated T4 antibody to 0.2 mg/mL with magnetic bead buffer to prepare R1.
  • R2 Dilute ALP-T4 stock solution 8000 times with enzyme labeling buffer to prepare R2.
  • R3 dissociating agent ANS, 0.5 mg/mL.
  • Linear range in the range of [10.0 ⁇ 300.0]nmol/L, with four-parameter fitting, the linear correlation coefficient (r) of the dose-response curve is ⁇ 0.9900.
  • the fitted curve is:
  • Minimum detection limit test 20 zero-concentration calibrators, and calculate the average of the luminescence values and the standard deviation SD, will Substitute into the equation of dose-response curve, the obtained concentration value is the minimum detection limit, which should not be higher than 10.0nmol/L. The results are shown in Table 5
  • Table 8 The results are shown in Table 8:

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Abstract

La présente invention relève du domaine technique de la détection biochimique, et concerne en particulier un procédé de préparation de la thyroxine marquée par une phosphatase alcaline. Dans le procédé de préparation selon l'invention, après activation, la phosphatase alcaline est directement liée à la thyroxine sans qu'il soit nécessaire d'activer T4 et sans avoir besoin de coupler des macromolécules telles que la BSA. Le complexe ALP-T4 préparé a une bonne activité et ne contient pas de sous-produits. Le procédé de préparation a des procédés simples et est peu coûteux. Il a été vérifié que l'ALP-T4 préparé à l'aide du présent procédé détecte la thyroxine libre ou la thyroxine totale avec de bonnes sensibilité, exactitude, précision et spécificité.
PCT/CN2021/117137 2021-05-06 2021-09-08 Procédé de préparation de thyroxine marquée par une phosphatase alcaline WO2022233106A1 (fr)

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CN113214380B (zh) * 2021-05-06 2023-05-09 三诺生物传感股份有限公司 碱性磷酸酶标记的甲状腺素的制备方法

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