WO1995013544A1 - Reactif permettant de determiner l'insaturation de la capacite de fixation du fer insature - Google Patents

Reactif permettant de determiner l'insaturation de la capacite de fixation du fer insature Download PDF

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Publication number
WO1995013544A1
WO1995013544A1 PCT/JP1993/001656 JP9301656W WO9513544A1 WO 1995013544 A1 WO1995013544 A1 WO 1995013544A1 JP 9301656 W JP9301656 W JP 9301656W WO 9513544 A1 WO9513544 A1 WO 9513544A1
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WO
WIPO (PCT)
Prior art keywords
agent
reagent
binding ability
unsaturated iron
iron
Prior art date
Application number
PCT/JP1993/001656
Other languages
English (en)
Japanese (ja)
Inventor
Masahiro Sekiguchi
Toshikatsu Abe
Kenji Hosoi
Original Assignee
Daiichi Pure Chemicals Co., Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to JP26162692A priority Critical patent/JP3170066B2/ja
Priority claimed from JP26162692A external-priority patent/JP3170066B2/ja
Application filed by Daiichi Pure Chemicals Co., Ltd. filed Critical Daiichi Pure Chemicals Co., Ltd.
Priority to PCT/JP1993/001656 priority patent/WO1995013544A1/fr
Publication of WO1995013544A1 publication Critical patent/WO1995013544A1/fr

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Classifications

    • 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
    • G01N33/90—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving iron binding capacity of blood

Definitions

  • the present invention relates to a reagent for measuring unsaturated iron binding ability in serum, which can maintain reagent performance stably for a long time in a solution state.
  • the total amount of iron in the body is about 400 mg. Approximately 2Z3 of these are present as hemoglobin iron in erythrocytes, and the remaining 13 are stored in tissues such as liver, spleen and bone marrow as ferritin or hemosiderin.
  • Transflurin is a carrier of iron in serum produced mainly by the liver.In particular, it transports iron and stored iron absorbed from the gastrointestinal tract to the bone marrow and supplies iron to hemoglobin-producing cells. Plays an important role.
  • Serum transfluulin levels are clinically indicated by its ability to bind iron.
  • SI serum iron
  • UIBC unsaturated iron binding capacity
  • the binding of transfrin to iron is reversible and strongly dependent on pH, It shows a very stable bond between 7 and 9, but it is easy to dissociate under acidic conditions and strong pH of 9 or more. Therefore, the measurement of unsaturated iron binding ability is carried out by adding an alkaline buffer solution containing a certain excess amount of iron ions (around pH 7 to 9) to serum to bind iron with transferrin and chelate the remaining iron.
  • a widely used method is to perform colorimetric determination using an agent and subtract it from the amount of iron added to calculate the amount.
  • most of the known chelating agents for which the iron ion concentration is colorimetrically determined by chemical coloring in this measurement are specific to divalent iron. Combination is required.
  • reducing agents examples include those that have no odor and are required for UIBC measurement.
  • Ascorbic acid is the most widely used because of its strong reducing power.
  • an object of the present invention is to provide a UIBC measurement reagent which can maintain the reagent performance stably for a long period of time.
  • the present inventors have result of ⁇ study, the alkaline buffer containing F e 2 + a first agent, a chelating agent, an acid solution containing completion ascorbic acid and a stabilizer second agent It has been found that, when these are used in combination, a UIBC measurement reagent that can maintain the reagent performance stably for a long period of time can be obtained, and the present invention has been completed.
  • the present invention relates to an unsaturated iron-binding agent comprising a first agent having a pH of 7 to 9 containing Fe 2+ and a buffer, and a second agent having a pH of less than 7 containing a chelating agent, ascorbic acid and a stabilizer. It relates to a measurement reagent.
  • the present invention further comprises adding a first agent of PH 7 to 9 containing Fe 2+ and a buffer to the sample, binding transphorin in the sample with F e 2+ in the first agent, , not characterized by this chelate agent, the second agent is less than pH 7 to ⁇ the Asukorubin acid and a stabilizing agent is added, the excess of F e 2 + is sharp Ichito color, colorimetry It relates to a method for measuring the saturated iron binding ability.
  • the present invention further comprises adding a first agent of PH 7 to 9 having Fe 2+ and a buffer solution to the sample, binding transphorin in the sample with F e 2+ in the first agent, A second agent having a pH of less than 7 containing a chelating agent, ascorbic acid and a stabilizing agent is added thereto, and the excess Fe2 + is chelated and colorimetrically measured to determine the unsaturated iron binding ability.
  • the present invention relates to a method for diagnosing anemia or hepatitis.
  • the first agent used in the present invention is one having a pH of 7 to 9 containing Fe 2+ and a buffer.
  • the source of Fe 2+ is not particularly limited.
  • ferrous sulfate ammonium, ferrous chloride, ferrous sulfate, and the like can be used, and the concentration of Fe 2+ in the first agent is 10%. It is preferred that the compounding be made in a range of 550 O jugZ, particularly 50 ⁇ 20 O ⁇ ugZc ⁇ .
  • the buffer solution is PH of the first agent?
  • a tris buffer a triethanolamine buffer, a borate buffer, a veronal buffer, a phosphate buffer and the like can be used.
  • the first agent is PH? 99, and within this range, Fe 2+ and transpelin can be stably bound.
  • the second agent is a product having a pH of less than 7 containing a chelating agent, ascorbic acid and a stabilizer.
  • the chelating agent is not particularly limited as long as it is not easily reduced by scorbic acid.
  • 3- (2-pyridyl) 1,5,6-bis (4-phenylsulfonic acid) 1,2,4-Triazine [Phenylene S ] Bathophenanthroline,, dipyridyl and the like.
  • the concentration of these chelating agents in the second agent is preferably 0.5 to 20 ⁇ , particularly preferably 1 to 5 mM.
  • the stabilizer is not particularly limited as long as it has a reducing property and is stable in an acidic solution.
  • the concentration of the stabilizer is 1 to 100 mM, particularly 2 to 5 OmM.
  • concentration of ascorbic acid is preferably from 0.02 to 3.0%, particularly preferably from 0.05 to 1.0%.
  • the pH of the second agent must be less than 7, preferably 1 to 5, and within this range, ascorbic acid can be stably maintained.
  • Ascorbic acid it is necessary to use a combination of ascorbic acid and a stabilizing agent as a reducing agent, and ascorbic acid alone is liable to be decomposed, and the stabilizing agent alone produces color. It will be incomplete, making accurate measurement impossible.
  • the first agent is added to the sample, and the transfusin in the sample and the F in the first agent are added.
  • the e 2+ is bound, and then a second agent is added thereto to chelate excess F e 2+, and colorimetrically determined.
  • the reaction is performed by measuring the absorbance of the reaction solution at 570 runs.
  • anemias such as iron deficiency anemia, aplastic anemia, pernicious anemia, chronic hemorrhagic anemia, polycythemia vera, infectious anemia, etc. can be diagnosed by measuring the unsaturated iron binding ability.
  • anemias such as iron deficiency anemia, aplastic anemia, pernicious anemia, chronic hemorrhagic anemia, polycythemia vera, infectious anemia, etc.
  • the liver is a transferrin production and iron storage organ, a decrease in hepatic function due to hepatocellular injury due to liver disease causes a change in transferrin production and the spread of iron into the blood. Varying the unsaturated iron binding ability. Therefore, liver disease such as acute hepatitis, chronic hepatitis, and cirrhosis can be diagnosed by measuring the unsaturated iron binding ability.
  • the reagent of Comparative Example 1 shows an increase in the measured value on the 8th day of storage of 10 t, whereas the reagent of the present invention shows that the value of the reagent of the present invention is 10 and that after storage for 18 days. No increase in measured values was observed.
  • Test example 3 The reagents for measuring unsaturated iron binding ability obtained in Examples 3 to 5 and Comparative Example 1 were stored at 37 t, and the UIBC value of the same specimen was measured over time in the same manner as in Test Example 1. Table 3 shows the results.
  • the reagent of Comparative Example 1 showed an increase in UIBC value on the second day of storage at 37 t and was unable to maintain the reagent performance, whereas / 9-mercaptopropion was used as a stabilizer.
  • the reagent of the present invention using an acid (Example 3), dithiothreitol (Example 4) or N-acetylsyl cysteine (Example 5), the UIBC value even after 30 days under the extreme condition of 37 No increase was observed, confirming that the reagent performance was maintained.
  • the reagent for measuring unsaturated iron binding ability of the present invention can maintain the reagent performance stably for a long period of time. Therefore, it can be supplied as a stable reagent, and is useful in test centers and hospitals that measure a large number of samples in large quantities.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Hematology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne un réactif permettant de déterminer l'insaturation de la capacité de fixation du fer et se composant d'un premier agent présentant un pH de 7 à 9 et comprenant Fe2+, d'une solution tampon et d'un deuxième agent représentant un pH au-dessous de 7, et contenant un agent de chélation, de l'acide ascorbique et un stabilisant. Les propriétés dudit réactif restent stables longtemps.
PCT/JP1993/001656 1992-09-30 1993-11-12 Reactif permettant de determiner l'insaturation de la capacite de fixation du fer insature WO1995013544A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
JP26162692A JP3170066B2 (ja) 1992-09-30 1992-09-30 不飽和鉄結合能測定試薬
PCT/JP1993/001656 WO1995013544A1 (fr) 1992-09-30 1993-11-12 Reactif permettant de determiner l'insaturation de la capacite de fixation du fer insature

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP26162692A JP3170066B2 (ja) 1992-09-30 1992-09-30 不飽和鉄結合能測定試薬
PCT/JP1993/001656 WO1995013544A1 (fr) 1992-09-30 1993-11-12 Reactif permettant de determiner l'insaturation de la capacite de fixation du fer insature

Publications (1)

Publication Number Publication Date
WO1995013544A1 true WO1995013544A1 (fr) 1995-05-18

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP1993/001656 WO1995013544A1 (fr) 1992-09-30 1993-11-12 Reactif permettant de determiner l'insaturation de la capacite de fixation du fer insature

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WO (1) WO1995013544A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102323430A (zh) * 2011-08-15 2012-01-18 北京利德曼生化股份有限公司 稳定的不饱和铁结合力测定试剂盒
CN110568206A (zh) * 2019-09-12 2019-12-13 苏州普瑞斯生物科技有限公司 一种总铁结合力检测试剂盒及其制备方法
CN111257549A (zh) * 2018-12-03 2020-06-09 深圳迈瑞生物医疗电子股份有限公司 检测血清中的不饱和铁结合力的试剂盒及方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS529160B2 (fr) * 1972-04-12 1977-03-14
JPS6232364A (ja) * 1985-08-06 1987-02-12 Shinotesuto Kenkyusho:Kk 血清中の不飽和鉄結合能の測定方法
JPH02167476A (ja) * 1988-12-21 1990-06-27 Internatl Reagents Corp 不飽和鉄結合能の測定方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS529160B2 (fr) * 1972-04-12 1977-03-14
JPS6232364A (ja) * 1985-08-06 1987-02-12 Shinotesuto Kenkyusho:Kk 血清中の不飽和鉄結合能の測定方法
JPH02167476A (ja) * 1988-12-21 1990-06-27 Internatl Reagents Corp 不飽和鉄結合能の測定方法

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102323430A (zh) * 2011-08-15 2012-01-18 北京利德曼生化股份有限公司 稳定的不饱和铁结合力测定试剂盒
CN111257549A (zh) * 2018-12-03 2020-06-09 深圳迈瑞生物医疗电子股份有限公司 检测血清中的不饱和铁结合力的试剂盒及方法
CN110568206A (zh) * 2019-09-12 2019-12-13 苏州普瑞斯生物科技有限公司 一种总铁结合力检测试剂盒及其制备方法

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