WO2020093305A1 - Procédé et kit pour détecter la teneur en fer dans un échantillon de sang - Google Patents

Procédé et kit pour détecter la teneur en fer dans un échantillon de sang Download PDF

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Publication number
WO2020093305A1
WO2020093305A1 PCT/CN2018/114545 CN2018114545W WO2020093305A1 WO 2020093305 A1 WO2020093305 A1 WO 2020093305A1 CN 2018114545 W CN2018114545 W CN 2018114545W WO 2020093305 A1 WO2020093305 A1 WO 2020093305A1
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WO
WIPO (PCT)
Prior art keywords
mmol
reagent
interference
metal ion
iron
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PCT/CN2018/114545
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English (en)
Chinese (zh)
Inventor
杜少卿
王嘉鹏
张裕平
Original Assignee
深圳迈瑞生物医疗电子股份有限公司
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Application filed by 深圳迈瑞生物医疗电子股份有限公司 filed Critical 深圳迈瑞生物医疗电子股份有限公司
Priority to CN201880097735.7A priority Critical patent/CN112714870A/zh
Priority to PCT/CN2018/114545 priority patent/WO2020093305A1/fr
Publication of WO2020093305A1 publication Critical patent/WO2020093305A1/fr

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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
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements

Definitions

  • the invention relates to a method and a kit for detecting the iron content in a blood sample, in particular to a method and a kit capable of resisting the interference of a chelating agent when detecting the iron content in a blood sample.
  • Iron plays a very important role in the living system. It participates in various life processes in the body, such as cell oxidation mechanism and transportation and supply of oxygen to body cells. At the same time, iron is a component of oxygen-carrying pigment proteins, hemoglobin and myoglobin, and various enzymes (such as cytochrome oxidase and peroxidase). The rest of the iron in the body is present in flavin protein, iron-sulfur protein, ferritin that stores iron, and transferrin that transports iron.
  • the detection of iron content in serum can be used for iron-deficiency anemia and hemochromatosis (two kinds of iron-containing pigments in tissues: hemosiderin and hemofuscin disseminated , Often manifested as skin pigmentation) and the diagnosis and treatment of chronic kidney disease.
  • the detection of iron content can also be used for small cell anemia (causes such as iron metabolism disorders and hemoglobin diseases), large cell anemia (such as vitamin B12 and folic acid deficiency, or metabolic disorders due to drugs) and positive cell anemia such as Diagnosis of renal anemia (promoting erythropoietin deficiency), hemolytic anemia, hemoglobin disease, bone marrow disease, and toxic bone marrow injury.
  • small cell anemia causes such as iron metabolism disorders and hemoglobin diseases
  • large cell anemia such as vitamin B12 and folic acid deficiency, or metabolic disorders due to drugs
  • positive cell anemia such as Diagnosis of renal anemia (promoting erythropoietin deficiency), hemolytic anemia, hemoglobin disease, bone marrow disease, and toxic bone marrow injury.
  • the colorimetric method is usually used to detect the iron content in blood samples, that is: the iron combined with transferrin is dissociated into ferric ions and transferrin in an acid medium; the ferric ions are reduced with a reducing agent It is a divalent iron ion; finally, the divalent iron ion complexes with a complexing agent (also called a color developer) to produce a color change.
  • a complexing agent also called a color developer
  • the present invention provides a method for detecting the iron content in the blood sample and a corresponding kit.
  • the present invention resists the interference caused by the chelating agent by using metal ions with stronger binding ability to EDTA than divalent iron ions under acidic environment conditions with a pH of 3.0 to 6.0.
  • the present invention also aims to solve the problem of how to remove the interference caused by iron dextran in the detection of iron content.
  • the present invention provides a method for detecting iron content in a blood sample, the method comprising:
  • a detection reagent containing the following components to the blood sample: a buffer solution with a pH of about 3.0 to 6.0, a reducing agent that reduces Fe 3+ to Fe 2+ , an anti-interference metal ion, and a reagent for binding to Fe 2+ .
  • Colored complexing agent wherein the iron bound to transferrin dissociates into Fe 3+ in an acidic environment with a pH of 3.0 to 6.0, and the anti-interference metal ion has a stronger binding capacity to EDTA than Fe 2+ and EDTA's binding ability;
  • the colorimetric method is used to detect the iron content.
  • the metal ion may be selected from one or more of the following metal ions: Al 3+ , Bi 3+ , Cd 2+ , Co 2+ , Cr 3+ , Ga 3+ , Hg 2 + , Ni 2+ , Pb 2+ , Sc 2+ , Sn 2+ , Th 4+ , TiO 2+ , Tl 3+ , U 4+ , VO 2+ , Y 3+ , Zn 2+ and Zr 4+ .
  • the metal ion may be selected from one or more of the following metal ions: Cd 2+ , Co 2+ , Hg 2+ , Ni 2+ , Pb 2+ , Sc 2+ , Sn 2+ , Th 4+ , TiO 2+ , U 4+ , VO 2+ , Zn 2+ and Zr 4+ .
  • the metal ion may be at least one of Zn 2+ and Cd 2+ .
  • the concentration of the metal ion may be about 0.1 mmol / L to 50 mmol / L, preferably about 1 mmol / L to 50 mmol / L, and more preferably about 10 mmol / L to 50 mmol / L.
  • the pH of the buffer may be about 3.0 to 5.0, preferably about 3.5 to 5.0, and more preferably about 3.5 to 4.0.
  • the concentration of the reducing agent may be about 0.1 mmol / L to 100 mmol / L.
  • the reducing agent may be selected from at least one of ascorbic acid, hydroxylamine hydrochloride, sodium sulfite, and dithiothreitol.
  • the reducing agent may be ascorbic acid.
  • the detection reagent may further include at least one of a surfactant, a preservative, and an anti-copper ion interference reagent.
  • the surfactant may be a nonionic surfactant.
  • the blood sample may be serum or plasma.
  • the present invention also provides a kit comprising the following components:
  • the pH of the buffer is about 3.0 to 6.0, and the iron bound to transferrin dissociates to Fe 3+ in an acidic environment with a pH of 3.0 to 6.0;
  • Reducing agent which can reduce Fe 3+ to Fe 2+ ;
  • Anti-interference metal ions the binding capacity of the metal ion to EDTA is stronger than that of Fe 2+ to EDTA;
  • the kit of the present invention may include the following reagents:
  • the first reagent includes a buffer and an anti-interference metal ion, wherein the pH of the buffer is about 3.0 to 6.0, and the iron bound to transferrin dissociates to Fe 3+ in an acidic environment with a pH of 3.0 to 6.0
  • the binding ability of the anti-interference metal ion to EDTA is stronger than that of Fe 2+ to EDTA;
  • the second reagent includes a reducing agent and a complexing agent.
  • the reducing agent can reduce Fe 3+ to Fe 2+ .
  • the complexing agent can combine with Fe 2+ to develop color, so that the colorimetric method can be used to detect blood. The iron content in the sample.
  • the metal ion may be selected from one or more of the following metal ions: Cd 2+ , Co 2+ , Hg 2+ , Ni 2+ , Pb 2+ , Sc 2+ , Sn 2 + , Th 4+ , TiO 2+ , U 4+ , VO 2+ , Zn 2+, and Zr 4+ .
  • the metal ion may be at least one of Zn 2+ and Cd 2+ .
  • the concentration of the metal ion may be about 0.1 mmol / L to 50 mmol / L, preferably about 1 mmol / L to 50 mmol / L, and more preferably about 10 mmol / L to 50 mmol / L.
  • the pH of the buffer may be about 3.0 to 5.0, preferably about 3.5 to 5.0, and more preferably about 3.5 to 4.0.
  • the concentration of the reducing agent may be about 0.1 mmol / L to 100 mmol / L.
  • the reducing agent may be selected from at least one of ascorbic acid, hydroxylamine hydrochloride, sodium sulfite, and dithiothreitol.
  • the reducing agent may be ascorbic acid.
  • the kit of the present invention may further include at least one of a surfactant, a preservative, and an anti-copper ion interference reagent.
  • a surfactant e.g., a preservative
  • an anti-copper ion interference reagent e.g., a surfactant, a preservative, and an anti-copper ion interference reagent.
  • the kit of the present invention includes a first reagent and a second reagent
  • one or more of a surfactant, a preservative, and an anti-copper ion interference reagent may be included in the first reagent or the first reagent. Two reagents or these two reagents.
  • the surfactant may be a nonionic surfactant.
  • the blood sample may be serum or plasma.
  • the beneficial effects of the present invention are: compared with the prior art, the method and kit for detecting iron content according to the present invention can better resist chelating agents, while maintaining the basic performance of the basic performance is unchanged, and the reagent stability is good
  • the interference of EDTA and the interference of similar drugs such as deferoxamine make the EDTA blood collection tube samples and the samples of patients taking deferoxamine drugs more accurate, and will not cause other iron supplement drugs and hemolysis due to changes in the reaction system. Increased impact.
  • colorimetric methods are usually used to detect iron content in blood samples, that is: iron bound to transferrin is dissociated into ferric iron ions and transferrin in an acid medium; ferric iron is reduced using a reducing agent The ions are reduced to divalent iron ions; the complexing agent is then combined with the divalent iron ions to form a colored substance, thereby realizing the detection of iron content.
  • the present invention provides a solution for detecting the iron content in blood samples, that is, in an acidic environment with a pH of 3.0 to 6.0 Next, metal ions with a stronger coordination constant with EDTA than ferrous ions are introduced.
  • this solution can obtain more accurate results when testing blood samples of EDTA blood collection tubes and blood samples of subjects taking deferoxamine drugs. Further, this solution does not add other interference while removing the interference of the chelating agent. Specifically, although some solutions can also achieve the effect of anti-chelator interference, it will also cause the iron ions in iron dextran to be more easily detected, thereby affecting the detection results.
  • the present invention has good resistance to EDTA interference. It does not cause interference by iron dextran, and the accuracy is better.
  • the present invention provides a kit for detecting iron content in a blood sample, the kit includes:
  • the first reagent R1 the first reagent includes a buffer solution and a metal ion
  • the buffer solution has a pH of 3.0 to 6.0
  • iron bound to transferrin dissociates to Fe 3 in an acidic environment with a pH of 3.0 to 6.0 + ,
  • the binding ability of the metal ion to EDTA is stronger than that of Fe 2+ to EDTA;
  • the second reagent includes a reducing agent and a complexing agent.
  • the reducing agent is used to reduce Fe 3+ to Fe 2+ .
  • the complexing agent can combine with Fe 2+ to develop color, thereby Colorimetric method can be used to detect iron content in blood samples.
  • An exemplary detection process can be:
  • Iron content measured absorbance (A2-A1) ⁇ calibration solution / calibrated absorbance (A2-A1).
  • the first reagent R1 may also include other reagent components, such as surfactants, preservatives, and / or anti-interference components.
  • the second reagent R2 may also include other reagent components, such as surfactants, preservatives, and / or anti-interference components.
  • the first agent R1 of the present invention may further include a surfactant and an anti-interference component
  • the second agent R2 of the present invention may also include a preservative
  • metal ions and “anti-interference metal ions” refer to metal ions that have a stronger binding capacity to EDTA than ferric iron ions or that the coordination stability constant with EDTA is stronger than ferric iron ions Metal ions.
  • the binding ability of the metal ion and the complexing agent is weaker than the binding ability of divalent iron ion and the complexing agent.
  • Exemplary metal ions can be: Cd 2+ , Co 2+ , Hg 2+ , Ni 2+ , Pb 2+ , Sc 2+ , Sn 2+ , Th 4+ , TiO 2+ , U 4+ , VO 2 + , Zr 4+ or Zn 2+ , but the invention is not limited thereto.
  • Zn 2+ or Cd 2+ are selected as metal ions resistant to EDTA interference, so that the reagent cost can be reduced.
  • the metal ion may be provided in the form of a soluble salt.
  • the metal ion when the metal ion is Cd 2+ , it can be provided in the form of cadmium acetate; when the metal ion is Sn 2+ , it can be provided in the form of stannous chloride; when the metal ion is Co 2+ , it It can be provided in the form of cobalt acetate; when the metal ion is Zn 2+ , it can be provided in the form of zinc sulfate heptahydrate.
  • the concentration of metal ions may be, for example, about 0.1 mmol / L to 50 mmol / L, preferably about 1 mmol / L to 50 mmol / L, more preferably about 10 mmol / L to 50 mmol / L, for example 20 mmol / L, 30 mmol / L and 40mmol / L.
  • reducing agent refers to a reagent capable of reducing ferric ion to divalent iron ion.
  • the concentration of the reducing agent may preferably be 0.1 mmol / L to 100 mmol / L; in this case, the present invention can remove the interference caused by the iron dextran while removing the interference of the chelating agent. .
  • reducing agent capable of reducing ferric ions in the blood sample to ferric ions
  • exemplary reducing agents may include at least one of ascorbic acid, hydroxylamine hydrochloride, sodium sulfite, dithiothreitol.
  • the buffer solution may include acetate buffer solution, succinate buffer solution, citric acid buffer solution, tartaric acid buffer solution or glycine buffer solution, but the present invention is not limited thereto.
  • the pH of the buffer may be, for example, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0; or the pH is about 3.0 to 6.0, preferably about 3.0 to 5.0, more preferably about 3.5 to 5.0, and most preferably about 3.5 to 4.0 .
  • the pH range with only one endpoint or the pH range without any endpoint is within the protection scope of the present invention.
  • the pH of the buffer of the present invention is greater than 3.0 and less than 6.0, greater than 3.0 and less than or equal to 5.0, or greater than 3.5 and less than or equal to 5.0.
  • the complexing agent refers to an agent that can combine with divalent iron ions to form a colored substance.
  • exemplary complexing agents may be ferrousazine, 2,4,6-tris (2-pyridyl) triazine (TPTZ), orthophenanthroline, and the like.
  • surfactant suitable for blood sample detection and selectively use it in the solution of the present invention.
  • the present invention is not particularly limited.
  • Exemplary surfactants may be Brij35, Triton X-100, Tween 20 or Tween 80, but the invention is not limited thereto.
  • non-ionic surfactants can be used to achieve the purpose of solubilizing or eliminating blood lipid interference.
  • preservatives suitable for blood sample detection and selectively use it in the solution of the present invention.
  • the present invention is not particularly limited.
  • Exemplary preservatives are sodium azide, Proclin 300, but the invention is not limited thereto.
  • an anti-other type interference reagent suitable for iron content detection For the purpose of eliminating other types of interference present in the blood sample, a person skilled in the art can select an anti-other type interference reagent suitable for iron content detection.
  • the present invention is not particularly limited.
  • an agent resistant to copper ion interference such as thiourea, can be used in the solution of the present invention, but the present invention is not limited to this.
  • the blood sample of the present invention may be a serum or plasma sample.
  • the measured main wavelength is 570 nm
  • the auxiliary wavelength is 700 nm
  • the sample (calibrator) dosage is 20 ⁇ L
  • reagent 1 dosage is 250 ⁇ L
  • reagent 2 dosage is 50 ⁇ L.
  • a reagent composition (representing seven groups) including reagent 1 and reagent 2 was prepared according to the following formula.
  • the pH value and ascorbic acid concentration in the reagent composition of each group are shown in Table 1 and Table 2.
  • the standard sample and the high-concentration EDTA interference sample were tested three times respectively according to the above-mentioned iron content detection method. The results are shown in Table 1.
  • the standard sample and the high-concentration iron dextran interference sample were tested three times respectively according to the above-mentioned iron content detection method. The results are shown in Table 2.
  • a reagent composition (a total of eighteen groups) including reagent 1 and reagent 2 is prepared according to the following formula.
  • the values of pH and types of metal ions in each group of reagent compositions are shown in Table 3 and Table 4.
  • the standard sample and the high-concentration EDTA interference sample were tested three times respectively according to the above-mentioned iron content detection method. The results are shown in Table 3.
  • the standard sample and the high-concentration iron dextran interference sample were tested three times respectively according to the above-mentioned iron content detection method. The results are shown in Table 4.
  • a reagent composition (four groups in total) including reagent 1 and reagent 2 is prepared according to the following formula, and the concentration of metal ions in the reagent composition of each group is shown in Table 5 and Table 6.
  • the standard sample and the high-concentration EDTA interference sample were tested three times respectively according to the above iron content detection method, and the results are shown in Table 5.
  • the standard sample and the high-concentration iron dextran interference sample were tested three times respectively according to the above-mentioned iron content detection method. The results are shown in Table 6.
  • the present invention when using the method and kit according to the present invention to detect the iron content in blood samples, it can effectively avoid the interference of chelating agents, especially EDTA, on the iron content detection, and at the same time, it can not increase the iron pair of dextran. Interference with iron content detection results.
  • the present invention can further improve the effects of anti-chelating agent interference and anti-dextran iron interference.

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Abstract

L'invention concerne un procédé et un kit pour détecter la teneur en fer dans un échantillon de sang, le procédé comprenant : ajouter un agent de détection dans l'échantillon de sang, l'agent de détection comprenant un tampon ayant un pH de 3,0 à 6,0, un agent réducteur qui réduit Fe3+ en Fe2+, un ion métallique anti-interférence et un agent de complexation pour une liaison à Fe2+ pour développer une couleur, le fer lié à une transferrine se dissociant en Fe3+ dans un environnement acide de pH de 3,0 à 6,0, et la capacité de liaison entre l'ion métallique anti-interférence et l'EDTA est plus forte que celle de Fe2+ et EDTA ; et après l'ajout de l'agent de détection dans l'échantillon de sang, utiliser un procédé colorimétrique pour détecter la teneur en fer dans l'échantillon de sang.
PCT/CN2018/114545 2018-11-08 2018-11-08 Procédé et kit pour détecter la teneur en fer dans un échantillon de sang WO2020093305A1 (fr)

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CN201880097735.7A CN112714870A (zh) 2018-11-08 2018-11-08 检测血液样本中的铁含量的方法及试剂盒
PCT/CN2018/114545 WO2020093305A1 (fr) 2018-11-08 2018-11-08 Procédé et kit pour détecter la teneur en fer dans un échantillon de sang

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CN114460163B (zh) * 2022-02-11 2023-03-03 河北师范大学 一种检测生物组织铁含量的maldi质谱成像方法

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