JP2013141453A - Measuring method and measuring reagent for measuring object substance, and method for avoiding effects derived from bilirubin - Google Patents

Abstract

PROBLEM TO BE SOLVED: To avoid effects on measurement, which are derived from bilirubin contained in a sample, in a measuring method and measuring reagent for measuring a measuring object substance in the sample by optically measuring hydrogen peroxide generated by oxidase by use of chromogen.SOLUTION: The method (or reagent) for measuring the measuring object substance in the sample by optically measuring hydrogen peroxide generated by oxidase by use of chromogen includes: performing the measurement in the presence of at least one polyoxyethylene alkyl nitrogen-containing compound selected from polyoxyethylene alkylamine compounds and polyoxyethylene alkylamide compounds (or including the compound).

Description

The present invention relates to a method and a reagent for measuring a substance to be measured in a sample by optically measuring hydrogen peroxide generated by an oxidase using a chromogen, which is derived from bilirubin contained in the sample. The present invention relates to a measurement method and a measurement reagent for a substance to be measured in a sample that can avoid an influence on the measurement.
In addition, the present invention avoids an influence on measurement derived from bilirubin contained in a sample in measurement of a measurement target substance in a sample in which hydrogen peroxide generated by an oxidase is optically measured using a chromogen. It is about how to do.
The present invention is useful in the field of life science such as clinical examination, clinical pathology and medicine, and in the field of chemistry such as analytical chemistry.

  A method and a reagent for measuring a substance to be measured in a sample by optically measuring hydrogen peroxide generated by an oxidase using a chromogen are frequently used because the measurement is accurate. .

  However, it is also known that the measurement of a measurement target substance in a sample using these measurement methods and measurement reagents is easily influenced by various reducing substances, drugs, dyes, and the like contained in the sample.

  Among these influential substances, bilirubin exists as a free or conjugated form in biological samples, and it has been found that it causes errors in the measured values of substances to be measured in samples and affects the measurement. Yes.

  As a method of avoiding the influence on the measurement derived from bilirubin contained in the sample, for example, a method using a chromogen that has a high affinity for hydrogen peroxide and develops a color by reacting with hydrogen peroxide faster than bilirubin ( Non-patent document 1), a method of degrading bilirubin using an oxidizing agent such as potassium ferrocyanide, iodic acid or periodic acid (see non-patent document 1), lactate oxidase or uricase reaction using an endogenous substrate A method of oxidatively degrading bilirubin with hydrogen peroxide generated by a pretreatment enzyme reaction such as (see Non-Patent Document 1), a method using bilirubin oxidase (see Patent Document 1), and a divalent copper ion compound Method using surfactant and / or cyanide compound (see Patent Document 2), cationic surfactant and / or amphoteric surfactant A method using an agent (see Patent Document 3), a method using an amphoteric surfactant (see Patent Document 4), a nonionic surfactant comprising a ferrocyanated compound and an aglycone such as a polysaccharide and an alkyl group. Method of use (see Patent Document 5), method of using a surfactant containing an iron complex and an alkyl group having a steroid skeleton and a polyoxyalkylene group (see Patent Document 6), and using zinc ions at pH 7 or higher A method (see Patent Document 7) or the like has been proposed, but it has not always been perfect.

  That is, in the measurement of the measurement target substance in the sample, in order to obtain an accurate measurement result (measurement value), further improvement of the method for avoiding the influence on the measurement derived from bilirubin contained in the sample has been desired. .

Japanese Patent Publication No.55-25840 JP 59-159798 A Japanese Patent Laid-Open No. 3-10696 Japanese Patent Laid-Open No. 7-39394 Japanese Patent Laid-Open No. 9-224697 JP-A-11-243993 JP 2004-271440 A

Proposal for Clinical Laboratory Methods, 33rd edition, page 37, supervised by Masamitsu Kanai, published by Kanbara Publishing, April 1, 2010

In measurement of a measurement target substance in a sample, the measurement is affected by bilirubin contained in the sample, and an error may occur in the measurement result (measurement value).
For this reason, for example, when a measurement result (measurement value) of a measurement target substance in a sample is used for diagnosis in a clinical test in a medical field, there is a possibility that a diagnosis of a disease or the like may be mistaken.

  Therefore, in the measurement of the measurement target substance in the sample, there is a measurement method, a measurement reagent, and a method for avoiding the influence on the measurement derived from bilirubin, which has further improved the avoidance of the influence on the measurement derived from the bilirubin contained in the sample. It was desired.

  On the other hand, an object of the present invention is a method and a reagent for measuring a measurement target substance in a sample by optically measuring hydrogen peroxide generated by an oxidase using a chromogen. It is to provide a measuring method and a measuring reagent for a substance to be measured in a sample that can avoid the influence on the measurement derived from bilirubin contained in the sample.

  Also, a method for avoiding the influence on the measurement derived from bilirubin contained in the sample in the measurement of the measurement target substance in the sample by optically measuring hydrogen peroxide generated by the oxidase using a chromogen. Is to provide.

  The inventors of the present invention have repeatedly investigated the avoidance of the influence on the measurement derived from bilirubin contained in the sample, and found that the above-mentioned problems can be solved by performing the measurement in the presence of a specific compound. It came to complete.

That is, this invention consists of the following invention.
(1) In a method for measuring a substance to be measured in a sample by optically measuring hydrogen peroxide generated by an oxidase using a chromogen, a polyoxyethylene alkylamine compound and a polyoxyethylene alkylamide compound A method for measuring a substance to be measured in a sample, wherein the measurement is performed in the presence of at least one polyoxyethylene alkyl nitrogen-containing compound selected from the group consisting of:
(2) By measuring in the presence of at least one polyoxyethylene alkyl nitrogen-containing compound selected from a polyoxyethylene alkylamine compound and a polyoxyethylene alkylamide compound, the measurement is derived from bilirubin contained in the sample. The method for measuring a substance to be measured in a sample according to (1), wherein the influence of the above is avoided.
(3) In a reagent for measuring a substance to be measured in a sample by optically measuring hydrogen peroxide generated by an oxidase using a chromogen, a polyoxyethylene alkylamine compound and a polyoxyethylene alkylamide compound A measuring reagent for a substance to be measured in a sample, comprising at least one polyoxyethylene alkyl nitrogen-containing compound selected from the group consisting of:
(4) At least one polyoxyethylene alkyl nitrogen-containing compound selected from a polyoxyethylene alkylamine compound and a polyoxyethylene alkylamide compound is included as an agent for avoiding an influence on measurement derived from bilirubin contained in a sample. The reagent for measuring the substance to be measured in the sample according to (3) above.
(5) In measuring the substance to be measured in a sample by optically measuring hydrogen peroxide generated by oxidase using a chromogen, polyoxyethylene alkylamine compound and polyoxyethylene alkylamide compound A method for avoiding an influence on measurement derived from bilirubin contained in a sample, wherein the measurement is carried out in the presence of at least one polyoxyethylene alkyl nitrogen-containing compound selected from the group consisting of:

  The method for measuring a substance to be measured in a sample according to the present invention, a reagent for measuring a substance to be measured in a sample, and a method for avoiding an influence on measurement derived from bilirubin contained in a sample are provided by measuring a substance to be measured contained in a sample In this case, it is possible to prevent the measurement from being affected by the bilirubin contained in the sample, and to prevent an error from occurring in the measurement result (measurement value).

  That is, in the measurement method of the measurement target substance in the sample of the present invention, the measurement reagent of the measurement target substance in the sample, and the method of avoiding the influence on the measurement derived from bilirubin contained in the sample, the bilirubin contained in the sample An accurate measurement result (measurement value) that is not affected can be obtained.

  Thereby, the measurement method of the measurement target substance in the sample of the present invention, the measurement reagent of the measurement target substance in the sample, and the method for avoiding the influence on the measurement derived from bilirubin contained in the sample are, for example, in the medical field. It is possible to prevent misdiagnosis.

[1] Method for measuring a substance to be measured in a sample General The method for measuring a substance to be measured in a sample of the present invention is a method for measuring a substance to be measured in a sample by optically measuring hydrogen peroxide generated by an oxidase using a chromogen. The measurement is performed in the presence of at least one polyoxyethylene alkyl nitrogen-containing compound selected from an oxyethylene alkylamine compound and a polyoxyethylene alkylamide compound.

  And in the measuring method of the substance to be measured in the sample of the present invention, the measurement is performed in the presence of at least one polyoxyethylene alkyl nitrogen-containing compound selected from a polyoxyethylene alkylamine compound and a polyoxyethylene alkylamide compound. By performing, the influence on the measurement derived from bilirubin contained in the sample can be avoided.

2. Polyoxyethylene alkyl nitrogen-containing compound (1) General description In the method for measuring a substance to be measured in a sample of the present invention, hydrogen peroxide generated by an oxidase is optically measured using a chromogen in the sample. In the method of measuring a measurement target substance, measurement is performed in the presence of at least one polyoxyethylene alkyl nitrogen-containing compound selected from a polyoxyethylene alkylamine compound and a polyoxyethylene alkylamide compound.

  In the present invention, the polyoxyethylene alkylamine compound and the polyoxyethylene alkylamide compound are collectively referred to as a polyoxyethylene alkyl nitrogen-containing compound.

(2) Polyoxyethylene alkylamine compound Although it is a polyoxyethylene alkylamine compound in this invention, if it is a polyoxyethylene alkylamine compound, it can be used without limitation.

In the present invention, the polyoxyethylene alkylamine compound is represented by the general formula (I) [wherein R ₁ is a saturated or unsaturated alkyl group having 5 to 30 carbon atoms, and m and n are each an ethylene oxide addition mole number. And is an integer of 1 or more, and the sum of m and n is 2 to 30].

The lower limit of the carbon number of the saturated or unsaturated alkyl group in R ₁ of the polyoxyethylene alkylamine compound represented by the general formula (I) is more preferably 8 or more, particularly preferably. It has 12 or more carbon atoms.

The upper limit of the carbon number of the saturated or unsaturated alkyl group in R ₁ of the polyoxyethylene alkylamine compound represented by the general formula (I) is more preferably 20 or less, particularly preferably. It has 18 or less carbon atoms.

  The lower limit of the sum of m and n in the polyoxyethylene alkylamine compound represented by the general formula (I) is more preferably 5 or more, and particularly preferably 10 or more. (M and n are each an integer of 1 or more.)

  Further, the upper limit of the sum of m and n in the polyoxyethylene alkylamine compound represented by the general formula (I) is more preferably 25 or less, and particularly preferably 20 or less. (M and n are each an integer of 1 or more.)

More specifically, examples of the polyoxyethylene alkylamine compound include polyoxyethylene oleylamine (alkyl group R ₁ is an oleyl group having 18 carbon atoms and ₁ double bond), polyoxyethylene stearylamine (alkyl group R _1). carbon atoms 18, stearyl group of saturated), polyoxyethylene laurylamine (the alkyl group R ₁ is 12 carbon atoms, lauryl group, saturated), or polyoxyethylene octyl amine (the alkyl group R ₁ is 8 carbon atoms, a saturated Octyl group) and the like.

  In the present invention, examples of the polyoxyethylene alkylamine compound also include polyoxyethylene coconut oil alkylamine.

The general fatty acid composition of coconut oil is: caprylic acid (C ₇ H ₁₅ COOH; saturated) 8%, capric acid (C ₉ H ₁₉ COOH; saturated) 6%, lauric acid (C ₁₁ H ₂₃ COOH; saturated) ) 49%, myristic acid (C ₁₃ H ₂₇ COOH; saturated) 18%, palmitic acid (C ₁₅ H ₃₁ COOH; saturated) 8%, stearic acid (C ₁₇ H ₃₅ COOH; saturated) 3%, and oleic acid ( C ₁₇ H ₃₄ COOH; number of double bonds 1) 7%.

  In addition, in this invention, a polyoxyethylene beef tallow alkylamine can also be mentioned as a polyoxyethylene alkylamine compound.

More specifically, as a polyoxyethylene alkylamine compound, for example, polyoxyethylene oleylamine (an alkyl group R ₁ is an oleyl group having 18 carbon atoms and ₁ double bond), “Nymeen O” [NOF Corporation (Japan)].

In addition, as polyoxyethylene stearylamine (alkyl group R ₁ has 18 carbon atoms and a saturated stearyl group), for example, “Nimine S” [Nippon Oil Corporation (Japan)] or “Amate” [Kao Corporation (Japan) Country)] and the like.

Examples of polyoxyethylene laurylamine (alkyl group R ₁ is a saturated lauryl group having 12 carbon atoms) include, for example, “Naimine L” [Nippon Oil Corporation (Japan)], “Amate” [Kao Corporation (Japan) Country)], or “Nikkor tamnd” [Nikko Chemicals Co., Ltd. (Japan)].

  Examples of the polyoxyethylene coconut oil alkylamine include “Naymin F” [Nippon Oil Co., Ltd. (Japan)].

  Moreover, as polyoxyethylene beef tallow alkylamine, for example, “Naymin T2” [NOF Corporation (Japan)] can be exemplified. ("Naymin" is a registered trademark.)

More specifically, as a polyoxyethylene alkylamine compound, for example, as polyoxyethylene oleylamine (alkyl group R ₁ is an oleyl group having 18 carbon atoms and ₁ double bond), “Nymeen O-205” (ethylene oxide addition mole) is used. The sum of m and n which is a number: 5) [NOF Corporation (Japan)] and the like.

In addition, as polyoxyethylene stearylamine (alkyl group R ₁ has 18 carbon atoms and a saturated stearyl group), for example, “Nymine S-202” (sum of m and n, which is the number of moles of ethylene oxide added: 2) or “Nymine "S-204" (sum of ethylene oxide addition moles and n: 4) or "Naimine S-210" (sum of ethylene oxide addition moles and n: 10) or "Naimine S-215" (ethylene oxide) The sum of m and n, which is the number of added moles: 15) or “Nymeen S-220” (sum of m and n, the number of moles of added ethylene oxide: 20) [both NOF Corporation (Japan)], or “ “Amate 302” or “Amate 308” or “Amate 320” (methylene oxide addition moles and n: 20) [all are Kao Corporation (Japan)].

Further, as polyoxyethylene laurylamine (alkyl group R ₁ is a saturated lauryl group having 12 carbon atoms), for example, “Naymin L-202” or “Naymine L-207” [both NOF Corporation (Japan) ], Or "Amate 102" or "Amate 105" [both Kao Corporation (Japan)], or "Nikkor tamnd-15" (sum of m and n, which is the number of moles of ethylene oxide added: 15) [Nikko Chemicals Corporation Company (Japan)].

In addition, as polyoxyethylene octylamine (alkyl group R ₁ has 8 carbon atoms and a saturated octyl group), for example, “polyoxyethylene octylamine” (sum of m and n, which is the number of moles of added ethylene oxide: 10), etc. Can be mentioned.

  In addition, as polyoxyethylene coconut oil alkylamine, for example, “Naymeen F-202” (sum of m and n added ethylene oxide number: 2), “Nymeen F-203” (methylene oxide added mole number) Sum of n: 3), “Naymeen F-205” (sum of m and n added to ethylene oxide: 5), “Nymeen F-210” (sum of added m and n of ethylene oxide: 10) Or “Naymine F-215” (sum of m and n, which is the number of moles of ethylene oxide added: 15) [all NOF Corporation (Japan)].

  Moreover, as polyoxyethylene beef tallow alkylamine, for example, “Naymeen T2-202”, “Nymeen T2-210”, or “Nymeen T2-230” [all are NOF Corporation (Japan)] and the like can be mentioned. it can.

(3) Polyoxyethylene alkylamide compound Although it is a polyoxyethylene alkylamide compound in this invention, if it is a polyoxyethylene alkylamide compound, it can use without limitation.

In the present invention, the polyoxyethylene alkylamide compound has a general formula (II) [wherein R ₂ —CO—N is a saturated or unsaturated fatty acid residue having 5 to 30 carbon atoms, and p and q are Each of them is an ethylene oxide addition mole number and an integer of 1 or more, and the sum of p and q is 2 to 30].

In the polyoxyethylene alkylamide compound represented by the general formula (II), the lower limit of the carbon number of the saturated or unsaturated fatty acid residue in R ₂ —CO—N is more preferably 8 or more. Yes, particularly preferably it has 12 or more carbon atoms.

In the polyoxyethylene alkylamide compound represented by the general formula (II), the upper limit of the carbon number of the saturated or unsaturated fatty acid residue in R ₂ —CO—N is more preferably 20 or less. Yes, particularly preferably it has 18 or less carbon atoms.

  The lower limit of the sum of p and q in the polyoxyethylene alkylamide compound represented by the general formula (II) is more preferably 5 or more, and particularly preferably 10 or more. (Note that p and q are each an integer of 1 or more.)

  Further, the upper limit of the sum of p and q in the polyoxyethylene alkylamide compound represented by the general formula (II) is more preferably 25 or less, and particularly preferably 20 or less. (Note that p and q are each an integer of 1 or more.)

In the present invention, the polyoxyethylene alkylamide compound is represented by the general formula (III) [wherein R ₃ —CO—NH is a saturated or unsaturated fatty acid residue having 5 to 30 carbon atoms, and r is Also preferred is a polyoxyethylene alkylamide compound represented by the number of moles of ethylene oxide added and an integer of 1 to 30].

In the polyoxyethylene alkylamide compound represented by the general formula (III), the lower limit of the carbon number of the saturated or unsaturated fatty acid residue in R ₃ —CO—NH is more preferably 8 or more. Yes, particularly preferably it has 12 or more carbon atoms.

In the polyoxyethylene alkylamide compound represented by the general formula (III), the upper limit of the carbon number of the saturated or unsaturated fatty acid residue in R ₃ —CO—NH is more preferably 20 or less. Yes, particularly preferably it has 18 or less carbon atoms.

  The lower limit of r in the polyoxyethylene alkylamide compound represented by the general formula (III) is more preferably 5 or more, and particularly preferably 10 or more. (R is an integer.)

  In addition, the upper limit of r in the polyoxyethylene alkylamide compound represented by the general formula (III) is more preferably 25 or less, and particularly preferably 20 or less. (R is an integer.)

More specifically, as a polyoxyethylene alkylamide compound, for example, polyoxyethylene oleic acid amide (fatty acid which is a fatty acid residue group is oleic acid having 18 carbon atoms and 1 double bond), or polyoxyethylene Examples thereof include lauric acid amide (the fatty acid serving as the group of the fatty acid residue R ₃ —CO—NH is a saturated lauric acid having 12 carbon atoms).

In the present invention, examples of the polyoxyethylene alkylamide compound include polyoxyethylene coconut oil fatty acid amide.
The general fatty acid composition of coconut oil is: caprylic acid (C ₇ H ₁₅ COOH; saturated) 8%, capric acid (C ₉ H ₁₉ COOH; saturated) 6%, lauric acid (C ₁₁ H ₂₃ COOH; saturated) ) 49%, myristic acid (C ₁₃ H ₂₇ COOH; saturated) 18%, palmitic acid (C ₁₅ H ₃₁ COOH; saturated) 8%, stearic acid (C ₁₇ H ₃₅ COOH; saturated) 3%, and oleic acid ( C ₁₇ H ₃₄ COOH; number of double bonds 1) 7%.

  More specifically, as a polyoxyethylene alkylamide compound, for example, polyoxyethylene oleic acid amide (fatty acid residue fatty acid is oleic acid having 18 carbon atoms and 1 double bond), “TAMDO” [Nikko Chemicals Corporation (Japan)] and the like.

In addition, as polyoxyethylene lauric acid amide (fatty acid which is a group of fatty acid residue R ₃ —CO—NH is a lauric acid having 12 carbon atoms and saturated), for example, “Nymid ML” [NOF Corporation (Japan) And the like.

  Further, as polyoxyethylene coconut oil fatty acid amide, for example, “Nymid DF” [Nippon Co., Ltd. (Japan)], “Nymid MF” [Nippon Oil Co., Ltd. (Japan)], or “Nymid F” [Japan Oil Co., Ltd. (Japan)]. ("Nymid" is a registered trademark.)

  More specifically, as a polyoxyethylene alkylamide compound, for example, polyoxyethylene oleic acid amide (fatty acid which is a fatty acid residue group is oleic acid having 18 carbon atoms and 1 double bond), “TAMDO-10” (Ethylene oxide addition mole number: 10) [Nikko Chemicals Co., Ltd. (Japan)] etc. can be mentioned.

In addition, as polyoxyethylene lauric acid amide (fatty acid which is a group of fatty acid residue R ₃ —CO—NH is carbon number 12 and saturated lauric acid), for example, “Nymid ML-203” (ethylene oxide addition mole number) The number of r: 3), or “Nymid ML-211” (number of r as the number of moles of ethylene oxide added: 11) [all are NOF Corporation (Japan)].

  Moreover, as polyoxyethylene coconut oil fatty acid amide, for example, “Nymid DF-203” (sum of addition of p and q, which is the number of moles of ethylene oxide added: 3) [Nippon Oil Co., Ltd. (Japan)] and the like can be mentioned. .

  Moreover, as polyoxyethylene coconut oil fatty acid amide, for example, “Nymid MF-203” (number of r as ethylene oxide addition moles) or “Nymid MF-210” (number of r as ethylene oxide addition moles) : 11) [both NOF Corporation (Japan)] and the like.

  Examples of the polyoxyethylene coconut oil fatty acid amide include “Nymid F-205” (number of r as the number of moles of ethylene oxide added: 5) [Nippon Oil Co., Ltd. (Japan)].

(4) Details of concentration of polyoxyethylene alkyl nitrogen-containing compound In the present invention, at least one polyoxyethylene alkyl nitrogen-containing compound selected from a polyoxyethylene alkylamine compound and a polyoxyethylene alkylamide compound is present. The concentration at which the polyoxyethylene alkyl nitrogen-containing compound is present is not particularly limited, but is preferably 0.01% (w / v) or more in the measurement reaction.

  The lower limit of the preferred concentration at which the polyoxyethylene alkyl nitrogen-containing compound is present is more preferably 0.05% (w / v) or more, particularly preferably 0.1% (w / v) during the measurement reaction. v) Above.

  Moreover, although it is a density | concentration in which this polyoxyethylene alkyl nitrogen-containing compound exists, there is no upper limit in particular in the density | concentration at the time of a measurement reaction, but 2% (w / v) is enough considering cost etc. .

  The upper limit of the preferred concentration at which the polyoxyethylene alkyl nitrogen-containing compound is present is more preferably 1% (w / v) or less, particularly preferably 0.5% (w / v) during the measurement reaction. It is as follows.

  In addition, it is preferable that the measurement reagent of the measurement target substance in the sample of the present invention contains a polyoxyethylene alkyl nitrogen-containing compound having such a concentration as to be the above-described concentration during the measurement reaction.

  In the present invention, the polyoxyethylene alkyl nitrogen-containing compound may be measured in the presence of one type, or may be measured in the presence of a plurality of types.

  In the present invention, the polyoxyethylene alkyl nitrogen-containing compound may be measured in the presence of only the polyoxyethylene alkylamine compound, or the measurement may be performed in the presence of only the polyoxyethylene alkylamide compound. Alternatively, the measurement may be performed in the presence of both a polyoxyethylene alkylamine compound and a polyoxyethylene alkylamide compound.

  In the present invention, when the measurement reaction consists of only one stage, a polyoxyethylene alkyl nitrogen-containing compound is present in the measurement reaction at that stage.

  When the measurement reaction is composed of two or more stages, it is preferable that the polyoxyethylene alkyl nitrogen-containing compound is present at least at the final stage of the measurement reaction.

  In the present invention, the bilirubin contained in the sample is measured by performing measurement in the presence of at least one polyoxyethylene alkyl nitrogen-containing compound selected from a polyoxyethylene alkylamine compound and a polyoxyethylene alkylamide compound. The influence on the derived measurement can be avoided.

3. Measurement of a measurement target substance in a sample (1) General The measurement method of a measurement target substance in a sample of the present invention is based on optical measurement of hydrogen peroxide generated by an oxidase using a chromogen. In the method for measuring a substance to be measured, the measurement is performed in the presence of at least one polyoxyethylene alkyl nitrogen-containing compound selected from a polyoxyethylene alkylamine compound and a polyoxyethylene alkylamide compound. It is.

  In this method, the target substance in the sample is measured by optically measuring the hydrogen peroxide generated by this oxidase using the chromogen. There is no particular limitation as long as it is a method that can be used to measure the substance to be measured in the sample by optical measurement.

  As this method, for example, an oxidase is used to generate hydrogen peroxide directly or indirectly from a substance to be measured, and this is mixed with peroxidase and a chromogen and brought into contact with the color developing system to produce an oxidation reaction. Examples include a method of measuring the generated hydrogen peroxide by optically measuring the dye produced from the chromogen by the above, and thereby measuring the measurement target substance contained in the sample.

  More specifically, for example, oxidase is used to generate hydrogen peroxide directly or indirectly from the substance to be measured, and this is converted to peroxidase, a chromogen that is a hydrogen donor, and the chromogen of this chromogen. The resulting hydrogen peroxide is measured by mixing and bringing into contact with the coupler as the condensation target, leading to a color development system, and optically measuring the dye produced from the chromogen by the oxidative condensation reaction. And the like, and the like.

  In addition, for example, oxidase is used to generate hydrogen peroxide directly or indirectly from the substance to be measured, and this is mixed with peroxidase and chromogen and brought into contact with the coloring system without using the coupler. In addition, by measuring optically the dye produced from the chromogen by the oxidation reaction, the generated hydrogen peroxide can be measured, and the method of measuring the measurement target substance contained in the sample can also be mentioned. it can.

(2) Sample The sample in the present invention is a sample that may contain a substance to be measured and is intended to be measured.

  The sample is not particularly limited as long as it is as described above. For example, the sample may be a biological sample, meat, vegetables, grains, fruits, marine products, processed foods, beverages, drinking water, well water, river water, lake water. , Seawater, soil, air, or pharmaceuticals.

  For example, biological samples include human or animal blood, serum, plasma, urine, stool, cerebrospinal fluid, saliva, sweat, tears, ascites, amniotic fluid, brain and other organs, hair, skin, nails, muscles, nerves, etc. Examples include tissues and cells.

In the present invention, the sample is preferably a liquid.
If the sample containing the substance to be measured is not liquid, pretreatment such as extraction or solubilization may be performed according to a known method so that the substance to be measured is contained in the liquid.

  In the present invention, a biological sample is preferable as the sample, and blood, serum, plasma, or urine is more preferable as the sample.

(3) Substance to be measured The substance to be measured in the present invention is contained in a sample and is intended to measure the presence or absence (or concentration) in the sample. It is a substance that can be measured by optical measurement using a chromogen.
If it is as above-mentioned, there will be no limitation in particular as a measuring object substance

  Examples of this measurement target substance are shown in Table 1.

  In the present invention, the measurement target substance is preferably creatinine or uric acid, and the measurement target substance is more preferably creatinine.

(4) Oxidase In the present invention, an oxidase refers to a substance that catalyzes a reaction for generating hydrogen peroxide, which is an oxidizing substance, directly or indirectly from a measurement target substance contained in a sample. Any substance can be used without particular limitation.

  Table 1 also shows examples of “oxidase” and “other main components” used for measurement of the substance to be measured in this sample. (The peroxidase, chromogen, and coupler that generate pigment from hydrogen peroxide generated by the reaction with oxidase will be described in detail later. Is not listed.)

  That is, examples of the oxidase include sarcosine oxidase, cholesterol oxidase, glycerol-3-phosphate oxidase, uricase, glucose oxidase, choline oxidase, and acyl CoA oxidase.

  In the present invention, sarcosine oxidase or uricase is preferable as the oxidase, and sarcosine oxidase is more preferable as the oxidase.

  In the measurement of the measurement target substance in the sample of the present invention, the activity value of the oxidase is not particularly limited, but is preferably 100 units / L (Unit / L) or more in the measurement reaction.

  Further, the lower limit of the activity value of the oxidase is more preferably 1,000 units / L or more, and particularly preferably 5,000 units / L or more, in the measurement reaction.

  There is no particular upper limit to the activity value during the measurement reaction of this oxidase, but up to 100,000 units / L is sufficient in view of cost and the like.

  Further, the upper limit of the activity value of this oxidase is more preferably 80,000 units / L or less, particularly preferably 50,000 units / L or less, in the measurement reaction.

  In addition, it is preferable that the measurement reagent of the measurement target substance in the sample of the present invention contains an oxidase having an activity value that becomes the above activity value during the measurement reaction.

(5) Peroxidase In the present invention, a peroxidase (peroxidase, POD) is used together with a chromogen or with a chromogen and a coupler in order to produce a pigment from hydrogen peroxide generated by a reaction with an oxidase. Can do.

  This peroxidase can catalyze the reaction of oxidizing the chromogen with hydrogen peroxide to produce a dye, or catalyzing the reaction of oxidative condensation of the chromogen and coupler with hydrogen peroxide to produce a dye. Anything can be used as long as it can be used.

  As this peroxidase, those derived from any source can be used, for example, those derived from animals such as humans or cows, those derived from plants such as horseradish, or those derived from microorganisms such as bacteria or mold. it can.

  The peroxidase may be a peroxidase prepared from a genetic recombination technique in which a peroxidase gene such as a microorganism is incorporated into a microorganism such as Escherichia coli, or a peroxidase prepared from a microorganism whose properties have been improved by gene modification or the like. Good.

  In the measurement of the substance to be measured in the sample of the present invention, the activity value of peroxidase is not particularly limited, but is preferably 10 units / L (Unit / L) or more during the measurement reaction.

  Moreover, the lower limit of the activity value of the peroxidase is more preferably 50 units / L or more, and particularly preferably 100 units / L or more, in the measurement reaction.

  There is no particular upper limit to the activity value during the measurement reaction of peroxidase, but up to 500,000 units / L is sufficient in view of cost and the like.

  Moreover, the upper limit of the activity value of this peroxidase is more preferably 100,000 units / L or less, and particularly preferably 50,000 units / L or less, in the measurement reaction.

  In addition, it is preferable that the measurement reagent of the measurement target substance in the sample of the present invention contains peroxidase having an activity value that becomes the above activity value during the measurement reaction.

(6) Chromogen (a) In the present invention, the chromogen is a measurement of a measurement target substance in a sample by optically measuring hydrogen peroxide generated by an oxidase using the chromogen. It is a substance that can do. Any such substance can be used without any particular limitation.

(B) In the present invention, this chromogen is, for example, a substance that produces a dye by oxidative condensation with this coupler by mixing and contacting with hydrogen peroxide, peroxidase, and coupler produced by oxidase during measurement. It may be.
In this case, the chromogen is a hydrogen donor that releases hydrogen during the oxidative condensation reaction.

  In this case, examples of the chromogen include phenol, a phenol derivative, and an aniline derivative.

  Examples of this phenol derivative include 4-chlorophenol, 2,4-dichlorophenol, 2,4-dibromophenol, 2,4,6-trichlorophenol, and salts thereof.

  Examples of aniline derivatives include N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (HDAOS), N-sulfopropyl-3,5-dimethoxyaniline (HDAPS), and N-ethyl. -N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (DAOS), N-ethyl-N-sulfopropyl-3,5-dimethoxyaniline (DAPS), N-ethyl-N- ( 2-hydroxy-3-sulfopropyl) -3,5-dimethoxy-4-fluoroaniline (FDAOS), N-ethyl-N-sulfopropyl-3,5-dimethoxy-4-fluoroaniline (FDAPS), N- ( 2-carboxyethyl) -N-ethyl-3,5-dimethoxyaniline (CEDB), N-ethyl-N- (2-hydroxy -3-Sulfopropyl) -3-methoxyaniline (ADOS), N-ethyl-N- (3-sulfopropyl) -3-methoxyaniline (ADPS), N-ethyl-N- (2-hydroxy-3-sulfo) Propyl) aniline (ALOS), N-ethyl-N- (3-sulfopropyl) aniline (ALPS), N- (3-sulfopropyl) aniline (HALPS), N-ethyl-N- (2-hydroxy-3- Sulfopropyl) -3,5-dimethylaniline (MAOS), N-ethyl-N- (3-sulfopropyl) -3,5-dimethylaniline (MAPS), N-ethyl-N- (2-hydroxy-3- Sulfopropyl) -3-methoxyaniline (TOOS), N- (2-carboxyethyl) -N-ethyl-m-toluidine (CEMB), N- (2-cal Kishiechiru) -N- ethyl-3-methoxy aniline (Cemo), or N- (3- sulfopropyl) -3-methoxy-5-methylaniline (HMMPS), or may be their salts, and the like.

  In the measurement of the substance to be measured in the sample of the present invention, the concentration of the chromogen is not particularly limited, but is preferably 0.005 mM or more during the measurement reaction.

  Further, the lower limit of the concentration of the chromogen is more preferably 0.05 mM or more, and particularly preferably 0.5 mM or more, in the measurement reaction.

  There is no particular upper limit to the concentration of the chromogen during the measurement reaction, but up to 50 mM is sufficient in view of cost and the like.

  In addition, the upper limit of the concentration of the chromogen is more preferably 20 mM or less, and particularly preferably 10 mM or less, during the measurement reaction.

  In addition, it is preferable that the measurement reagent of the measurement target substance in the sample of the present invention contains the chromogen at a concentration such that the concentration becomes the concentration during the measurement reaction.

(C) In the present invention, the chromogen is mixed and contacted with, for example, hydrogen peroxide generated by an oxidase and peroxidase at the time of measurement (that is, without being mixed and contacted with a coupler). Further, it may be a substance that produces a pigment by an oxidation reaction.

  In this case, examples of the chromogen include bis [3-bis (4-chlorophenyl) methyl-4-dimethyl-aminophenyl] amine (BCMA) or a salt thereof.

  In addition, it is preferable that the measurement reagent of the measurement target substance in the sample of the present invention contains the chromogen at a concentration such that the concentration becomes the concentration during the measurement reaction.

(7) Coupler In the present invention, a coupler is a mixture of hydrogen peroxide generated by an oxidase with the coupler, peroxidase, and a chromogen (phenol, phenol derivative, aniline derivative, etc.) as the hydrogen donor. , A substance that can be oxidatively condensed with the chromogen to form a dye. Any such substance can be used without any particular limitation.

Examples of the coupler include 4-aminoantipyrine or a derivative thereof, phenylenediamine, or 3-methyl-2-benzothiazolinone hydrazone (MBTH).
In the present invention, this coupler is preferably 4-aminoantipyrine.

  In the measurement of the measurement target substance in the sample of the present invention, the concentration of the coupler is not particularly limited, but is preferably 0.005 mM or more during the measurement reaction.

  Further, the lower limit of the concentration of the coupler is more preferably 0.05 mM or more, and particularly preferably 0.5 mM or more during the measurement reaction.

  There is no particular upper limit to the concentration during the measurement reaction of the coupler, but up to 50 mM is sufficient in view of cost and the like.

  In addition, the upper limit of the concentration of the coupler is more preferably 20 mM or less, and particularly preferably 10 mM or less, during the measurement reaction.

  In addition, it is preferable that the measurement reagent of the measurement target substance in the sample of the present invention contains the above-described coupler having a concentration such that the above-described concentration is obtained during the measurement reaction.

(8) pH
In the measurement of the measurement target substance in the sample of the present invention, the pH is not particularly limited, but the pH at the time of the measurement reaction may be in the pH range in which the measurement reaction proceeds.
In addition, it is preferable that pH at the time of this measurement reaction exists in the optimal pH range of the said measurement reaction, or its vicinity.

  For example, the pH during the measurement reaction is preferably pH 6.0 (20 ° C.) or higher.

  In addition, the lower limit of the pH during the measurement reaction is more preferably pH 6.5 (20 ° C.) or more, and particularly preferably pH 7.0 (20 ° C.) or more.

  And it is preferable that the pH at the time of this measurement reaction is pH 9.0 (20 degreeC) or less.

  Further, the upper limit of the pH during the measurement reaction is more preferably pH 8.5 (20 ° C.) or less, and particularly preferably pH 8.0 (20 ° C.) or less during the measurement reaction.

  In the measurement reagent of the measurement target substance in the sample of the present invention, it is preferable to set the pH of the measurement reagent so as to be the pH in the above pH range during the measurement reaction.

(9) Buffering agent In the present invention, a buffering agent can be appropriately present as necessary during the measurement reaction.

  For example, a buffering agent having a buffering capacity in the pH range or the like described in “(8) pH” described above can be present in the measurement reaction at a concentration having a buffering capacity.

  Examples of such a buffer include MES, Bis-Tris, Bis-Tris propane, ADA, PIPES, ACES, MOPSO, MOPS, MES, BES, TES, HEPES, DIPSO, TAPSO, POPSO, HEPPSO, HEPPS, EPPS. , Tricine, Bicine, TAPS, CHES, CAPSO, CAPS, phosphoric acid, phosphate, boric acid, borate, glycine, glycylglycine, imidazole, or tris (hydroxymethyl) aminomethane [Tris] Can do.

In the measurement reagent of the substance to be measured in the sample of the present invention, the pH of the measurement reagent may be set so that the pH in the pH range described in “(8) pH” is obtained during the measurement reaction. preferable.
For this reason, it is preferable to contain in this measuring reagent the buffer which has a buffer capacity in the pH range etc. which are described in said (8) pH in the density | concentration which has a buffer capacity at the time of a measurement reaction.

(10) Other components In the present invention, during the measurement reaction, the stability of enzyme substrates, conjugate enzymes, coenzymes, metal ions or metal salts containing these, chelating agents, proteins such as albumin, saccharides or polymer compounds, etc. Agents, preservatives such as sodium azide or antibiotics, elimination agents or influence inhibitors of measurement interfering substances contained in other samples (for example, potassium ferrocyanide for bilirubin, ascorbate oxidase for ascorbic acid, etc.), A surfactant, an activator, or the like can be appropriately present as necessary.

  In addition, although the density | concentration at the time of making these exist is not specifically limited, In a measurement reaction, it is preferable that it is 0.001% (w / v) or more.

  In addition, the lower limit of the concentration when these are present is more preferably 0.01% (w / v) or more, and particularly preferably 0.1% (w / v) or more during the measurement reaction.

  There is no particular upper limit to the concentration when these are present, but up to 10% (w / v) is sufficient in view of cost and the like.

  Moreover, the upper limit of the concentration when these are present is more preferably 5% (w / v) or less, and particularly preferably 2.5% (w / v) or less, in the measurement reaction.

  Specific examples of the surfactants include, for example, sorbitan fatty acid ester, glycerin fatty acid ester, decaglycerin fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene glycerin fatty acid ester, polyethylene glycol fatty acid ester, polyoxy Nonionic surfactants such as ethylene alkyl ether, polyoxyethylene phytosterol, phytostanol, polyoxyethylene polyoxypropylene alkyl ether, polyoxyethylene alkylphenyl ether, polyoxyethylene castor oil, hydrogenated castor oil or polyoxyethylene lanolin An amphoteric surfactant such as betaine acetate; or polyoxyethylene alkyl ether sulfate or polyoxyethylene alkyl ester; It can be cited anionic surfactants such as ether acetates.

  In addition, it is preferable that the measurement reagent of the measurement target substance in the sample of the present invention contains the above-described component at a concentration such that the above-described concentration is obtained during the measurement reaction.

(11) Measurement When measuring a measurement target substance in a sample according to the present invention, the measurement may be performed by appropriately selecting a method such as an end point method (end point method) or a reaction rate method (rate method).

  This measurement is performed by appropriately selecting a one-step method (one-reagent method) performed in one step or a multi-step method (multi-reagent method) performed in two or more steps. Just do it.

  Moreover, in this measurement, the measurement wavelength when measuring the produced | generated pigment | dye can select and use the suitable wavelength of a visible part suitably. In this case, a single wavelength method or a dual wavelength method may be used.

  In this measurement, the temperature during the measurement reaction is set to a temperature within a range where the measurement reaction proceeds such as 30 ° C. or 37 ° C. and the reaction components such as enzymes involved in the measurement reaction are not inactivated, denatured or altered by heat. do it.

  In this measurement, the method for starting the measurement reaction may be any method such as a method performed by adding a substrate or the like, or a method performed by adding a sample.

  Further, in this measurement, the measurement may be performed by a method or using an apparatus such as an automatic analyzer.

(12) Specific example of measurement of measurement target substance in sample As a specific example of measurement of a measurement target substance in a sample according to the present invention, a method for measuring creatinine in a sample using sarcosine oxidase, which is an oxidase, is described below. Describe.

(A) Preparation of measurement reagent A fixed amount of each of the following components was dissolved in pure water, and the pH was adjusted to a constant pH, thereby preparing a first reagent and a second reagent of the creatinine measurement reagent.

(I) first reagent polyoxyethylene alkylamine compound or polyoxyethylene alkylamide compound sarcosine oxidase creatinase catalase N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline, sodium salt, 2 Hydrate [ADOS] <Chromogen (hydrogen donor)>
Buffer

(Ii) Second reagent creatininase 4-aminoantipyrine peroxidase sodium azide buffer

(B) Sample Human serum was used as a sample.

(C) Measurement (i) First Stage The sample and the first reagent are mixed to prepare a mixed solution.

The amount of each of the sample and the first reagent to be mixed is the amount of the second reagent, the concentration of the measurement target substance (creatinine) contained in the sample, the extinction coefficient of the pigment derived from the chromogen and coupler, the measurement wavelength, and the like. What is necessary is just to determine suitably according to the specification etc. of an analyzer, and other conditions.
In general, for example, the amount of the sample is preferably 0.5 to 100 μL, and the amount of the first reagent is preferably in the range of 20 to 1,000 μL.

Incubation is performed after preparation of this mixture.
The incubation time is not particularly limited, but is usually preferably within 20 minutes, more preferably within 10 minutes, and particularly preferably within 5 minutes.

Moreover, the temperature at the time of incubation should just be the temperature above the temperature which the said liquid mixture freezes.
In general, the higher the temperature during the measurement reaction, the higher the reaction rate.
However, if the temperature is too high, the enzyme involved in the reaction is denatured and inactivated, and therefore the incubation temperature needs to be lower than the temperature at which the enzyme involved in the measurement reaction is denatured and deactivated.
The temperature during the incubation is usually 2 to 70 ° C, preferably 20 to 37 ° C, and more preferably 30 to 37 ° C.
If the enzyme involved in the measurement reaction is a thermostable enzyme, it may be at a higher temperature.

  By preparing and incubating the mixed solution of the sample and the first reagent, the creatine contained in the sample comes into contact with the reagent components contained in the first reagent, and the creatine contained in the sample by these components is erased. The reaction starts.

That is, first, creatine and water contained in the sample are converted into sarcosine and urea by creatinase.
Next, sarcosine, oxygen and water are converted into glycine, formaldehyde and hydrogen peroxide by sarcosine oxidase (oxidase).
Next, these two hydrogen peroxide molecules are decomposed by catalase into water and oxygen.
By the above reaction, creatine contained in the sample is erased.

(Ii) Second Stage The second reagent is mixed with the “mixture of sample and first reagent” prepared in the first stage. This is the final reaction solution.

The amount of the second reagent to be mixed is the amount of the sample, the amount of the first reagent, the concentration of the measurement target substance (creatinine) contained in the sample, the extinction coefficient of the pigment derived from the chromogen and coupler, the measurement wavelength, and the use What is necessary is just to determine suitably according to the specification etc. of an analyzer to perform, and other conditions.
In general, for example, the amount of the second reagent is preferably in the range of 10 to 1,000 μL.

Incubation is performed after preparation of this final reaction solution.
The incubation time is not particularly limited, but is usually preferably within 20 minutes, more preferably within 10 minutes, and particularly preferably within 5 minutes.

Moreover, the temperature at the time of incubation should just be the temperature above the temperature which freezes the said last reaction liquid.
In general, the higher the temperature during the measurement reaction, the higher the reaction rate.
However, if the temperature is too high, the enzyme involved in the measurement reaction is denatured and inactivated. Therefore, the incubation temperature must be lower than the temperature at which the enzyme involved in the measurement reaction is denatured and inactivated.
The temperature during the incubation is usually 2 to 70 ° C, preferably 20 to 37 ° C, and more preferably 30 to 37 ° C.
If the enzyme involved in the measurement reaction is a thermostable enzyme, it may be at a higher temperature.

  By preparing and incubating the final reaction solution, the measurement reaction in the second stage starts following the measurement reaction in the first stage, and the measurement reaction of creatinine contained in the sample is advanced.

That is, first, the activity of the catalase used in the elimination reaction of creatine contained in the sample in the first stage is inhibited by sodium azide contained in the second reagent, and hydrogen peroxide is converted into water and oxygen. Can no longer be disassembled.
The creatinine and water contained in the sample are converted into creatine by creatininase.

Next, the creatine and water are converted into sarcosine and urea by creatinase.
Next, sarcosine, oxygen and water are converted into glycine, formaldehyde and hydrogen peroxide by sarcosine oxidase (oxidase).

  Next, these two hydrogen peroxide molecules, chromogen (ADOS), and coupler (4-aminoantipyrine) are converted into a dye and four water molecules by peroxidase. [The chromogen and coupler were oxidized and condensed to form a dye. ]

Next, the absorbance (or transmittance) of the final reaction solution derived from the produced dye is measured.
The absorbance (or transmittance) may be measured by a one-wavelength method performed at only one wavelength or a two-wavelength method performed at two wavelengths, respectively, and may be selected as appropriate.
Moreover, what is necessary is just to select suitably the wavelength which measures an absorbance (or transmittance | permeability) according to the absorption wavelength of the pigment | dye.

  The absorbance (or transmittance) may be measured by appropriately selecting a method such as an end point method (end point method) or a reaction rate method (rate method).

  Note that calculating the concentration or activity value of the substance to be measured contained in the sample from the measured absorbance (or transmittance) or change in absorbance (or transmittance) is based on the molar extinction coefficient of the dye. Select a method such as a method of calculating from the measured absorbance (or transmittance) or a method of calculating by comparing with the absorbance (or transmittance) of the measurement target substance (standard substance) whose concentration or activity value is known. Just do it.

  In the present invention, the reagent blank (reagent blank) is subtracted from the absorbance (or transmittance) obtained by measuring the sample to calculate the concentration or activity value of the measurement target substance contained in the sample. Is preferred.

  The operation of measuring the measurement target substance in the sample may be performed by the measurer himself or herself, or may be performed using an apparatus such as an automatic analyzer.

  Further, when the measurement reagent used for measurement of the measurement target substance in the sample is composed of the first reagent, the second reagent, and another reagent (one or two or more reagents), that is, composed of three or more reagents. In this case, the measurement reaction is performed through the number of steps necessary to perform the measurement using these reagents (two steps or three or more steps as necessary), and the measurement target substance in the sample is measured. Just do it.

[2] Measuring reagent for the substance to be measured in the sample General Remarks A reagent for measuring a substance to be measured in a sample of the present invention is a reagent for measuring a substance to be measured in a sample by optically measuring hydrogen peroxide generated by an oxidase using a chromogen. At least one polyoxyethylene alkyl nitrogen-containing compound selected from an oxyethylene alkylamine compound and a polyoxyethylene alkylamide compound is contained.

  And in the measuring reagent of the substance to be measured in the sample of the present invention, by containing at least one polyoxyethylene alkyl nitrogen-containing compound selected from a polyoxyethylene alkylamine compound and a polyoxyethylene alkylamide compound, The influence on the measurement derived from bilirubin contained in the sample can be avoided.

  In addition, in the measurement reagent of the measurement target substance in the sample of the present invention, a polyoxyethylene alkylamine compound and a polyoxyethylene alkylamide compound are selected as an avoidance agent for influence on measurement derived from bilirubin contained in the sample. It is preferable to contain at least one polyoxyethylene alkyl nitrogen-containing compound.

2. Polyoxyethylene alkyl nitrogen-containing compound The polyoxyethylene alkyl nitrogen-containing compound is as described in “2. Polyoxyethylene alkyl nitrogen-containing compound” in “[1] Measuring method of measurement target substance in sample”. .

3. Reagents for Measuring Substances in Samples (1) General The measuring reagent for measuring substances in samples of the present invention is obtained by optically measuring hydrogen peroxide generated by oxidase using a chromogen. The reagent for measuring the substance to be measured contains at least one polyoxyethylene alkyl nitrogen-containing compound selected from a polyoxyethylene alkylamine compound and a polyoxyethylene alkylamide compound.

  It is a reagent that measures the target substance in a sample by optically measuring the hydrogen peroxide generated by this oxidase using the chromogen. There is no particular limitation as long as it is a reagent that can be used and optically measured to measure a measurement target substance in a sample.

  As this measurement reagent, for example, an oxidase is used to generate hydrogen peroxide directly or indirectly from a substance to be measured, and this is mixed with peroxidase and a chromogen and brought into contact with each other to lead to a color development system. By measuring optically the pigment | dye produced | generated from the chromogen by reaction, the produced | generated hydrogen peroxide is measured, The reagent etc. which measure the measuring object substance contained in a sample by this can be mentioned.

  More specifically, for example, oxidase is used to generate hydrogen peroxide directly or indirectly from the substance to be measured, and this is converted to peroxidase, a chromogen that is a hydrogen donor, and the chromogen of this chromogen. The resulting hydrogen peroxide is measured by mixing and bringing into contact with the coupler as the condensation target, leading to a color development system, and optically measuring the dye produced from the chromogen by the oxidative condensation reaction. And a reagent for measuring a substance to be measured contained in the sample.

  In addition, for example, oxidase is used to generate hydrogen peroxide directly or indirectly from the substance to be measured, and this is mixed with peroxidase and chromogen and brought into contact with the coloring system without using the coupler. In addition, by optically measuring the dye produced from the chromogen by the oxidation reaction, the generated hydrogen peroxide can be measured, thereby including a reagent etc. for measuring the measurement target substance contained in the sample. it can.

(2) Sample The sample is as described in “(2) Sample” in “3. Measurement of measurement target substance in sample” of “[1] Measurement method of measurement target substance in sample”.

(3) Substance to be measured Regarding substance to be measured, refer to “(3) Substance to be measured” in “3. Measurement of substance to be measured in sample” in “[1] Method for measuring substance to be measured in sample” above. As described.

(4) Oxidase As for the oxidase, as described in “(4) Oxidase” in “3. Measurement of substance to be measured in sample” in “[1] Method for measuring substance to be measured in sample”. It is.

(5) Peroxidase The peroxidase is as described in “(5) Peroxidase” in “3. Measurement of a measurement target substance in a sample” in “[1] Measurement method of a measurement target substance in a sample”.

(6) Chromogen For the chromogen, refer to “(6) Chromogen” in “3. Measurement of the measurement target substance in the sample” in “[1] Measurement method of the measurement target substance in the sample”. As described.

(7) Coupler The coupler is as described in “(7) Coupler” in “3. Measurement of measurement target substance in sample” in “[1] Measurement method of measurement target substance in sample”.

(8) pH
The pH is as described in “(8) pH” in “3. Measurement of measurement target substance in sample” in “[1] Measurement method of measurement target substance in sample”.

(9) Buffering agent The buffering agent is as described in “(9) Buffering agent” in “3. Measurement of measuring substance in sample” in “[1] Measuring method of measuring substance in sample”. It is.

(10) Other components For other components, refer to “(10) Other components” in “3. Measurement of the measurement target substance in the sample” in “[1] Measurement method of the measurement target substance in the sample”. As described.

(11) Measuring reagent The measuring reagent of the substance to be measured in the sample of the present invention may be one measured by the end point method (end point method) or one measured by the reaction rate method (rate method). May be selected as appropriate.

  In addition, the measurement reagent of the present invention may be a one-step method (one-reagent method) in which measurement is performed in one step, or a multi-step measurement in two or more steps. A step method (multi-reagent method) may be used and may be selected as appropriate.

  In the measurement reagent of the present invention, an appropriate wavelength in the visible region can be appropriately selected as the measurement wavelength when the produced dye is optically measured. In this case, a single wavelength method or a dual wavelength method may be used.

  In the measurement reagent of the present invention, the temperature during the measurement reaction is within a range in which the measurement reaction such as 30 ° C. or 37 ° C. proceeds and the reaction components such as enzymes involved in the measurement reaction are not inactivated, denatured or altered by heat. What is necessary is just to set the temperature.

  In the measurement reagent of the present invention, the measurement reaction may be started by any method such as a method performed by adding a substrate or the like, or a method performed by adding a sample.

  Further, in the measurement reagent of the present invention, the measurement may be performed by a method, or may be performed using an apparatus such as an automatic analyzer.

  Further, in the measurement reagent of the present invention, all or a part of the constituent reagents may be a liquid reagent.

  In addition, the measurement reagent of the present invention can be sold alone or used for measurement of a measurement target substance in a sample.

In addition, the measurement reagent of the present invention can be sold in combination with other reagents other than the above-described measurement reagent, or can be used for measurement of a measurement target substance in a sample.
Examples of other reagents other than the above-described measurement reagents include, for example, a buffer solution, a sample diluent, a reagent diluent, a reagent containing a substance for performing calibration (calibration), or a substance for performing quality control. And the like.

  The measurement reagent of the present invention may be a measurement reagent kit comprising a plurality of constituent reagents such as the first reagent and the second reagent, or other reagents.

[3] Method for avoiding influence derived from bilirubin contained in sample General The method for avoiding the influence derived from bilirubin contained in the sample of the present invention is to measure the measurement target substance in the sample by optically measuring hydrogen peroxide generated by the oxidase using a chromogen. The measurement is carried out in the presence of at least one polyoxyethylene alkyl nitrogen-containing compound selected from a polyoxyethylene alkylamine compound and a polyoxyethylene alkylamide compound.

  The method for avoiding the influence derived from bilirubin contained in the sample of the present invention is carried out in the presence of at least one polyoxyethylene alkyl nitrogen-containing compound selected from a polyoxyethylene alkylamine compound and a polyoxyethylene alkylamide compound. By doing so, it is possible to avoid the influence on the measurement derived from bilirubin contained in the sample.

2. Polyoxyethylene alkyl nitrogen-containing compound The polyoxyethylene alkyl nitrogen-containing compound is as described in “2. Polyoxyethylene alkyl nitrogen-containing compound” in “[1] Measuring method of measurement target substance in sample”. .

3. Measurement of the substance to be measured in the sample (1) General The method for avoiding the effects derived from bilirubin contained in the sample of the present invention is to optically measure hydrogen peroxide generated by the oxidase using a chromogen. When measuring the measurement target substance in the sample, the measurement is performed in the presence of at least one polyoxyethylene alkyl nitrogen-containing compound selected from a polyoxyethylene alkylamine compound and a polyoxyethylene alkylamide compound. It is what.

  By measuring the hydrogen peroxide generated by this oxidase optically using a chromogen, the substance to be measured in the sample is measured. There is no particular limitation as long as it can measure the substance to be measured in the sample by optical measurement using the material.

  For this measurement, for example, oxidase is used to generate hydrogen peroxide directly or indirectly from the substance to be measured, and this is mixed with peroxidase and chromogen and brought into contact with the chromogenic system, leading to the oxidation reaction. Examples include a method of measuring the generated hydrogen peroxide by optically measuring the dye produced from the chromogen by the above, and thereby measuring the measurement target substance contained in the sample.

  More specifically, for example, oxidase is used to generate hydrogen peroxide directly or indirectly from the substance to be measured, and this is converted to peroxidase, a chromogen that is a hydrogen donor, and the chromogen of this chromogen. The resulting hydrogen peroxide is measured by mixing and bringing into contact with the coupler as the condensation target, leading to a color development system, and optically measuring the dye produced from the chromogen by the oxidative condensation reaction. And the like, and the like.

  In addition, for example, oxidase is used to generate hydrogen peroxide directly or indirectly from the substance to be measured, and this is mixed with peroxidase and chromogen and brought into contact with the coloring system without using the coupler. In addition, by measuring optically the dye produced from the chromogen by the oxidation reaction, the generated hydrogen peroxide can be measured, and the method of measuring the measurement target substance contained in the sample can also be mentioned. it can.

(2) Sample The sample is as described in “(2) Sample” in “3. Measurement of measurement target substance in sample” of “[1] Measurement method of measurement target substance in sample”.

(3) Substance to be measured Regarding substance to be measured, refer to “(3) Substance to be measured” in “3. Measurement of substance to be measured in sample” in “[1] Method for measuring substance to be measured in sample” above. As described.

(4) Oxidase As for the oxidase, as described in “(4) Oxidase” in “3. Measurement of substance to be measured in sample” in “[1] Method for measuring substance to be measured in sample”. It is.

(5) Peroxidase The peroxidase is as described in “(5) Peroxidase” in “3. Measurement of a measurement target substance in a sample” in “[1] Measurement method of a measurement target substance in a sample”.

(6) Chromogen For the chromogen, refer to “(6) Chromogen” in “3. Measurement of the measurement target substance in the sample” in “[1] Measurement method of the measurement target substance in the sample”. As described.

(7) Coupler The coupler is as described in “(7) Coupler” in “3. Measurement of measurement target substance in sample” in “[1] Measurement method of measurement target substance in sample”.

(8) pH
The pH is as described in “(8) pH” in “3. Measurement of measurement target substance in sample” in “[1] Measurement method of measurement target substance in sample”.

(9) Buffering agent The buffering agent is as described in “(9) Buffering agent” in “3. Measurement of measuring substance in sample” in “[1] Measuring method of measuring substance in sample”. It is.

(10) Other components For other components, refer to “(10) Other components” in “3. Measurement of the measurement target substance in the sample” in “[1] Measurement method of the measurement target substance in the sample”. As described.

(11) Measurement Other measurements are as described in “(11) Measurement” in “3. Measurement of measurement target substance in sample” in “[1] Measurement method of measurement target substance in sample”. .

(12) Specific example of measurement of measurement target substance in sample For a specific example of measurement of a measurement target substance in a sample according to the present invention, “3. As described in “(12) Specific example of measurement of measurement target substance in sample” in “Measurement of measurement target substance in sample”.

  EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited by these Examples.

[Example 1] (Confirmation of effect of avoiding influence derived from bilirubin contained in sample-1)
The effect of avoiding the influence on the measurement derived from the bilirubin contained in the sample in the measurement method and measurement reagent of the measurement target substance in the sample of the present invention and the method for avoiding the influence on the measurement derived from the bilirubin contained in the sample confirmed.
Specifically, in the present invention, when a polyoxyethylene alkylamine compound is used as the polyoxyethylene alkyl nitrogen-containing compound, the effect of avoiding the influence on the measurement of creatinine in the sample derived from bilirubin contained in the sample is achieved. confirmed.

1. Creatinine measuring reagent (1) First reagent

(A) Preparation of First Reagent [Invention (0.05% Naimine S-210)] The following reagent components were dissolved in pure water to the respective concentrations described above, and the pH was adjusted to pH 7.4 (20 ° C.). The first reagent [the present invention (0.05% Naimine S-210)] was prepared.

Naimine S-210 [NOF Corporation (Japan)] <Polyoxyethylene alkylamine compound> 0.05% (w / v)
Sarcosine oxidase 13,000Unit / L
Creatinase 40,000Unit / L
Catalase 200,000Unit / L
N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline, sodium salt, dihydrate [ADOS] <chromogen (hydrogen donor)> 2 mM
Ascorbate oxidase 3,000Unit / L
TES [N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid] <Buffer> 20 mM

(B) Preparation of First Reagent [Invention (0.05% Naimine O-205)] The following reagent components were dissolved in pure water to the respective concentrations described above, and the pH was adjusted to pH 7.4 (20 ° C.). The first reagent [the present invention (0.05% Naimine O-205)] was prepared.

Naimine O-205 [NOF Corporation (Japan)] <Polyoxyethylene alkylamine compound> 0.05% (w / v)
Sarcosine oxidase 13,000Unit / L
Creatinase 40,000Unit / L
Catalase 200,000Unit / L
N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline, sodium salt, dihydrate [ADOS] <chromogen (hydrogen donor)> 2 mM
Ascorbate oxidase 3,000Unit / L
TES [N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid] <Buffer> 20 mM

(C) Preparation of first reagent [conventional invention (0%)] The following reagent components were dissolved in pure water so as to have the respective concentrations described above, and the pH was adjusted to pH 7.4 (20 ° C.). A first reagent [conventional invention (0%)] containing no oxyethylenealkyl nitrogen-containing compound (concentration 0%) was prepared.

Sarcosine oxidase 13,000Unit / L
Creatinase 40,000Unit / L
Catalase 200,000Unit / L
N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline, sodium salt, dihydrate [ADOS] <chromogen (hydrogen donor)> 2 mM
Ascorbate oxidase 3,000Unit / L
TES [N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid] <Buffer> 20 mM

(2) Second reagent

Preparation of Second Reagent The following reagent components were dissolved in pure water so as to have the indicated concentrations, and the pH was adjusted to 7.8 (20 ° C.) to prepare a second reagent.

Creatininase 400,000Unit / L
4-aminoantipyrine 2 mM
Peroxidase 15,000Unit / L
Sodium azide 0.15% (w / v)
Potassium ferrocyanide 0.2 mM
TES [N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid] <Buffer> 20 mM

2. Sample (1) “Bilirubin C (conjugated type)” of “Interference Check A Plus” (Sysmex Corporation (Japan)), which is a commercially available control substance for examining interference substances, has a bilirubin (conjugated type) concentration of 500 mg. / DL was dissolved in pure water to prepare a bilirubin solution.

(2) To 1 volume of the bilirubin solution prepared in (1) above, 9 volumes of human serum were added and mixed to prepare a serum sample with a bilirubin (conjugated) concentration of 50 mg / dL.

(3) The “bilirubin C“ blank ”” of the “interference check A plus” was dissolved in the same amount of pure water as the amount used for the dissolution in (1) to prepare a blank solution.

(4) To 1 volume of the solution (blank solution) prepared in (3) above, 9 volumes of the human serum used in (2) above are added and mixed, and the blank solution is 1/10 of the human serum. A blank sample diluted in 1 was prepared.

(5) The serum sample prepared in (2) is diluted with the blank sample prepared in (4), and a serum sample having a bilirubin (conjugated) concentration of 40 mg / dL, a serum sample of 30 mg / dL, A 20 mg / dL serum sample and a 10 mg / dL serum sample were prepared, respectively.
The blank sample used for this dilution was a serum sample with a bilirubin (conjugated) concentration of 0 mg / dL.

That is, the following six types of serum samples (a) to (f) were used as samples.
(A) Serum sample-1 [Bilirubin (conjugated) concentration 0 mg / dL]
(B) Serum sample-2 [Bilirubin (conjugate) concentration 10 mg / dL]
(C) Serum sample-3 [Bilirubin (conjugate) concentration 20 mg / dL]
(D) Serum sample-4 [Bilirubin (conjugate) concentration 30 mg / dL]
(E) Serum sample-5 [Bilirubin (conjugate) concentration 40 mg / dL]
(F) Serum sample-6 [Bilirubin (conjugate) concentration 50 mg / dL]

3. Measurement of Creatinine in Sample The measurement of creatinine in each of the two samples was performed using a 7180 Hitachi automatic analyzer [Hitachi High-Technologies Corporation (Japan)].

(1) In each of the serum samples of (2) (a) to (f) above, 6.0 μL of the sample was added to the first reagent of (a) of (1) above [the present invention (0.05% 150 μL of Naimine S-210)] was added and allowed to react at 37 ° C.

(2) Next, the absorbance at 15 points (254.193 seconds after the addition of the first reagent) and the absorbance at 16 points (270.093 seconds after the addition of the first reagent) were measured at a main wavelength of 600 nm and a subwavelength of 800 nm, respectively. Thus, the average value of the absorbance at 15 points and the absorbance at 16 points was calculated [“photometry 1”]. (Measurement of absorbance of sample blank)

(3) Next, between 16 points (270.093 seconds after the addition of the first reagent) and 17 points (286.977 seconds after the addition of the first reagent), 50 μL of the second reagent in (1) above is added. Added and allowed to react at 37 ° C.

(4) Next, the absorbance at 33 points (56.582 seconds after the addition of the first reagent) and the absorbance at 34 points (587.426 seconds after the addition of the first reagent) were measured at a main wavelength of 600 nm and a sub wavelength of 800 nm, respectively. Thus, the average value of the absorbance at 33 points and the absorbance at 34 points was calculated [“photometry 2”]. (Measurement of absorbance according to the concentration of creatinine contained in the sample)

(5) Next, the measured value of the concentration of creatinine contained in the sample was determined as follows.
That is, for each sample of (2) in (2) above, from the average absorbance (“photometry 2”) measured in (4) above, the average absorbance (“photometry 1”) measured in (2) above. Was subtracted to determine the value of the absorbance difference.
Next, a standard sample whose creatinine concentration is known is measured as described in the above (1) to (4), and the average value of absorbance measured in the above (4) (“photometry 2”) By comparing the value of the difference in absorbance obtained by subtracting the average value of absorbance measured in (2) (“photometry 1”) with the value of the absorbance difference in each of the above samples, and performing proportional calculation, A measurement of creatinine concentration was obtained.

(6) Further, in the above (1), in place of the first reagent (a) of (1) in (1) above (the present invention (0.05% Naimine S-210)), Except for using the first reagent (the present invention (0.05% Naimine O-205)) of (b), the operation was carried out as in the above (1) to (5), and “serum sample-1”, “ The final measured values of the creatinine concentrations of “serum sample-2”, “serum sample-3”, “serum sample-4”, “serum sample-5”, and “serum sample-6” were determined.

(7) In the above (1), instead of the first reagent (a) of (1) in (1) above (the present invention (0.05% Naimine S-210)), Except for using the first reagent (c) (conventional invention (0%)) of (c), the operation was carried out as in (1) to (5) above, and “serum sample-1”, “serum sample-2”, The final measured values of the creatinine concentrations of “serum sample-3”, “serum sample-4”, “serum sample-5”, and “serum sample-6” were determined.

4). Measurement Results Table 2 shows the results obtained by measuring the creatinine concentration of each sample in 3 above.

  In this table, the horizontal column represents the type and concentration of the polyoxyethylene alkylamine compound contained in the first reagent. [The 1st reagent which does not contain a polyoxyethylene alkyl nitrogen-containing compound was expressed as "0%". ]

  In this table, the vertical column represents the concentration of bilirubin (conjugated) in the sample.

  And the value shown in each column of this table | surface shows the measured value [mg / dL] of the density | concentration of the creatinine in the said sample.

  In addition, the numerical value in (parentheses) is the value obtained by dividing each measured value by the measured value when the concentration of bilirubin (conjugated) in the sample when the same first reagent is used is 0 mg / dL. It is expressed as a percentage.

5. Consideration (1) From this table, when the first reagent does not contain a polyoxyethylene alkyl nitrogen-containing compound (0%), the concentration of creatinine in the sample increases as the concentration of bilirubin (conjugated) in the sample increases. It can be seen that the measured value of the concentration decreases, and when the concentration of bilirubin (conjugated) in the sample is 50 mg / dL, the measured value has decreased to 79% of the original value.

  That is, it can be seen that the measurement value of the concentration of creatinine in the sample is affected by the presence of bilirubin contained in the sample, and a large negative error occurs.

(2) From this table, the measured value of the concentration of creatinine in the sample when the first reagent contains a polyoxyethylene alkylamine compound does not contain the polyoxyethylene alkyl nitrogen-containing compound (0%) It can be seen that the decrease in the measurement value derived from bilirubin contained in the sample is suppressed with respect to the measurement value in the first reagent.

(3) That is, in measuring a measurement target substance in a sample by optically measuring hydrogen peroxide generated by an oxidase using a chromogen, a polyoxyethylene alkylamine compound and a polyoxyethylene alkylamide are measured. By performing the measurement in the presence of at least one polyoxyethylene alkyl nitrogen-containing compound selected from the compounds, it is possible to avoid the influence on the measurement derived from bilirubin contained in the sample, and the measurement result (measurement value) It has been confirmed that it is possible to prevent the occurrence of errors.

[Example 2] (Confirmation of effect of avoiding influence derived from bilirubin contained in sample-2)
The effect of avoiding the influence on the measurement derived from the bilirubin contained in the sample in the measurement method and measurement reagent of the measurement target substance in the sample of the present invention and the method for avoiding the influence on the measurement derived from the bilirubin contained in the sample Further confirmation.
Specifically, in the present invention, when a polyoxyethylene alkylamine compound is used as the polyoxyethylene alkyl nitrogen-containing compound, the effect of avoiding the influence on the measurement of creatinine in the sample derived from bilirubin contained in the sample is achieved. confirmed.

1. Creatinine measuring reagent (1) First reagent

(A) Preparation of First Reagent [Invention (0.05% Naimine S-215)] The following reagent components were dissolved in pure water so as to have the respective concentrations, and the pH was adjusted to pH 7.4 (20 ° C.). The first reagent [the present invention (0.05% Naimine S-215)] was prepared.

Naimine S-215 [NOF Corporation (Japan)] <Polyoxyethylene alkylamine compound> 0.05% (w / v)
Sarcosine oxidase 13,000Unit / L
Creatinase 40,000Unit / L
Catalase 200,000Unit / L
N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline, sodium salt, dihydrate [ADOS] <chromogen (hydrogen donor)> 2 mM
Ascorbate oxidase 3,000Unit / L
TES [N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid] <Buffer> 20 mM

(B) Preparation of First Reagent [Invention (0.10% Naimine S-215)] The following reagent components were dissolved in pure water so as to have the respective concentrations described above, and the pH was adjusted to pH 7.4 (20 ° C.). The first reagent [the present invention (0.10% Naimine S-215)] was prepared.

Naimine S-215 [NOF Corporation (Japan)] <Polyoxyethylene alkylamine compound> 0.10% (w / v)
Sarcosine oxidase 13,000Unit / L
Creatinase 40,000Unit / L
Catalase 200,000Unit / L
N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline, sodium salt, dihydrate [ADOS] <chromogen (hydrogen donor)> 2 mM
Ascorbate oxidase 3,000Unit / L
TES [N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid] <Buffer> 20 mM

(C) Preparation of First Reagent [Invention (0.05% Naimine S-220)] The following reagent components were dissolved in pure water so as to have the respective concentrations, and the pH was adjusted to pH 7.4 (20 ° C.). The first reagent [the present invention (0.05% Naimine S-220)] was prepared.

Naimine S-220 [NOF Corporation (Japan)] <Polyoxyethylene alkylamine compound> 0.05% (w / v)
Sarcosine oxidase 13,000Unit / L
Creatinase 40,000Unit / L
Catalase 200,000Unit / L
N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline, sodium salt, dihydrate [ADOS] <chromogen (hydrogen donor)> 2 mM
Ascorbate oxidase 3,000Unit / L
TES [N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid] <Buffer> 20 mM

(D) Preparation of First Reagent [Invention (0.10% Naimine S-220)] The following reagent components were dissolved in pure water so as to have the respective concentrations described above, and the pH was adjusted to pH 7.4 (20 ° C.). The first reagent [the present invention (0.10% Naimine S-220)] was prepared.

Naimine S-220 [NOF Corporation (Japan)] <Polyoxyethylene alkylamine compound> 0.10% (w / v)
Sarcosine oxidase 13,000Unit / L
Creatinase 40,000Unit / L
Catalase 200,000Unit / L
N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline, sodium salt, dihydrate [ADOS] <chromogen (hydrogen donor)> 2 mM
Ascorbate oxidase 3,000Unit / L
TES [N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid] <Buffer> 20 mM

(E) Preparation of First Reagent [Invention (0.05% Naimine F-215)] The following reagent components were dissolved in pure water to the respective concentrations described above, and the pH was adjusted to pH 7.4 (20 ° C.). The first reagent [the present invention (0.05% Naimine F-215)] was prepared.

Naimine F-215 [NOF Corporation (Japan)] <Polyoxyethylene alkylamine compound> 0.05% (w / v)
Sarcosine oxidase 13,000Unit / L
Creatinase 40,000Unit / L
Catalase 200,000Unit / L
N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline, sodium salt, dihydrate [ADOS] <chromogen (hydrogen donor)> 2 mM
Ascorbate oxidase 3,000Unit / L
TES [N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid] <Buffer> 20 mM

(F) Preparation of First Reagent [Invention (0.10% Naimine F-215)] The following reagent components were dissolved in pure water so as to have the respective concentrations, and the pH was adjusted to pH 7.4 (20 ° C.). The first reagent [the present invention (0.10% Naimine F-215)] was prepared.

Naimine F-215 [NOF Corporation (Japan)] <Polyoxyethylene alkylamine compound> 0.10% (w / v)
Sarcosine oxidase 13,000Unit / L
Creatinase 40,000Unit / L
Catalase 200,000Unit / L
N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline, sodium salt, dihydrate [ADOS] <chromogen (hydrogen donor)> 2 mM
Ascorbate oxidase 3,000Unit / L
TES [N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid] <Buffer> 20 mM

(G) Preparation of First Reagent [Conventional Invention (0%)] The following reagent components were dissolved in pure water so as to have the respective concentrations described above, and the pH was adjusted to pH 7.4 (20 ° C.). A first reagent [conventional invention (0%)] containing no oxyethylenealkyl nitrogen-containing compound (concentration 0%) was prepared.

Sarcosine oxidase 13,000Unit / L
Creatinase 40,000Unit / L
Catalase 200,000Unit / L
N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline, sodium salt, dihydrate [ADOS] <chromogen (hydrogen donor)> 2 mM
Ascorbate oxidase 3,000Unit / L
TES [N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid] <Buffer> 20 mM

(2) Second reagent

Preparation of Second Reagent The following reagent components were dissolved in pure water so as to have the indicated concentrations, and the pH was adjusted to 7.8 (20 ° C.) to prepare a second reagent.

Creatininase 400,000Unit / L
4-aminoantipyrine 2 mM
Peroxidase 15,000Unit / L
Sodium azide 0.15% (w / v)
Potassium ferrocyanide 0.2 mM
TES [N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid] <Buffer> 20 mM

2. Sample (1) “Bilirubin C (conjugated type)” of “Interference Check A Plus” (Sysmex Corporation (Japan)), which is a commercially available control substance for examining interference substances, has a bilirubin (conjugated type) concentration of 500 mg. / DL was dissolved in pure water to prepare a bilirubin solution.

(2) To 1 volume of the bilirubin solution prepared in (1) above, 9 volumes of human serum were added and mixed to prepare a serum sample with a bilirubin (conjugated) concentration of 50 mg / dL.

(3) The “bilirubin C“ blank ”” of the “interference check A plus” was dissolved in the same amount of pure water as the amount used for the dissolution in (1) to prepare a blank solution.

(4) To 1 volume of the solution (blank solution) prepared in (3) above, 9 volumes of the human serum used in (2) above are added and mixed, and the blank solution is 1/10 of the human serum. A blank sample diluted in 1 was prepared.

(5) The serum sample prepared in (2) is diluted with the blank sample prepared in (4), and a serum sample having a bilirubin (conjugated) concentration of 40 mg / dL, a serum sample of 30 mg / dL, A 20 mg / dL serum sample and a 10 mg / dL serum sample were prepared, respectively.
The blank sample used for this dilution was a serum sample with a bilirubin (conjugated) concentration of 0 mg / dL.

That is, the following six types of serum samples (a) to (f) were used as samples.
(A) Serum sample-1 [Bilirubin (conjugated) concentration 0 mg / dL]
(B) Serum sample-2 [Bilirubin (conjugate) concentration 10 mg / dL]
(C) Serum sample-3 [Bilirubin (conjugate) concentration 20 mg / dL]
(D) Serum sample-4 [Bilirubin (conjugate) concentration 30 mg / dL]
(E) Serum sample-5 [Bilirubin (conjugate) concentration 40 mg / dL]
(F) Serum sample-6 [Bilirubin (conjugate) concentration 50 mg / dL]

3. Measurement of Creatinine in Sample The measurement of creatinine in each of the two samples was performed using a 7180 Hitachi automatic analyzer [Hitachi High-Technologies Corporation (Japan)].

(1) In each of the serum samples of (2) (a) to (f) above, 6.0 μL of the sample was added to the first reagent of (a) of (1) above [the present invention (0.05% 150 μL of Naimine S-215)] was added and allowed to react at 37 ° C.

(2) Next, the absorbance at 15 points (254.193 seconds after the addition of the first reagent) and the absorbance at 16 points (270.093 seconds after the addition of the first reagent) were measured at a main wavelength of 600 nm and a subwavelength of 800 nm, respectively. Thus, the average value of the absorbance at 15 points and the absorbance at 16 points was calculated [“photometry 1”]. (Measurement of absorbance of sample blank)

(3) Next, between 16 points (270.093 seconds after the addition of the first reagent) and 17 points (286.977 seconds after the addition of the first reagent), 50 μL of the second reagent in (1) above is added. Added and allowed to react at 37 ° C.

(4) Next, the absorbance at 33 points (56.582 seconds after the addition of the first reagent) and the absorbance at 34 points (587.426 seconds after the addition of the first reagent) were measured at a main wavelength of 600 nm and a sub wavelength of 800 nm, respectively. Thus, the average value of the absorbance at 33 points and the absorbance at 34 points was calculated [“photometry 2”]. (Measurement of absorbance according to the concentration of creatinine contained in the sample)

(5) Next, the measured value of the concentration of creatinine contained in the sample was determined as follows.
That is, for each sample of (2) in (2) above, from the average absorbance (“photometry 2”) measured in (4) above, the average absorbance (“photometry 1”) measured in (2) above. Was subtracted to determine the value of the absorbance difference.
Next, a standard sample whose creatinine concentration is known is measured as described in the above (1) to (4), and the average value of absorbance measured in the above (4) (“photometry 2”) By comparing the value of the difference in absorbance obtained by subtracting the average value of absorbance measured in (2) (“photometry 1”) with the value of the absorbance difference in each of the above samples, and performing proportional calculation, A measurement of creatinine concentration was obtained.

(6) Further, in the above (1), instead of the first reagent (a) of (1) of (1) above (the present invention (0.05% Naimine S-215)), Except for using the first reagent (the present invention (0.10% Naimine S-215)) of (b), the operation was carried out as in the above (1) to (5), and “serum sample-1”, “ The final measured values of the creatinine concentrations of “serum sample-2”, “serum sample-3”, “serum sample-4”, “serum sample-5”, and “serum sample-6” were determined.

(7) Further, in the above (1), in place of the first reagent (a) of (1) in (1) above (the present invention (0.05% Naimine S-215)), (C) Except using the 1st reagent [this invention (0.05% Naimine S-220)], operation is performed as said (1)-(5), and "serum sample-1", " The final measured values of the creatinine concentrations of “serum sample-2”, “serum sample-3”, “serum sample-4”, “serum sample-5”, and “serum sample-6” were determined.

(8) Further, in the above (1), instead of the first reagent of (1) (a) in the above [1] (the present invention (0.05% Naimine S-215)), Except for using the first reagent (d) of the present invention (the present invention (0.10% Naimine S-220)), the operation was carried out as in the above (1) to (5), and “serum sample-1”, “ The final measured values of the creatinine concentrations of “serum sample-2”, “serum sample-3”, “serum sample-4”, “serum sample-5”, and “serum sample-6” were determined.

(9) Further, in the above (1), in place of the first reagent (a) of (1) in (1) above (the present invention (0.05% Naimine S-215)), (E) Except using the 1st reagent [this invention (0.05% Naimine F-215)], operation was performed as said (1)-(5), and "serum sample-1", " The final measured values of the creatinine concentrations of “serum sample-2”, “serum sample-3”, “serum sample-4”, “serum sample-5”, and “serum sample-6” were determined.

(10) Further, in the above (1), in place of the first reagent (a) of (1) of the above [1] (the present invention (0.05% Naimine S-215)), Except for using the first reagent (f) of the present invention (the present invention (0.10% Naimine F-215)), the operation was performed as described in the above (1) to (5), and “serum sample-1”, “ The final measured values of the creatinine concentrations of “serum sample-2”, “serum sample-3”, “serum sample-4”, “serum sample-5”, and “serum sample-6” were determined.

(11) Further, in the above (1), in place of the first reagent (1) of (1) (a) [the present invention (0.05% Naimine S-215)], Except for using the first reagent (conventional invention (0%)) of (g), the operation was carried out as in (1) to (5) above, and “serum sample-1”, “serum sample-2”, The final measured values of the creatinine concentrations of “serum sample-3”, “serum sample-4”, “serum sample-5”, and “serum sample-6” were determined.

4). Measurement Results Table 3 shows the results obtained by measuring the creatinine concentration of each sample in 3 above.

  In this table, the horizontal column represents the type and concentration of the polyoxyethylene alkylamine compound contained in the first reagent. [The 1st reagent which does not contain a polyoxyethylene alkyl nitrogen-containing compound was expressed as "0%". ]

  In this table, the vertical column represents the concentration of bilirubin (conjugated) in the sample.

  And the value shown in each column of this table | surface shows the measured value [mg / dL] of the density | concentration of the creatinine in the said sample.

  In addition, the numerical value in (parentheses) is the value obtained by dividing each measured value by the measured value when the concentration of bilirubin (conjugated) in the sample when the same first reagent is used is 0 mg / dL. It is expressed as a percentage.

5. Consideration (1) From this table, when the first reagent does not contain a polyoxyethylene alkyl nitrogen-containing compound (0%), the concentration of creatinine in the sample increases as the concentration of bilirubin (conjugated) in the sample increases. It can be seen that the measured value of the concentration decreases, and when the concentration of bilirubin (conjugated) in the sample is 50 mg / dL, the measured value has decreased to 79% of the original value.

  That is, it can be seen that the measurement value of the concentration of creatinine in the sample is affected by the presence of bilirubin contained in the sample, and a large negative error occurs.

(2) From this table, the measured value of the concentration of creatinine in the sample when the first reagent contains a polyoxyethylene alkylamine compound does not contain the polyoxyethylene alkyl nitrogen-containing compound (0%) It can be seen that the decrease in the measurement value derived from bilirubin contained in the sample is suppressed with respect to the measurement value in the first reagent.

  In addition, from this table, when the concentration of the polyoxyethylene alkylamine compound contained in the first reagent is 0.05% (w / v), the concentration is 0.10% (w / v). It can be seen that the decrease in the measurement value derived from bilirubin is further suppressed.

(3) That is, in measuring a measurement target substance in a sample by optically measuring hydrogen peroxide generated by an oxidase using a chromogen, a polyoxyethylene alkylamine compound and a polyoxyethylene alkylamide are measured. By performing the measurement in the presence of at least one polyoxyethylene alkyl nitrogen-containing compound selected from the compounds, it is possible to avoid the influence on the measurement derived from bilirubin contained in the sample, and the measurement result (measurement value) It has been confirmed that it is possible to prevent the occurrence of errors.

[Example 3] (Confirmation of effect of avoiding influence derived from bilirubin contained in sample-3)
The effect of avoiding the influence on the measurement derived from the bilirubin contained in the sample in the measurement method and measurement reagent of the measurement target substance in the sample of the present invention and the method for avoiding the influence on the measurement derived from the bilirubin contained in the sample Further confirmation.
Specifically, in the present invention, when a polyoxyethylene alkylamine compound is used as the polyoxyethylene alkyl nitrogen-containing compound, the effect of avoiding the influence on the measurement of creatinine in the sample derived from bilirubin contained in the sample is achieved. confirmed.

1. Creatinine measuring reagent (1) First reagent

(A) Preparation of First Reagent [Invention (0.04% Amite 320)] The following reagent components were dissolved in pure water so as to have the respective concentrations, and the pH was adjusted to pH 7.4 (20 ° C.). Thus, the first reagent [the present invention (0.04% Amit 320)] was prepared.

Amit 320 [Kao Corporation (Japan)] <Polyoxyethylene alkylamine compound> 0.04% (w / v)
Sarcosine oxidase 13,000Unit / L
Creatinase 40,000Unit / L
Catalase 200,000Unit / L
N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline, sodium salt, dihydrate [ADOS] <chromogen (hydrogen donor)> 2 mM
Ascorbate oxidase 3,000Unit / L
TES [N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid] <Buffer> 20 mM

(B) Preparation of First Reagent [Invention (0.08% Amite 320)] The following reagent components were dissolved in pure water so that each concentration was as described, and the pH was adjusted to pH 7.4 (20 ° C.). Then, the first reagent [the present invention (0.08% Amit 320)] was prepared.

Amit 320 [Kao Corporation (Japan)] <Polyoxyethylene alkylamine compound> 0.08% (w / v)
Sarcosine oxidase 13,000Unit / L
Creatinase 40,000Unit / L
Catalase 200,000Unit / L
N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline, sodium salt, dihydrate [ADOS] <chromogen (hydrogen donor)> 2 mM
Ascorbate oxidase 3,000Unit / L
TES [N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid] <Buffer> 20 mM

(C) Preparation of First Reagent [Invention (0.12% Amite 320)] The following reagent components were dissolved in pure water so as to have the concentrations indicated, and the pH was adjusted to pH 7.4 (20 ° C.). Thus, the first reagent [the present invention (0.12% Amit 320)] was prepared.

Amit 320 [Kao Corporation (Japan)] <Polyoxyethylene alkylamine compound> 0.12% (w / v)
Sarcosine oxidase 13,000Unit / L
Creatinase 40,000Unit / L
Catalase 200,000Unit / L
N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline, sodium salt, dihydrate [ADOS] <chromogen (hydrogen donor)> 2 mM
Ascorbate oxidase 3,000Unit / L
TES [N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid] <Buffer> 20 mM

(D) Preparation of First Reagent [Invention (0.16% Amite 320)] The following reagent components were dissolved in pure water so as to have the concentrations indicated, and the pH was adjusted to pH 7.4 (20 ° C.). Thus, the first reagent [the present invention (0.16% Amit 320)] was prepared.

Amit 320 [Kao Corporation (Japan)] <Polyoxyethylene alkylamine compound> 0.16% (w / v)
Sarcosine oxidase 13,000Unit / L
Creatinase 40,000Unit / L
Catalase 200,000Unit / L
N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline, sodium salt, dihydrate [ADOS] <chromogen (hydrogen donor)> 2 mM
Ascorbate oxidase 3,000Unit / L
TES [N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid] <Buffer> 20 mM

(E) Preparation of First Reagent [Conventional Invention (0%)] The following reagent components were dissolved in pure water so that each concentration was as described, and the pH was adjusted to pH 7.4 (20 ° C.). A first reagent [conventional invention (0%)] containing no oxyethylenealkyl nitrogen-containing compound (concentration 0%) was prepared.

Sarcosine oxidase 13,000Unit / L
Creatinase 40,000Unit / L
Catalase 200,000Unit / L
N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline, sodium salt, dihydrate [ADOS] <chromogen (hydrogen donor)> 2 mM
Ascorbate oxidase 3,000Unit / L
TES [N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid] <Buffer> 20 mM

(2) Second reagent

Preparation of Second Reagent The following reagent components were dissolved in pure water so as to have the indicated concentrations, and the pH was adjusted to 7.8 (20 ° C.) to prepare a second reagent.

Creatininase 400,000Unit / L
4-aminoantipyrine 2 mM
Peroxidase 15,000Unit / L
Sodium azide 0.15% (w / v)
Potassium ferrocyanide 0.2 mM
TES [N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid] <Buffer> 20 mM

2. Sample (1) “Bilirubin C (conjugated type)” of “Interference Check A Plus” (Sysmex Corporation (Japan)), which is a commercially available control substance for examining interference substances, has a bilirubin (conjugated type) concentration of 500 mg. / DL was dissolved in pure water to prepare a bilirubin solution.

(2) To 1 volume of the bilirubin solution prepared in (1) above, 9 volumes of human serum were added and mixed to prepare a serum sample with a bilirubin (conjugated) concentration of 50 mg / dL.

(3) The “bilirubin C“ blank ”” of the “interference check A plus” was dissolved in the same amount of pure water as the amount used for the dissolution in (1) to prepare a blank solution.

(4) To 1 volume of the solution (blank solution) prepared in (3) above, 9 volumes of the human serum used in (2) above are added and mixed, and the blank solution is 1/10 of the human serum. A blank sample diluted in 1 was prepared.

(5) The serum sample prepared in the above (2) was diluted with the blank sample prepared in the above (4) to prepare a serum sample having a bilirubin (conjugated) concentration of 25 mg / dL.
The blank sample used for this dilution was a serum sample with a bilirubin (conjugated) concentration of 0 mg / dL.

That is, the following three types of serum samples (a) to (c) were used as samples.
(A) Serum sample-1 [Bilirubin (conjugated) concentration 0 mg / dL]
(B) Serum sample-2 [Bilirubin (conjugate) concentration 25 mg / dL]
(C) Serum sample-3 [Bilirubin (conjugate) concentration 50 mg / dL]

3. Measurement of Creatinine in Sample The measurement of creatinine in each of the two samples was performed using a 7180 Hitachi automatic analyzer [Hitachi High-Technologies Corporation (Japan)].

(1) In each of the serum samples of (2) (a) to (c), 6.0 μL of the sample was added to the first reagent of (a) of (1) above [the present invention (0.04% 150 μL of Amit 320)] was added and reacted at 37 ° C.

(2) Next, the absorbance at 15 points (254.193 seconds after the addition of the first reagent) and the absorbance at 16 points (270.093 seconds after the addition of the first reagent) were measured at a main wavelength of 600 nm and a subwavelength of 800 nm, respectively. Thus, the average value of the absorbance at 15 points and the absorbance at 16 points was calculated [“photometry 1”]. (Measurement of absorbance of sample blank)

(3) Next, between 16 points (270.093 seconds after the addition of the first reagent) and 17 points (286.977 seconds after the addition of the first reagent), 50 μL of the second reagent in (1) above is added. Added and allowed to react at 37 ° C.

(4) Next, the absorbance at 33 points (56.582 seconds after the addition of the first reagent) and the absorbance at 34 points (587.426 seconds after the addition of the first reagent) were measured at a main wavelength of 600 nm and a sub wavelength of 800 nm, respectively. Thus, the average value of the absorbance at 33 points and the absorbance at 34 points was calculated [“photometry 2”]. (Measurement of absorbance according to the concentration of creatinine contained in the sample)

(5) Next, the measured value of the concentration of creatinine contained in the sample was determined as follows.
That is, for each sample of (2) in (2) above, from the average absorbance (“photometry 2”) measured in (4) above, the average absorbance (“photometry 1”) measured in (2) above. Was subtracted to determine the value of the absorbance difference.
Next, a standard sample whose creatinine concentration is known is measured as described in the above (1) to (4), and the average value of absorbance measured in the above (4) (“photometry 2”) By comparing the value of the difference in absorbance obtained by subtracting the average value of absorbance measured in (2) (“photometry 1”) with the value of the absorbance difference in each of the above samples, and performing proportional calculation, A measurement of creatinine concentration was obtained.

(6) Further, in the above (1), in place of the first reagent (a) of (1) of [1] [the present invention (0.04% Amit 320)], (1) to (5) except that the first reagent (the present invention (0.08% Amit 320)) is used, and “serum sample-1”, “serum sample-2” ”And“ Serum Sample-3 ”were finally measured for their respective creatinine concentrations.

(7) Further, in the above (1), instead of the first reagent (a) of (1) in the above (1) (the present invention (0.04% Amit 320)), (1) to (5) except that the first reagent [invention (0.12% Amiate 320)] is used, and "serum sample-1", "serum sample-2" ”And“ Serum Sample-3 ”were finally measured for their respective creatinine concentrations.

(8) Further, in the above (1), in place of the first reagent (a) of (1) in (1) above (the present invention (0.04% Amit 320)), (1) to (5) except that the first reagent (the present invention (0.16% Amit 320)) is used, and “serum sample-1”, “serum sample-2” ”And“ Serum Sample-3 ”were finally measured for their respective creatinine concentrations.

(9) Further, in the above (1), in place of the first reagent (a) of (1) in (1) above (the present invention (0.04% Amit 320)), ) Except that the first reagent [conventional invention (0%)] is used, the procedure is performed as in (1) to (5) above, and “serum sample-1”, “serum sample-2”, and “ The final measured value of each creatinine concentration of “serum sample-3” was determined.

4). Measurement Results Table 4 shows the results obtained by measuring the creatinine concentration of each sample in 3 above.

  In this table, the horizontal column represents the type and concentration of the polyoxyethylene alkylamine compound contained in the first reagent. [The 1st reagent which does not contain a polyoxyethylene alkyl nitrogen-containing compound was expressed as "0%". ]

  In this table, the vertical column represents the concentration of bilirubin (conjugated) in the sample.

  And the value shown in each column of this table | surface shows the measured value [mg / dL] of the density | concentration of the creatinine in the said sample.

  In addition, the numerical value in (parentheses) is the value obtained by dividing each measured value by the measured value when the concentration of bilirubin (conjugated) in the sample when the same first reagent is used is 0 mg / dL. It is expressed as a percentage.

5. Consideration (1) From this table, when the first reagent does not contain a polyoxyethylene alkyl nitrogen-containing compound (0%), the concentration of creatinine in the sample increases as the concentration of bilirubin (conjugated) in the sample increases. It can be seen that the measured value of the concentration decreases, and when the concentration of bilirubin (conjugated) in the sample is 50 mg / dL, the measured value has decreased to 73% of the original value.

  That is, it can be seen that the measurement value of the concentration of creatinine in the sample is affected by the presence of bilirubin contained in the sample, and a large negative error occurs.

(2) From this table, the measured value of the concentration of creatinine in the sample when the first reagent contains a polyoxyethylene alkylamine compound does not contain the polyoxyethylene alkyl nitrogen-containing compound (0%) It can be seen that the decrease in the measurement value derived from bilirubin contained in the sample is suppressed with respect to the measurement value in the first reagent.

  Further, from this table, when the concentration of the polyoxyethylene alkylamine compound contained in the first reagent is 0.04% (w / v), the concentration is 0.08% (w / v) or more. It can be seen that the decrease in the measurement value derived from bilirubin is further suppressed.

(3) That is, in measuring a measurement target substance in a sample by optically measuring hydrogen peroxide generated by an oxidase using a chromogen, a polyoxyethylene alkylamine compound and a polyoxyethylene alkylamide are measured. By performing the measurement in the presence of at least one polyoxyethylene alkyl nitrogen-containing compound selected from the compounds, it is possible to avoid the influence on the measurement derived from bilirubin contained in the sample, and the measurement result (measurement value) It was also confirmed from this experiment that it was possible to prevent the occurrence of errors in.

[Example 4] (Confirmation of effect of avoiding influence derived from bilirubin contained in sample-4)
The effect of avoiding the influence on the measurement derived from the bilirubin contained in the sample in the measurement method and measurement reagent of the measurement target substance in the sample of the present invention and the method for avoiding the influence on the measurement derived from the bilirubin contained in the sample Further confirmation.
Specifically, in the present invention, when a polyoxyethylene alkylamide compound is used as the polyoxyethylene alkyl nitrogen-containing compound, the effect of avoiding the influence on the measurement of creatinine in the sample derived from bilirubin contained in the sample is achieved. confirmed.

1. Creatinine measuring reagent (1) First reagent

(A) Preparation of First Reagent [Invention (0.05% TAMDO-10)] The following reagent components were dissolved in pure water so as to have the respective concentrations, and the pH was adjusted to pH 7.4 (20 ° C.). The first reagent [the present invention (0.05% TAMDO-10)] was prepared by adjusting.

TAMDO-10 [Nikko Chemicals Corporation (Japan)] <Polyoxyethylene alkylamide compound> 0.05% (w / v)
Sarcosine oxidase 13,000Unit / L
Creatinase 40,000Unit / L
Catalase 200,000Unit / L
N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline, sodium salt, dihydrate [ADOS] <chromogen (hydrogen donor)> 2 mM
Ascorbate oxidase 3,000Unit / L
TES [N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid] <Buffer> 20 mM

(B) Preparation of First Reagent [Invention (0.10% TAMDO-10)] The following reagent components were dissolved in pure water so as to have the respective concentrations, and the pH was adjusted to pH 7.4 (20 ° C.). The first reagent [the present invention (0.10% TAMDO-10)] was prepared by adjusting.

TAMDO-10 [Nikko Chemicals Corporation (Japan)] <Polyoxyethylene alkylamide compound> 0.10% (w / v)
Sarcosine oxidase 13,000Unit / L
Creatinase 40,000Unit / L
Catalase 200,000Unit / L
N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline, sodium salt, dihydrate [ADOS] <chromogen (hydrogen donor)> 2 mM
Ascorbate oxidase 3,000Unit / L
TES [N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid] <Buffer> 20 mM

(C) Preparation of First Reagent [Invention (0.50% TAMDO-10)] The following reagent components were dissolved in pure water so as to have the respective concentrations described above, and the pH was adjusted to pH 7.4 (20 ° C.). The first reagent [the present invention (0.50% TAMDO-10)] was prepared by adjusting.

TAMDO-10 [Nikko Chemicals Corporation (Japan)] <Polyoxyethylene alkylamide compound> 0.50% (w / v)
Sarcosine oxidase 13,000Unit / L
Creatinase 40,000Unit / L
Catalase 200,000Unit / L
N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline, sodium salt, dihydrate [ADOS] <chromogen (hydrogen donor)> 2 mM
Ascorbate oxidase 3,000Unit / L
TES [N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid] <Buffer> 20 mM

(D) Preparation of First Reagent [Invention (1.00% TAMDO-10)] The following reagent components were dissolved in pure water so as to have the respective concentrations described above, and the pH was adjusted to pH 7.4 (20 ° C.). The first reagent [the present invention (1.00% TAMDO-10)] was prepared by adjusting.

TAMDO-10 [Nikko Chemicals Corporation (Japan)] <Polyoxyethylene alkylamide compound> 1.00% (w / v)
Sarcosine oxidase 13,000Unit / L
Creatinase 40,000Unit / L
Catalase 200,000Unit / L
N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline, sodium salt, dihydrate [ADOS] <chromogen (hydrogen donor)> 2 mM
Ascorbate oxidase 3,000Unit / L
TES [N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid] <Buffer> 20 mM

(E) Preparation of First Reagent [Conventional Invention (0%)] The following reagent components were dissolved in pure water so that each concentration was as described, and the pH was adjusted to pH 7.4 (20 ° C.). A first reagent [conventional invention (0%)] containing no oxyethylenealkyl nitrogen-containing compound (concentration 0%) was prepared.

Sarcosine oxidase 13,000Unit / L
Creatinase 40,000Unit / L
Catalase 200,000Unit / L
N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline, sodium salt, dihydrate [ADOS] <chromogen (hydrogen donor)> 2 mM
Ascorbate oxidase 3,000Unit / L
TES [N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid] <Buffer> 20 mM

(2) Second reagent

Preparation of Second Reagent The following reagent components were dissolved in pure water so as to have the indicated concentrations, and the pH was adjusted to 7.8 (20 ° C.) to prepare a second reagent.

Creatininase 400,000Unit / L
4-aminoantipyrine 2 mM
Peroxidase 15,000Unit / L
Sodium azide 0.15% (w / v)
Potassium ferrocyanide 0.2 mM
TES [N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid] <Buffer> 20 mM

2. Sample (1) “Bilirubin C (conjugated type)” of “Interference Check A Plus” (Sysmex Corporation (Japan)), which is a commercially available control substance for examining interference substances, has a bilirubin (conjugated type) concentration of 500 mg. / DL was dissolved in pure water to prepare a bilirubin solution.

(2) To 1 volume of the bilirubin solution prepared in (1) above, 9 volumes of human serum were added and mixed to prepare a serum sample with a bilirubin (conjugated) concentration of 50 mg / dL.

(3) The “bilirubin C“ blank ”” of the “interference check A plus” was dissolved in the same amount of pure water as the amount used for the dissolution in (1) to prepare a blank solution.

(4) To 1 volume of the solution (blank solution) prepared in (3) above, 9 volumes of the human serum used in (2) above are added and mixed, and the blank solution is 1/10 of the human serum. A blank sample diluted in 1 was prepared.

(5) The serum sample prepared in the above (2) was diluted with the blank sample prepared in the above (4) to prepare a serum sample having a bilirubin (conjugated) concentration of 25 mg / dL.
The blank sample used for this dilution was a serum sample with a bilirubin (conjugated) concentration of 0 mg / dL.

That is, the following three types of serum samples (a) to (c) were used as samples.
(A) Serum sample-1 [Bilirubin (conjugated) concentration 0 mg / dL]
(B) Serum sample-2 [Bilirubin (conjugate) concentration 25 mg / dL]
(C) Serum sample-3 [Bilirubin (conjugate) concentration 50 mg / dL]

3. Measurement of Creatinine in Sample The measurement of creatinine in each of the two samples was performed using a 7180 Hitachi automatic analyzer [Hitachi High-Technologies Corporation (Japan)].

(1) In each of the serum samples of (2) (a) to (c) above, 6.0 μL of the sample was added to the first reagent of (a) of (1) above [the present invention (0.05% 150 μL of TAMDO-10)] was added and reacted at 37 ° C.

(2) Next, the absorbance at 15 points (254.193 seconds after the addition of the first reagent) and the absorbance at 16 points (270.093 seconds after the addition of the first reagent) were measured at a main wavelength of 600 nm and a subwavelength of 800 nm, respectively. Thus, the average value of the absorbance at 15 points and the absorbance at 16 points was calculated [“photometry 1”]. (Measurement of absorbance of sample blank)

(3) Next, between 16 points (270.093 seconds after the addition of the first reagent) and 17 points (286.977 seconds after the addition of the first reagent), 50 μL of the second reagent in (1) above is added. Added and allowed to react at 37 ° C.

(4) Next, the absorbance at 33 points (56.582 seconds after the addition of the first reagent) and the absorbance at 34 points (587.426 seconds after the addition of the first reagent) were measured at a main wavelength of 600 nm and a sub wavelength of 800 nm, respectively. Thus, the average value of the absorbance at 33 points and the absorbance at 34 points was calculated [“photometry 2”]. (Measurement of absorbance according to the concentration of creatinine contained in the sample)

(5) Next, the measured value of the concentration of creatinine contained in the sample was determined as follows.
That is, for each sample of (2) in (2) above, from the average absorbance (“photometry 2”) measured in (4) above, the average absorbance (“photometry 1”) measured in (2) above. Was subtracted to determine the value of the absorbance difference.
Next, a standard sample whose creatinine concentration is known is measured as described in the above (1) to (4), and the average value of absorbance measured in the above (4) (“photometry 2”) By comparing the value of the difference in absorbance obtained by subtracting the average value of absorbance measured in (2) (“photometry 1”) with the value of the absorbance difference in each of the above samples, and performing proportional calculation, A measurement of creatinine concentration was obtained.

(6) Further, in the above (1), in place of the first reagent (1) of (1) (a) [the present invention (0.05% TAMDO-10)], Except for using the first reagent b) of the present invention (the present invention (0.10% TAMDO-10)), the same procedures as in the above (1) to (5) were performed, and “serum sample-1”, “serum sample” -2 "and" serum sample-3 "were finally measured for their creatinine concentrations.

(7) Further, in the above (1), in place of the first reagent (1) of (1) (a) [the present invention (0.05% TAMDO-10)], c) The first reagent [the present invention (0.50% TAMDO-10)] is used except that the procedures described in (1) to (5) above are performed, and “serum sample-1” and “serum sample” -2 "and" serum sample-3 "were finally measured for their creatinine concentrations.

(8) Further, in the above (1), in place of the first reagent (1) of (1) (a) [the present invention (0.05% TAMDO-10)], Except for the use of the first reagent of d) [the present invention (1.00% TAMDO-10)], the operations were carried out as in the above (1) to (5), and "serum sample-1", "serum sample" -2 "and" serum sample-3 "were finally measured for their creatinine concentrations.

(9) Further, in the above (1), in place of the first reagent (1) of (1) (a) [the present invention (0.05% TAMDO-10)], e) Except for using the first reagent [conventional invention (0%)], the operation was carried out as in the above (1) to (5), and “serum sample-1”, “serum sample-2”, and The final measured value of each creatinine concentration of “serum sample-3” was determined.

4). Measurement Results Table 5 shows the results obtained by measuring the creatinine concentration of each sample in 3 above.

  In this table, the horizontal column represents the type and concentration of the polyoxyethylene alkylamide compound contained in the first reagent. [The 1st reagent which does not contain a polyoxyethylene alkyl nitrogen-containing compound was expressed as "0%". ]

  In this table, the vertical column represents the concentration of bilirubin (conjugated) in the sample.

  And the value shown in each column of this table | surface shows the measured value [mg / dL] of the density | concentration of the creatinine in the said sample.

  In addition, the numerical value in (parentheses) is the value obtained by dividing each measured value by the measured value when the concentration of bilirubin (conjugated) in the sample when the same first reagent is used is 0 mg / dL. It is expressed as a percentage.

5. Consideration (1) From this table, when the first reagent does not contain a polyoxyethylene alkyl nitrogen-containing compound (0%), the concentration of creatinine in the sample increases as the concentration of bilirubin (conjugated) in the sample increases. The measured value of the concentration decreases, and it can be seen that when the concentration of bilirubin (conjugated) in the sample is 50 mg / dL, the measured value has decreased to 71% of the original value.

  That is, it can be seen that the measurement value of the concentration of creatinine in the sample is affected by the presence of bilirubin contained in the sample, and a large negative error occurs.

(2) From this table, the measured value of the concentration of creatinine in the sample when the first reagent contains a polyoxyethylene alkylamide compound does not contain the above-mentioned polyoxyethylene alkyl nitrogen-containing compound (0%) It can be seen that the decrease in the measurement value derived from bilirubin contained in the sample is suppressed with respect to the measurement value in the first reagent.

  In addition, it can be seen from this table that the decrease in the measured value derived from bilirubin is further suppressed when the concentration of the polyoxyethylene alkylamide compound contained in the first reagent is higher.

(3) That is, in measuring a measurement target substance in a sample by optically measuring hydrogen peroxide generated by an oxidase using a chromogen, a polyoxyethylene alkylamine compound and a polyoxyethylene alkylamide are measured. By performing the measurement in the presence of at least one polyoxyethylene alkyl nitrogen-containing compound selected from the compounds, it is possible to avoid the influence on the measurement derived from bilirubin contained in the sample, and the measurement result (measurement value) It was also confirmed from this experiment that it was possible to prevent the occurrence of errors in.

[Example 5] (Confirmation of effect of avoiding influence derived from bilirubin contained in sample-5)
The effect of avoiding the influence on the measurement derived from the bilirubin contained in the sample in the measurement method and measurement reagent of the measurement target substance in the sample of the present invention and the method for avoiding the influence on the measurement derived from the bilirubin contained in the sample Further confirmation.
Specifically, in the present invention, when a polyoxyethylene alkylamine compound is used as the polyoxyethylene alkyl nitrogen-containing compound, the effect of avoiding the influence on the measurement of creatinine in the sample derived from bilirubin contained in the sample is achieved. confirmed.

1. Creatinine measuring reagent (1) First reagent

(A) Preparation of first reagent [present invention (3.50% polyoxyethylene (10) octylamine)] The following reagent components were dissolved in pure water so as to have the respective concentrations described above, and the pH was adjusted to pH 7.4. The first reagent [the present invention (3.50% polyoxyethylene (10) octylamine)] was prepared by adjusting to (20 ° C).

Polyoxyethylene octylamine (sum of m and n as ethylene oxide addition moles: 10) <Polyoxyethylene alkylamine compound> 3.50% (w / v)
Sarcosine oxidase 13,000Unit / L
Creatinase 40,000Unit / L
Catalase 200,000Unit / L
N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline, sodium salt, dihydrate [ADOS] <chromogen (hydrogen donor)> 2 mM
Ascorbate oxidase 3,000Unit / L
TES [N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid] <Buffer> 20 mM

(B) Preparation of First Reagent [Invention (0.15% Naimine S-220)] The following reagent components were dissolved in pure water so as to have the respective concentrations, and the pH was adjusted to pH 7.4 (20 ° C.). The first reagent [the present invention (0.15% Naimine S-220)] was prepared.

Naimine S-220 [NOF Corporation (Japan)] <Polyoxyethylene alkylamine compound> 0.15% (w / v)
Sarcosine oxidase 13,000Unit / L
Creatinase 40,000Unit / L
Catalase 200,000Unit / L
N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline, sodium salt, dihydrate [ADOS] <chromogen (hydrogen donor)> 2 mM
Ascorbate oxidase 3,000Unit / L
TES [N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid] <Buffer> 20 mM

(C) Preparation of First Reagent [Conventional Invention (0.10% BT-9)] The following reagent components were dissolved in pure water so as to have the respective concentrations, and the pH was adjusted to pH 7.4 (20 ° C.). The first reagent [conventional invention (0.10% BT-9)] was prepared.

BT-9 [Nikko Chemicals Corporation (Japan)] <Polyoxyethylene (9) secondary alkyl ether> 0.10% (w / v)
Sarcosine oxidase 13,000Unit / L
Creatinase 40,000Unit / L
Catalase 200,000Unit / L
N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline, sodium salt, dihydrate [ADOS] <chromogen (hydrogen donor)> 2 mM
Ascorbate oxidase 3,000Unit / L
TES [N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid] <Buffer> 20 mM

(D) Preparation of First Reagent [Conventional Invention (1.00% BT-9)] The following reagent components were dissolved in pure water so as to have the respective concentrations, and the pH was adjusted to pH 7.4 (20 ° C.). The first reagent [conventional invention (1.00% BT-9)] was prepared by adjusting.

BT-9 [Nikko Chemicals Corporation (Japan)] <Polyoxyethylene (9) secondary alkyl ether> 1.00% (w / v)
Sarcosine oxidase 13,000Unit / L
Creatinase 40,000Unit / L
Catalase 200,000Unit / L
N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline, sodium salt, dihydrate [ADOS] <chromogen (hydrogen donor)> 2 mM
Ascorbate oxidase 3,000Unit / L
TES [N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid] <Buffer> 20 mM

(E) Preparation of First Reagent [Conventional Invention (0.10% Sannix GP-400)] The following reagent components were dissolved in pure water so as to have the respective concentrations described above, and the pH was adjusted to pH 7.4 (20 ° C. The first reagent [conventional invention (0.10% Sannix GP-400)] was prepared.

SANNICS GP-400 [Sanyo Chemical Industries, Ltd. (Japan)] <Polyoxypropylene glyceryl ether> 0.10% (w / v)
Sarcosine oxidase 13,000Unit / L
Creatinase 40,000Unit / L
Catalase 200,000Unit / L
N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline, sodium salt, dihydrate [ADOS] <chromogen (hydrogen donor)> 2 mM
Ascorbate oxidase 3,000Unit / L
TES [N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid] <Buffer> 20 mM

(F) Preparation of First Reagent [Conventional Invention (1.00% Sannix GP-400)] The following reagent components were dissolved in pure water so as to have the respective concentrations described above, and the pH was adjusted to pH 7.4 (20 ° C. The first reagent [conventional invention (1.00% Sannix GP-400)] was prepared.

SANNICS GP-400 [Sanyo Chemical Industries, Ltd. (Japan)] <Polyoxypropylene glyceryl ether> 1.00% (w / v)
Sarcosine oxidase 13,000Unit / L
Creatinase 40,000Unit / L
Catalase 200,000Unit / L
N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline, sodium salt, dihydrate [ADOS] <chromogen (hydrogen donor)> 2 mM
Ascorbate oxidase 3,000Unit / L
TES [N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid] <Buffer> 20 mM

(G) Preparation of First Reagent [Conventional Invention (0%)] The following reagent components were dissolved in pure water so as to have the respective concentrations described above, and the pH was adjusted to pH 7.4 (20 ° C.). A first reagent [conventional invention (0%)] containing neither an oxyethylenealkyl-containing nitrogen compound nor other surfactants (such as BT-9 and Sanniks GP-400) (concentration 0%) was prepared.

Sarcosine oxidase 13,000Unit / L
Creatinase 40,000Unit / L
Catalase 200,000Unit / L
N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline, sodium salt, dihydrate [ADOS] <chromogen (hydrogen donor)> 2 mM
Ascorbate oxidase 3,000Unit / L
TES [N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid] <Buffer> 20 mM

(2) Second reagent

Preparation of Second Reagent The following reagent components were dissolved in pure water so as to have the indicated concentrations, and the pH was adjusted to 7.8 (20 ° C.) to prepare a second reagent.

Creatininase 400,000Unit / L
4-aminoantipyrine 2 mM
Peroxidase 15,000Unit / L
Sodium azide 0.15% (w / v)
Potassium ferrocyanide 0.2 mM
TES [N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid] <Buffer> 20 mM

2. Sample (1) “Bilirubin C (conjugated type)” of “Interference Check A Plus” (Sysmex Corporation (Japan)), which is a commercially available control substance for examining interference substances, has a bilirubin (conjugated type) concentration of 500 mg. / DL was dissolved in pure water to prepare a bilirubin solution.

(2) To 1 volume of the bilirubin solution prepared in (1) above, 9 volumes of human serum were added and mixed to prepare a serum sample with a bilirubin (conjugated) concentration of 50 mg / dL.

(3) The “bilirubin C“ blank ”” of the “interference check A plus” was dissolved in the same amount of pure water as the amount used for the dissolution in (1) to prepare a blank solution.

(4) To 1 volume of the solution (blank solution) prepared in (3) above, 9 volumes of the human serum used in (2) above are added and mixed, and the blank solution is 1/10 of the human serum. A blank sample diluted in 1 was prepared.

(5) The serum sample prepared in (2) is diluted with the blank sample prepared in (4), and a serum sample having a bilirubin (conjugated) concentration of 40 mg / dL, a serum sample of 30 mg / dL, A 20 mg / dL serum sample and a 10 mg / dL serum sample were prepared, respectively.
The blank sample used for this dilution was a serum sample with a bilirubin (conjugated) concentration of 0 mg / dL.

That is, the following six types of serum samples (a) to (f) were used as samples.
(A) Serum sample-1 [Bilirubin (conjugated) concentration 0 mg / dL]
(B) Serum sample-2 [Bilirubin (conjugate) concentration 10 mg / dL]
(C) Serum sample-3 [Bilirubin (conjugate) concentration 20 mg / dL]
(D) Serum sample-4 [Bilirubin (conjugate) concentration 30 mg / dL]
(E) Serum sample-5 [Bilirubin (conjugate) concentration 40 mg / dL]
(F) Serum sample-6 [Bilirubin (conjugate) concentration 50 mg / dL]

3. Measurement of Creatinine in Sample The measurement of creatinine in each of the two samples was performed using a 7180 Hitachi automatic analyzer [Hitachi High-Technologies Corporation (Japan)].

(1) In each of the serum samples of (2) (a) to (f) above, 6.0 μL of the sample was added to the first reagent of (a) of (1) above [the present invention (3.50% Polyoxyethylene (10) octylamine)] was added and reacted at 37 ° C.

(2) Next, the absorbance at 15 points (254.193 seconds after the addition of the first reagent) and the absorbance at 16 points (270.093 seconds after the addition of the first reagent) were measured at a main wavelength of 600 nm and a subwavelength of 800 nm, respectively. Thus, the average value of the absorbance at 15 points and the absorbance at 16 points was calculated [“photometry 1”]. (Measurement of absorbance of sample blank)

(3) Next, between 16 points (270.093 seconds after the addition of the first reagent) and 17 points (286.977 seconds after the addition of the first reagent), 50 μL of the second reagent in (1) above is added. Added and allowed to react at 37 ° C.

(4) Next, the absorbance at 33 points (56.582 seconds after the addition of the first reagent) and the absorbance at 34 points (587.426 seconds after the addition of the first reagent) were measured at a main wavelength of 600 nm and a sub wavelength of 800 nm, respectively. Thus, the average value of the absorbance at 33 points and the absorbance at 34 points was calculated [“photometry 2”]. (Measurement of absorbance according to the concentration of creatinine contained in the sample)

(5) Next, the measured value of the concentration of creatinine contained in the sample was determined as follows.
That is, for each sample of (2) in (2) above, from the average absorbance (“photometry 2”) measured in (4) above, the average absorbance (“photometry 1”) measured in (2) above. Was subtracted to determine the value of the absorbance difference.
Next, a standard sample whose creatinine concentration is known is measured as described in the above (1) to (4), and the average value of absorbance measured in the above (4) (“photometry 2”) By comparing the value of the difference in absorbance obtained by subtracting the average value of absorbance measured in (2) (“photometry 1”) with the value of the absorbance difference in each of the above samples, and performing proportional calculation, A measurement of creatinine concentration was obtained.

(6) Further, in the above (1), instead of the first reagent (a) of (1) in the above [1] (the present invention (3.50% polyoxyethylene (10) octylamine)), (1) Except for using the first reagent (b) of the present invention (the present invention (0.15% Naimine S-220)), the procedure was carried out as in (1) to (5) above. 1 ”,“ serum sample-2 ”,“ serum sample-3 ”,“ serum sample-4 ”,“ serum sample-5 ”, and“ serum sample-6 ”. Asked.

(7) Further, in the above (1), instead of the first reagent (a) of (1) in the above [1] (the present invention (3.50% polyoxyethylene (10) octylamine)), (1) (c) The first reagent [Conventional invention (0.10% BT-9)] is used except that the procedure is performed as in (1) to (5) above. ”,“ Serum sample-2 ”,“ serum sample-3 ”,“ serum sample-4 ”,“ serum sample-5 ”, and“ serum sample-6 ”, the final measured values of creatinine are obtained. It was.

(8) Further, in the above (1), instead of the first reagent (1) of (1) in (1) above (the present invention (3.50% polyoxyethylene (10) octylamine)), (1) Except for using the first reagent (d) of [d] [conventional invention (1.00% BT-9)], the procedure was carried out as in the above (1) to (5), and “serum sample-1” ”,“ Serum sample-2 ”,“ serum sample-3 ”,“ serum sample-4 ”,“ serum sample-5 ”, and“ serum sample-6 ”, the final measured values of creatinine are obtained. It was.

(9) Further, in the above (1), instead of the first reagent (1) of (1) in (1) above (the present invention (3.50% polyoxyethylene (10) octylamine)), (1) Except for using the first reagent (e) of [e] [conventional invention (0.10% Sannix GP-400)], the procedure was performed as described in (1) to (5) above. -1 ”,“ serum sample-2 ”,“ serum sample-3 ”,“ serum sample-4 ”,“ serum sample-5 ”, and“ serum sample-6 ”, the final measured value of each creatinine concentration Asked.

(10) Further, in the above (1), instead of the first reagent (a) of (1) in the above [1] (the present invention (3.50% polyoxyethylene (10) octylamine)), (1) Except for using the first reagent (f) of [f] (conventional invention (1.00% Sannix GP-400)), the operation was carried out as in (1) to (5) above. -1 ”,“ serum sample-2 ”,“ serum sample-3 ”,“ serum sample-4 ”,“ serum sample-5 ”, and“ serum sample-6 ”, the final measured value of each creatinine concentration Asked.

(11) Further, in the above (1), instead of the first reagent (a) of (1) in the above (1) (the present invention (3.50% polyoxyethylene (10) octylamine)), Except for using the first reagent (conventional invention (0%)) of (g) in (1), the operation was performed as in the above (1) to (5), and “serum sample-1”, “serum sample” -2 "," serum sample-3 "," serum sample-4 "," serum sample-5 ", and" serum sample-6 "were finally measured for their respective creatinine concentrations.

4). Measurement Results Table 6 shows the results obtained by measuring the creatinine concentration of each sample in 3 above.

  In this table, the horizontal column represents the type and concentration of the polyoxyethylene alkylamine compound or other surfactant (BT-9 or Sannics GP-400) contained in the first reagent. [The 1st reagent which does not contain a polyoxyethylene alkyl nitrogen-containing compound and other surfactants (BT-9, Sanniks GP-400, etc.) was expressed as "0%". ]

  In this table, the vertical column represents the concentration of bilirubin (conjugated) in the sample.

  And the value shown in each column of this table | surface shows the measured value [mg / dL] of the density | concentration of the creatinine in the said sample.

  In addition, the numerical value in (parentheses) is the value obtained by dividing each measured value by the measured value when the concentration of bilirubin (conjugated) in the sample when the same first reagent is used is 0 mg / dL. It is expressed as a percentage.

5. Consideration (1) From this table, when the first reagent is a polyoxyethylene alkyl-containing nitrogen compound and does not contain other surfactants (such as BT-9 and Sanniks GP-400) (0%), the sample As the concentration of bilirubin (conjugated) in the sample increases, the measured value of the concentration of creatinine in the sample decreases. When the concentration of bilirubin (conjugated) in the sample is 50 mg / dL, the original value It can be seen that the measured value has decreased to 73%.

  That is, it can be seen that the measurement value of the concentration of creatinine in the sample is affected by the presence of bilirubin contained in the sample, and a large negative error occurs.

(2) From this table, when the first reagent contains a polyoxyethylene alkylamine compound, the measured value of the concentration of creatinine in the sample is that the polyoxyethylene alkyl nitrogen-containing compound is also a surfactant ( It can be seen that the decrease in the measurement value derived from bilirubin contained in the sample is suppressed with respect to the measurement value in the first reagent that does not include (0%) such as BT-9 and Sannicks GP-400.

(3) On the other hand, the measured value of the concentration of creatinine in the sample when the first reagent contains another surfactant (BT-9 or Sannix GP-400) is the above-mentioned polyoxyethylene alkyl-containing value. Nitrogen compounds and other surfactants (BT-9, Sanniks GP-400, etc.) do not contain (0%) Degree of decrease in measured values derived from bilirubin contained in the sample relative to the measured value in the first reagent Can be seen to be equivalent or higher. That is, in this case, it can be seen that the decrease in the measurement value derived from bilirubin contained in the sample cannot be suppressed.

(4) That is, in measuring the substance to be measured in a sample by optically measuring hydrogen peroxide generated by an oxidase using a chromogen, a polyoxyethylene alkylamine compound and a polyoxyethylene alkylamide are used. By performing the measurement in the presence of at least one polyoxyethylene alkyl nitrogen-containing compound selected from the compounds, it is possible to avoid the influence on the measurement derived from bilirubin contained in the sample, and the measurement result (measurement value) It has been confirmed that it is possible to prevent the occurrence of errors.

  And even if it measures by making other surfactants (BT-9 or Sannix GP-400) other than this polyoxyethylene alkyl nitrogen-containing compound exist, the influence on the measurement derived from the bilirubin contained in a sample It was confirmed that it was not possible to avoid the occurrence of errors in the measurement results (measured values).

Claims (5)

  In a method for measuring a substance to be measured in a sample by optically measuring hydrogen peroxide generated by an oxidase using a chromogen, selected from a polyoxyethylene alkylamine compound and a polyoxyethylene alkylamide compound A method for measuring a substance to be measured in a sample, wherein the measurement is carried out in the presence of at least one polyoxyethylene alkyl nitrogen-containing compound.   By performing the measurement in the presence of at least one polyoxyethylene alkyl nitrogen-containing compound selected from a polyoxyethylene alkylamine compound and a polyoxyethylene alkylamide compound, the influence on the measurement derived from bilirubin contained in the sample is reduced. The method for measuring a substance to be measured in a sample according to claim 1 to avoid.   A reagent for measuring a substance to be measured in a sample by optically measuring hydrogen peroxide generated by an oxidase using a chromogen, selected from a polyoxyethylene alkylamine compound and a polyoxyethylene alkylamide compound A measuring reagent for a substance to be measured in a sample, comprising at least one polyoxyethylene alkyl nitrogen-containing compound.   The at least one polyoxyethylene alkyl nitrogen-containing compound selected from a polyoxyethylene alkylamine compound and a polyoxyethylene alkylamide compound is contained as an agent for avoiding an influence on measurement derived from bilirubin contained in a sample. 3. A reagent for measuring a substance to be measured in the sample according to 3.   When measuring the substance to be measured in a sample by optically measuring hydrogen peroxide generated by oxidase using a chromogen, it is selected from polyoxyethylene alkylamine compounds and polyoxyethylene alkylamide compounds A method for avoiding an influence on measurement derived from bilirubin contained in a sample, wherein the measurement is performed in the presence of at least one polyoxyethylene alkyl nitrogen-containing compound.