JP2006081471A - Reagent for assaying biological component and method for assaying the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a reagent and a method for assaying a specific component in a biological specimen while avoiding the influence of reducing substances including bilirubin and hemolytic factors. <P>SOLUTION: The method for assaying a component in a biological specimen comprises involving making 0.4 nmol/L or more of a peroxide present in a pre-treatment step. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for measuring a specific component in a biological sample such as serum, urine, plasma, etc. (1) caused by bilirubin and hemolyzed hemoglobin (1) influence on colorimetric measurement due to deterioration and coloration, (2 It relates to a reagent and a measurement method for measuring by avoiding both of the effects on the redox reaction.

  Component analysis by absorbance measurement is widely performed in the clinical laboratory field. As a method, dehydrogenase is used, NAD (P) H is used as an indicator (dehydrogenase method), hydrogen peroxide produced using oxidase and peroxidase are used to introduce color formers into dyes and colorimetrically determine. There are a method (oxidase method) and a method (synthetic substrate method) for measuring the rate of increase of the chromogen produced by the enzyme reaction using a substrate into which a chromogenic group is introduced. These methods are problematic because errors occur in measured values due to the effects of bilirubin and hemolysis that may be present in large amounts in biological samples. Factors that cause the influence include (1) the reduction action of bilirubin and hemolysis, and (2) the measurement wavelength or the subwavelength in the two-wavelength measurement in the vicinity of 400 to 450 nm, which is near the absorption maximum of bilirubin and hemolysis. Bilirubin, absorbance change caused by hemolysis.

In contrast, various methods for avoiding the effects of bilirubin and hemolysis have been reported so far. Methods for avoiding the effects of bilirubin include a method using ferrocyanide ions (see Patent Document 1), a method of degrading bilirubin using bilirubin oxidase (see Patent Document 2), and a large amount of excess before the main reaction. Methods of generating hydrogen oxide and degrading bilirubin using an oxidation reaction of peroxidase (see Patent Documents 3 and 4), methods using specific surfactants (see Patent Documents 5 to 8), and the like have been reported. Yes. As a method for avoiding the influence of hemolysis, a method using thiourea (see Patent Document 9), a method using pyridines, imidazoles and histamines (see Patent Document 10), and a method using an alkyl sulfonate (Patent Document). 11), a method using a specific surfactant (see Patent Document 12), and the like have been reported. However, these methods are not possible because the effects of bilirubin and hemolysis cannot be avoided all at once, have an inhibitory effect on the measurement system, and reduce the storage stability of the measurement reagent. It was enough.
JP 55-25840 A JP-A-57-71398 JP-A-2-49600 Japanese Patent Laid-Open No. 6-339397 Japanese Patent Laid-Open No. 3-10696 Japanese Patent Publication No.7-11519 Japanese Patent Laid-Open No. 9-224697 JP-A-11-243993 Japanese Patent Publication No. 6-12998 Japanese Examined Patent Publication No. 3-56425 Japanese Patent Publication No. 3-58467 JP 2000-189194 A

  An object of the present invention is to provide a reagent and a measurement method for measuring a specific component in a biological sample by avoiding the influence of a reducing substance such as bilirubin and hemolysis.

  In view of the above problems, the present invention relates to a method for measuring a biological component by allowing a peroxide to be present in an amount of 0.4 nmol / L or more in a pretreatment step in a method for measuring a component in a biological sample.

  According to the present invention, measurement can be performed while avoiding the influence of bilirubin, hemolyzed hemoglobin and the like.

  In the present invention, the biological sample contains a reducing contaminant (hereinafter referred to as a reducing substance), and the reducing substance interferes with the measurement of the measurement target component. Such biological samples include serum, urine, plasma and the like.

  A component to be measured in a biological sample is a hydrogen peroxide measurement system that conjugates an oxidase and a peroxidase, that is, an oxidase is allowed to act on an analyte or a reaction product thereof, and the generated hydrogen peroxide is converted into a peroxidase. Depending on the chromogen, for example, a quinoneimine dye may be used, and components measured by a colorimetric method by measuring the dye with absorbance may be mentioned. Specific examples include neutral fat, creatinine, calcium, creatine, free cholesterol, cholesterol ester, phospholipid, inorganic phosphorus, amylase, GOT, GPT, sialic acid, guanase and the like. When detecting an analyte, an oxidase may act directly on the analyte, but in many cases, one or more other enzymes are required in addition to the oxidase and peroxidase. To do.

  Various basic methods for measuring the analysis target are known, and the present invention can be applied to them without any particular limitation. The measurement principle of neutral fat, creatinine and calcium is illustrated below.

Various methods are known for the reagents for measuring these analytes. For example, the creatinine measurement reagent based on the above measurement principle includes those having the following composition.
First reagent R1: creatine amidinohydrolase, sarcosine oxidase, catalase,
Second reagent R2: creatinine amide hydrolase, peroxidase, chromogen.

  That is, in the first enzyme reaction used for the reagent R1, creatine in the sample is converted into a colorless substance by the reaction of creatine amidinohydrolase, sarcosine oxidase, and catalase, and is erased. Next, as a second enzyme reaction, reagent R2 is added to the sample and reagent R1 system, and a colored substance is generated from creatinine in the sample by the reaction of creatinine amide hydrolase, creatine amidinohydrolase, sarcosine oxidase, chromogen and peroxidase. The substance is colorimetrically determined. Catalase coexisting in the second enzyme reaction can be blocked by adding the catalase inhibitor. Similarly to creatinine, in the detection of neutral fat, it is necessary to eliminate hydrogen peroxide derived from other than the analyte.

  In the present invention, a pretreatment liquid (pretreatment reagent) and a detection start liquid (detection reagent) are used.

The pretreatment liquid contains a peroxide, and a pretreatment liquid is added to the biological sample so that a reducing substance (for example, bilirubin or hemolyzed hemoglobin) is processed so as not to affect the measurement.
The pretreatment step of the present invention means a step of incubation in a state in which a biological sample and a pretreatment liquid are mixed, and the measurement target substance is maintained as it is or converted to a previous stage of the detection substance, such as bilirubin. The reducing substance is decomposed in this step to reduce the influence on the measurement of the measurement target substance. The pretreatment step may include a step of removing interfering substances other than the reducing substance in the sample. For example, the process which decomposes | disassembles ascorbic acid which is an interfering substance using ascorbate oxidase is mentioned.
The pretreatment liquid contains a peroxide. The amount of peroxide may be any concentration that reduces bilirubin, hemolytic reducibility, and color change, and is usually 0.4 nmol / L or more in the pretreatment step, but considers reducing the reagent blank reaction. Then, 0.4 nmol / L to 5 nmol / L or less is preferable, and more preferably 0.5 to 2 nmol / L. If there is a concern about an increase in reagent blank due to the concentration of the substance to be measured, the presence of unreacted peroxide with reducing substances (bilirubin, hemolysis, etc.) in the pretreatment process can be prevented by allowing an antioxidant to coexist in the reagent. It can be activated and the rise of the reagent blank can be suppressed. The inactivation of the reducing substance (bilirubin, hemolysis, etc.) and unreacted residual peroxide is preferably carried out before the addition of the detection start reagent, and the detection start reagent may not contain any peroxide. This is desirable from the viewpoint of suppressing the rise of the reagent blank. The peroxide concentration in the detection initiation reagent is usually 1.5 nmol / L or less, preferably 1.0 nmol / L or less.

  The pretreatment can be performed by adding peroxide to the biological sample and treating at about 20 to 40 ° C. for about 2 to 10 minutes.

  The detection initiation reagent of the present invention means a reagent that leads to generation of a detection substance such as NADH, CNP, PNP, and quinone dye.

  The peroxide used for this invention will not be specifically limited if it is a peroxide except hydrogen peroxide, An organic peroxide is illustrated preferably.

  Organic peroxide (Organic peroxide is generally a derivative of hydrogen peroxide, that is, a compound in which the hydrogen atom of H—O—O—H is replaced by an organic atomic group, and its chemical characteristics are —O—O—. For example, the chemical structure is classified as follows. Hydroperoxide (R—O—O—H), dialkyl peroxide (R—O—O—R), peroxy ester (RCO—O—O—R ′), diacyl peroxide (RCO—O—O—) COR), peroxydicarbonate (ROCO—O—O—COOR), peroxyketal (R—O—O—C (—R ′) (— R ″) — O—O—R), ketone peroxide (H—O—O—C (—R ′) (— R ″) — O—O—H). In the present invention, lipid peroxide is preferably used.

  Further, the surfactant does not originally contain a peroxide and does not chemically change under proper distribution and storage. However, there are those that can cause peroxides by causing a chemical reaction by intentionally changing the environment. For example, by performing treatment such as coexistence with trace metals or irradiation with light, Can be generated. In the case of the treatment with a trace metal, the light irradiation treatment is more preferable because it is difficult to remove the metal after the treatment. Peroxides obtained from such surfactants can also be used in the present invention.

  In addition, when the surfactant is out of the quality control range of the manufacturer and distributor, for example, when the storage conditions are not appropriate for the user, a trace amount of peroxide may be generated. A surfactant obtained by further generating a peroxide can also be used.

  A specific processing method using light is as follows. For example, 10 g of a surfactant is put in a commercially available transparent test tube and sealed, and conditions for irradiation of 500 lux light at 10 ° C. for 2 weeks include the container of the irradiated sample, light source, illuminance, temperature, time, etc. Affects the amount of peroxide produced, so the amount of peroxide can be managed by managing these conditions. The container is preferably a transparent glass material with a lid in consideration of contamination of impurities, and it is desirable to irradiate the substance to be treated in a solid or flake form in an aqueous solution state. The light source is preferably a fluorescent lamp and white or D-65 daylight color is used. The illuminance is preferably about 300 to 1000 lux, the temperature is 2 to 30 ° C., and the time is preferably about 3 days to 3 weeks. These conditions are intended to treat the substance to be treated as mildly as peroxide is generated, and do not assume that the substance to be treated completely loses its original action.

  The peroxide obtained by these methods is difficult to identify as a substance, but the amount of peroxide can be measured by the method described later. Good. The kind of surfactant to be used is not particularly limited.

  As the surfactant, a nonionic surfactant and / or a zwitterionic surfactant is preferably used.

  Nonionic surfactants used in the present invention include polyoxyethylene lauryl ethers such as Emulgen 104P, Emulgen 105, Emulgen 106, Emulgen 108, Emulgen 109P, Emulgen 120, Emulgen 123P, Emulgen 147, Emulgen 130K, Nonion K- 204, Nonion K-215, Nonion K-220, Nonion K-230, NIKKOL BL-2, NIKKOL BL-4.2, NIKKOL BL-9EX, NIKKOL BL-21, NIKKOL BL-25, polyoxyethylene cetyl ethers Emulgen 210, Emulgen 220, NIKKOL BC-2, NIKKOL BC-5.5, NIKKOL BC-7, NIKKOL BC-10TX, NIKKOL B -15TX, NIKKOL BC-20TX, NIKKOL BC-23, NIKKOL BC-25TX, NIKKOL BC-30TX, NIKKOL BC-40TX, nonionic P-208, nonionic P-210, nonionic stearyl P-213, , Emulgen 306P, Emulgen 320P, NIKKOL BS-2, NIKKOL BS-4, NIKKOL BS-20, Nonion S-206, Nonion S-207, Nonion S-215, Nonion S-220, Polyoxyethylene oleyl ethers , Emulgen 404, Emulgen 408, Emulgen 409P, Emulgen 420, Emulgen 430, NIKKOL BO-2, NIKKOL BO-7, NIKKOL BO-10TX NIKKOL BO-20, NIKKOL BO-50, nonion E-206, nonion E-215, nonion E-230, and polyoxyethylene behenyl ethers include NIKKOL BB-5, NIKKOL BB-10, NIKKOL BB-20, NIKOL BB-20, NIKOL BB-30 etc. are mentioned.

  Examples of the polyoxyethylene secondary alkyl ethers include Emulgen 707, NIKKOL BT-5, NIKKOL BT-7, NIKKOL BT-9, Adekatol SO-80, Adecator SO-105, Adecator SO-120, Adecator SO-135. , Adekatol SO-145, Adekatol SO-160, Emulgen 705, Emulgen 707, Emulgen 709 and the like. Examples of polyoxyethylene alkylphenyl ethers include Emulgen 810, Emulgen 840S, Emulgen 909, Emulgen 910, Emulgen 930, Emulgen 950, Triton X-100, Triton X-114, NIKKOL NP-5, NIKKOL NP-7.5, NIKKOL NP-10, NIKKOL NP-15, NIKKOL NP-20, NIKKOL OP-10, NIKKOL OP-30, and the like. Examples of the oxyethylene / oxypropylene block copolymers include Emulgen PP-150, Emulgen PP-230, Emulgen PP-250, Emulgen PP-290, NIKKOL PBC-34, NIKKOL PBC-44, and the like. Examples of fatty acid esters include Leodol TW-L120, Leodol TW-L106, Leodol TW-P120, Leodol TW-S120, Leodol TW-O120, Leodol 460, Emanon 1112, Emanon 3115, Emanon 3170, Emanon 3299, Emanon 3130, etc. Can be mentioned. Examples of the polyoxyethylene sterols include NIKKOL BPS-10, NIKKOL BPS-20, NIKKOL BPS-30, NIKKOL BPSH-25, and NIKKOL DHC-30. In addition, n-octyl-β-D-glucoside, n-dodecyl-β-D-maltoside, n-octanoyl-N-methylglucoamide, n-nonanoyl-N-methylglucoamide, n-decanoyl-N- Examples include methylglucoamide, sucrose monocaprate, sucrose monolaurate, sucrose monocholate, digitonin and the like.

  Examples of the zwitterionic surfactant used in the present invention include alkylimidazolium betaine, alkylbetaine, alkylamidobetaine, alkylalanine, alkylamine oxide, and derivatives thereof. Specific examples of these include Amphithr 20BS, Amphithr 24B, Amphithr 86B, Amphithr 20Z, and the like.

The measurement of peroxide can be performed by the method described in Non-Patent Document 1, for example.
Fresenius J Anal Chem 365, 448-451 (1999) Alternatively, a commercially available peroxide measurement reagent can be used. (For example, see Non-Patent Document 2.) In-vitro diagnostic drug Determiner LPO (Kyowa Medex Co., Ltd., approval number (62AM) 0669) package insert, revised in March 2002 The antioxidant used in the present invention is not particularly limited, but is selected according to the characteristics of the measurement principle For example, α-tocopherol, thioglycerol, butylhydroxytoluene, methionine, and oxidase include catalase, glutathione peroxidase, superoxide dismutase, ascorbate peroxidase, NADH oxidase, alkyl hydroperoxidase, ascorbate oxidase, etc. .

  The amount of the antioxidant used is about 0.001 to 1% by weight, preferably about 0.001 to 0.1% by weight.

  The biological component measuring reagent of the present invention may contain an iron complex such as a preservative, a chelating agent, and a ferrocyanide.

Hereinafter, the present invention will be specifically described by way of examples. In addition, this invention is not specifically limited by an Example.
(Examples 1-4 and Comparative Examples 5-6)
Peroxides were added at different concentrations to the following creatinine measurement reagents. The peroxide was generated by putting 10 g of Triton X-100 in a transparent test tube and sealing it, and radiating 500 lux light at 10 ° C. for 2 weeks. The peroxide concentration in Triton X-100 was 0.07 μmol / g before the operation, but 5 μmol / g after the operation. The peroxide concentration level was prepared by mixing and preparing Triton X-100 before and after the operation, and added to the first or second reagent of the following creatinine measuring reagent so that the Triton X-100 concentration would be 1 g / L. After the preparation, the final concentrations of the peroxide of the first reagent and the second reagent of each reagent were measured. The peroxide concentration was measured using a commercially available lipid peroxide measuring reagent “Determiner LPO” (manufactured by Kyowa Medex Co., Ltd.) according to the dosage of this kit.

Using the thus prepared creatinine measurement reagent, purified water, bilirubin C (conjugated), bilirubin F (free form), and hemolysis (both Sysmex Inc., Interference Check A Plus) were used as samples. Pooled sera added to dL, 40 mg / dL, and 500 mg / dL, and pooled sera without addition of bilirubin C, bilirubin F, and hemolysis were used as controls. The measurement results of these samples were obtained by using purified water measurement absorbance as a reagent blank, subtracting the additive serum measurement value from the measurement value of bilirubin C, bilirubin F, and hemolyzed serum, and taking the measurement value of the additive serum as 100%. Relative% was calculated and used as the degree of influence of each reducing substance.
(Preparation of reagents)
A creatinine measuring reagent having the following composition was prepared.
First reagent PIPES-NaOH 80 mM pH 7.5
Ascorbate oxidase (ASO-311 manufactured by Toyobo Co., Ltd.) 5 U / mL
Sarcosine oxidase (SAO-351, Toyobo Co., Ltd.) 10 U / mL
Creatine amidinohydrolase (CRH-221, manufactured by Toyobo Co., Ltd.) 30 U / mL
Catalase (Toyobo CAO-509) 100 U / mL
TOOS 0.2g / L
Second reagent PIPES-NaOH 80 mM pH 7.5
Creatinine amide hydrolase (Toyobo CNH-311) 300 U / mL
Peroxidase (Toyobo PEO-301) 10 U / mL
Potassium ferrocyanide 50mg / L
(Measurement method)
A Hitachi 7170 automatic analyzer was used. 180 μL of the first reagent was added to 4 μL of the sample and incubated at 37 ° C. for 5 minutes to form the first reaction. Thereafter, 60 μL of the second reagent was added and incubated for 5 minutes to form a second reaction. Absorbance at 546 nm was measured by a two-point end method in which the absorbances of the first reaction and the second reaction were corrected for the liquid volume and the difference between the respective absorbances was taken.
The creatinine concentration of the sample with unknown creatinine concentration was calculated from the measured absorbance of purified water and a 5 mg / dL creatinine aqueous solution.

Results are shown in Table 1. In the comparative example, the effect of rising reagent blanks, bilirubin, and hemolysis increased, whereas in the examples, the reagent blanks were low, and the effects of bilirubin and hemolysis were reduced.
(Examples 7 to 10 and Comparative Examples 11 to 12)
Peroxides were added at different concentrations to the following neutral fat measurement reagent. The peroxide was generated by the procedure of Example 1 using Emulgen 108. As a result, the peroxide concentration in Emulgen 108 was 0.06 μmol / g before this operation, but became 3.4 μmol / g after the operation. The peroxide concentration level was prepared by mixing and preparing Emulgen 108 before and after the operation, and added to the first reagent or second reagent of the following neutral fat measurement reagent so that the concentration of Emulgen 108 was 1 g / L. After the preparation, the final concentrations of the peroxide of the first reagent and the second reagent of each reagent were measured.

Using the neutral fat measurement reagent thus prepared, the same sample as in Example 1 was measured, and the effects of reagent blank, bilirubin C, bilirubin F, and hemolysis were examined.
(Preparation of reagents)
Each neutral fat measurement reagent having the following composition was prepared.
First reagent PIPES 50 mM pH 7.0
MgCl2 0.2g / L
Adenosine triphosphate 2Na salt 1.2g / L
4-Aminoantipyrine 0.1g / L
Flavin adenine dinucleotide 2Na salt 8 μmol / L
Glycerol kinase (Toyobo GYK-311) 3 U / mL
Glycerophosphate oxidase (G3O-311 manufactured by Toyobo Co., Ltd.) 5 U / mL
Ascorbate oxidase (ASO-311 manufactured by Toyobo Co., Ltd.) 3 U / mL
Catalase (Toyobo) 200 U / mL
Second reagent HEPES-NaOH 50 mM pH 7.5
Magnesium chloride hexahydrate 0.2g / L
Calcium chloride 0.1g / L
ADPS 0.3g / L
Peroxidase (Toyobo PEO-301) 2.9 U / mL
Lipase (Toyobo LPL-314) 2U / mL
Potassium ferrocyanide 5mg / L
(Measurement method)
A Hitachi 7170 automatic analyzer was used. The first reaction was carried out by adding 180 μL of the first reagent to 2.1 μL of the sample and incubating at 37 ° C. for 5 minutes. Thereafter, 90 μL of the second reagent was added and incubated for 5 minutes to form a second reaction. Absorbance at 600 nm was measured by a two-point end method in which the absorbances of the first reaction and the second reaction were corrected for the liquid volume and the difference between the respective absorbances was taken.
The result was calculated from the measured absorbance of purified water and 200 mg / dL triolein aqueous solution, and obtained as the neutral fat concentration.

Results are shown in Table 2. In the comparative example, an increasing tendency of the reagent blank and an increase in the influence of hemolysis were observed, whereas in the example, the reagent blank was low and the influence of hemolysis was hardly observed. .
Examples 13-16 and Comparative Examples 17-18
Peroxides were added at different concentrations to the following calcium measurement reagents. The peroxide was generated according to the procedure of Example 1 using polyoxyethylene oleyl ether. As a result, the peroxide concentration in polyoxyethylene oleyl ether was 0.0 μmol / g before this operation, but became 2.2 μmol / g after the operation. Prepare a peroxide concentration level by mixing and preparing polyoxyethylene oleyl ether before and after the operation, and add it to the first or second reagent of the following calcium measurement reagent to a polyoxyethylene oleyl ether concentration of 1 g / L did. After the preparation, the final concentrations of the peroxide of the first reagent and the second reagent of each reagent were measured.
Using the calcium measurement reagent thus prepared, the same sample as in Example 1 was measured, and the effects of reagent blank, bilirubin C, bilirubin F, and hemolysis were examined.
First reagent Tris-HCl buffer (pH 7.1) 50 mM
NaCl 200 mM
1,2-bis (o-aminophenoxy) ethanetetraacetic acid 0.8 mM
Galactosyl maltose 2.3 mM
2-Chloro-4-nitrophenyl-4-o-β-D-galactopyranosyl-α-maltoside 0.9 mM
Second reagent Good buffer (pH 6.0) 300 mM
NaCl 200 mM
Galactosyl maltose 2.3 mM
Inactivated α-amylase (derived from human saliva) 20 IU / mL
(Measurement method)
A Hitachi 7170 automatic analyzer was used. After adding 180 μl of the first reagent to 3.5 μl of the sample volume and preheating for 5 minutes, 90 μl of the second reagent is further added to start the reaction. The change in absorbance was determined, and the amount of calcium in the sample was determined based on a 2-inspection curve with purified water and a 10 mg / dl standard solution of calcium. The measurement wavelength was a main wavelength of 405 nm and a sub wavelength of 546 nm, and the measurement temperature was 37 ° C.

Results are shown in Table 3. In the comparative example, the influence of hemolysis was large, whereas in the example, there was almost no influence.

  The biological component measuring reagent and measuring method of the present invention can be used in fields of application such as in vitro diagnostic drugs and contribute greatly to the industry.

Claims (4)

In a method for measuring a component in a biological sample,
(1) a step of pretreating a biological sample in the presence of 0.4 nmol / L or more peroxide; and (2) a pretreatment in step (1) in the presence of 1.5 nmol / L or less peroxide. A method comprising a step of measuring a component in a given biological sample. The method according to claim 1, wherein the pretreatment step (1) is carried out in the presence of 0.4 nmol / L or more peroxide and an antioxidant. A biological component measurement reagent comprising a pretreatment liquid for a biological sample containing 0.4 nmol / L or more peroxide and a biological component detection start liquid containing 1.5 nmol / L or less peroxide. The measurement reagent according to claim 3, wherein the pretreatment liquid further contains an antioxidant.