JP7298151B2 - How to reduce measurement error - Google Patents

Description

The present invention relates to a method for reducing measurement errors in biochemical test reagents.

In the field of biochemical testing in clinical testing, absorptiometry is widely used to quantitatively analyze the concentration of a substance by measuring the absorbance of a reaction solution obtained by mixing a sample such as serum with a desired reagent. It is In order to perform this analysis simply and quickly, an automatic biochemical analyzer is generally used in the field of clinical examination. This type of automated biochemical analyzer usually includes containers for storing samples and reagents, reaction cells for storing samples and reagents, a pipetting mechanism for automatically injecting samples and reagents into the reaction cells, and samples and reagents in the reaction cells. Equipped with an automatic stirring mechanism for mixing, a mechanism for measuring the spectroscopic spectrum of the sample during or after the reaction, and an automatic washing mechanism for aspirating and discharging the reaction solution after spectroscopic measurement is completed and washing the reaction cell (for example, Patent document 1).

Here, a mechanism for automatically dispensing reagents into reaction cells in such an automatic biochemical analyzer is generally called a dispensing nozzle, dispensing probe, reagent probe, etc., and is generally made of metal (for example, stainless steel). It consists of a shaped structure and requires regular maintenance. However, since all the reagents are dispensed with a single reagent probe, the reagent remains inside the cylindrical structure, etc., and when the next reagent is inhaled, it is mixed in the cylindrical structure, resulting in a mixture of the reagents. is dispensed into the reaction cell and used for analysis, there is an unavoidable problem due to the structure of the apparatus that the analysis may not be performed accurately. Such a problem is so-called contamination, which is regarded as a problem particularly in clinical practice because it causes measurement errors.

Various avoidance methods are known as countermeasures on the device side. For example, in order to measure various components of a plurality of samples, in an automatic analyzer that continuously dispenses a plurality of samples or reagents using a nozzle, the inner wall of a conductive tube is coated with polytetrafluoroethylene. An automatic analyzer (Patent Literature 2) is known, which is characterized by using a dispensing nozzle that has been designed for the purpose. In addition, as another method, a method for enhancing the cleaning function (for example, Patent Document 3) is disclosed. However, even if such countermeasures are taken, the coating peels off due to aged deterioration and cleaning conditions of the analyzer, and reagents or components tend to adhere to the inner wall of the reagent probe, and contamination frequently occurs over time.

On the other hand, as an operational countermeasure, when the occurrence of contamination is confirmed, a cleaning cycle is specified between combinations in which contamination occurs, and cleaning is performed. and is not a good idea. A method for changing the dispensing order of reagents (for example, Patent Document 4) has also been disclosed, but it cannot be implemented if the user of the device is not sufficiently skilled, and some devices do not have such a function in the first place. It is not included and may not be implemented.

Japanese Patent Publication No. 3-75827 JP-A-11-271329 Japanese Utility Model Laid-Open No. 5-50362 JP-A-5-126836

The problem to be solved by the present invention is to provide a measurement error reduction method and a reagent with reduced measurement error that reduces contamination with a metal reagent probe, suppresses the influence on other reagents, and obtains accurate analytical values. to do.

As a result of intensive studies to achieve the above object, the present inventors found that contamination in reagent probes can be suppressed by adding a certain surfactant to biochemical test reagents. , completed the present invention. That is, the present invention consists of the following configurations.

[Section 1]
A method for reducing measurement errors caused by using an enzyme-containing biochemical test reagent in an automatic biochemical analyzer equipped with a mechanism for dispensing different reagents with the same metal reagent probe, comprising at least one A method for reducing measurement errors, characterized by using a nonionic surfactant of
[Section 2]
The nonionic surfactant includes polyoxyethylene octylphenyl ether, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene alkyl ether, polyoxyethylene distyrenated phenyl ether, and polyoxyethylene Item 2. The method for reducing measurement error according to item 1, wherein the at least one nonionic surfactant is selected from the group consisting of ethylene tribenzylphenyl ether.
[Section 3]
Item 3. The method for reducing measurement error according to Item 1 or 2, wherein at least part of the reagent probe is made of stainless steel.
[Section 4]
Item 4. The method for reducing measurement error according to any one of Items 1 to 3, wherein the enzyme is cholesterol oxidase.
[Section 5]
Item 5. The method for reducing measurement error according to any one of Items 1 to 4, wherein the biochemical test reagent is a high-density lipoprotein cholesterol measurement reagent.
[Section 6]
Item 6. The method for reducing measurement error according to any one of Items 1 to 5, wherein the enzyme concentration in the biochemical test reagent is 0.01 to 30 U/mL.
[Section 7]
Item 7. The method for reducing measurement error according to any one of Items 1 to 6, wherein the biochemical test reagent has a pH of 5 to 10.
[Item 8]
The nonionic surfactant is Triton (registered trademark) X-100, Triton (registered trademark) X-114, Tween (registered trademark) 20, Tween (registered trademark) 80, Emulgen (registered trademark) 404, Emulgen (registered trademark) Emulgen® 408, Emulgen® 409PV, Emulgen® 420, Emulgen® 430, Emulgen® A60, Emulgen® A90, Emulgen® A500, and Emulgen® ( Item 8. The method according to any one of Items 1 to 7, wherein the surfactant is at least one nonionic surfactant selected from the group consisting of B66.
[Item 9]
A biochemical test reagent that reduces measurement errors that occur when used in a biochemical automatic analyzer equipped with a mechanism for dispensing different reagents with the same metal reagent probe, the reagent containing at least one kind of non-ion together with an enzyme. A biochemical test reagent, characterized by containing a surfactant.
[Item 10]
The nonionic surfactant includes polyoxyethylene octylphenyl ether, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene alkyl ether, polyoxyethylene distyrenated phenyl ether, and polyoxyethylene 10. The biochemical test reagent according to Item 9, which is at least one nonionic surfactant selected from the group consisting of ethylene tribenzylphenyl ether.
[Item 11]
Item 11. The reagent according to Item 9 or 10, wherein at least part of the reagent probe is made of stainless steel.
[Item 12]
Item 12. The reagent according to any one of items 9 to 11, wherein the biochemical test reagent contains cholesterol oxidase as the enzyme.
[Item 13]
Item 13. The reagent according to any one of Items 9 to 12, wherein the biochemical test is a cholesterol measurement test.
[Item 14]
Item 14. The reagent according to any one of Items 9 to 13, wherein the biochemical test reagent is a high-density lipoprotein cholesterol measurement reagent.
[Item 15]
Item 15. The reagent according to any one of Items 9 to 14, wherein the biochemical test reagent has an enzyme concentration of 0.01 to 30 U/mL.
[Item 16]
Item 16. The reagent according to any one of Items 9 to 15, wherein the biochemical test reagent has a pH of 5 to 10.
[Item 17]
The nonionic surfactant is Triton (registered trademark) X-100, Triton (registered trademark) X-114, Tween (registered trademark) 20, Tween (registered trademark) 80, Emulgen (registered trademark) 404, Emulgen (registered trademark) Emulgen® 408, Emulgen® 409PV, Emulgen® 420, Emulgen® 430, Emulgen® A60, Emulgen® A90, Emulgen® A500, and Emulgen® ( Item 17. The reagent according to any one of items 9 to 16, which is at least one nonionic surfactant selected from the group consisting of B66.

According to the present invention, it is possible to provide a biochemical test reagent that can easily avoid contamination with a metal reagent probe, and even when used in a biochemical analyzer that has been used for a long time or under severe conditions, other reagents It is possible to obtain accurate measurement values without causing measurement errors.

One of the embodiments of the present invention is the measurement error caused by using an enzyme-containing biochemical test reagent in an automatic biochemical analyzer equipped with a mechanism for dispensing different reagents with the same metal reagent probe. is a method for reducing measurement error, characterized by using at least one nonionic surfactant. It can be presumed that the present invention can reduce measurement errors by suppressing contamination caused by enzymes contained in biochemical test reagents adsorbing and remaining on metal reagent probes. Therefore, from another aspect, the present invention provides a method for reducing measurement error when using an enzyme-containing biochemical test reagent, which comprises using at least one nonionic surfactant to reduce biochemical autoanalysis. It can also be said to be a method for reducing measurement errors, characterized by suppressing adsorption of the enzyme to a metal reagent probe in the device.

A further embodiment of the present invention is a biochemical test in which measurement errors that occur when used in a biochemical automatic analyzer equipped with a mechanism for dispensing different reagents with the same metal reagent probe are reduced. A reagent for biochemical testing, characterized in that it contains at least one nonionic surfactant along with an enzyme. It can be presumed that the present invention can reduce measurement errors by suppressing contamination caused by enzymes contained in biochemical test reagents adsorbing and remaining on metal reagent probes. Therefore, from another point of view, the present invention suppresses the adsorption of an enzyme to a metallic reagent probe in an automatic biochemical analyzer, characterized by containing at least one nonionic surfactant together with the enzyme. It can also be said that it is a biochemical test reagent that reduces the occurrence of measurement errors.

In the present invention, in order to reduce the measurement error that occurs when used in a biochemical automatic analyzer equipped with a mechanism for dispensing different reagents with the same metal reagent probe, or to reduce the adsorption of enzymes to the metal reagent probe At least one kind of nonionic surfactant is used to suppress In this specification, the use of a nonionic surfactant means that in the operation of a biochemical automatic analyzer using an enzyme-containing biochemical test reagent, a nonionic surfactant is used so as to come into contact with the metal reagent probe. There is no particular limitation as long as it uses a surfactant. For example, at least one nonionic surfactant may be contained in the solution that contacts the metal reagent probe. It is preferable to contain a surfactant and allow both components to coexist. For example, when the biochemical test reagent is composed of a plurality of reagents (for example, when composed of a first reagent and a second reagent, or a first reagent, a second reagent, and a third reagent), the metal reagent probe When the first reagent contains an enzyme that tends to adsorb and cause contamination, it is preferable to add at least one kind of nonionic surfactant to the first reagent so that both components coexist. When the second reagent or the third reagent contains an enzyme that easily causes contamination, they can be coexisted in the same way.

The nonionic surfactant used in the present invention is not particularly limited as long as the effects of the present invention are exhibited. For example, polyoxyethylene-based nonionic surfactants, sorbitan fatty acid ester-based nonionic surfactants, and glycerin fatty acid ester-based nonionic surfactants can be used. Preferably, a polyoxyethylene-based nonionic surfactant is used.
The HLB value of the nonionic surfactant used in the present invention is also not particularly limited as long as the effects of the present invention are exhibited. For example, a nonionic surfactant with an HLB of about 10 to 20, preferably a nonionic surfactant with an HLB of about 12 to 18 can be used.

One of the embodiments of the present invention is polyoxyethylene octylphenyl ether, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene alkyl ether, polyoxyethylene oleyl ether, polyoxyethylene distyrene. Adsorption of an enzyme to a metal reagent probe, characterized by using at least one polyoxyethylene-based nonionic surfactant selected from the group consisting of triphenyl ether and polyoxyethylene tribenzylphenyl ether. It is a method for reducing measurement errors of biochemical test reagents by suppressing .
Measurement errors due to adsorption of reagents to probes in biochemical automatic analyzers are also called reagent contamination. Therefore, the present invention can also be said to be a method for avoiding contamination, characterized by using the nonionic surfactant as described above.

Also, one embodiment of the present invention is a biochemical test reagent using the present invention. Biochemical tests are tests that are used, along with blood tests and general tests, for differentiating diseases, monitoring treatment, determining prognosis, and the like. Serum is generally used as a test material, but depending on the purpose of the test, plasma, urine, pleural/ascites fluid, cerebrospinal fluid, blood cells, etc. are used and chemically analyzed. Inspection items (substances to be analyzed) include various enzymes, electrolytes/metals, proteins, nitrogen-containing components, lipids, and sugar-related substances. As various enzymes, AST (GOT), ALT (GPT), LDH (lactate dehydrogenase) and isozyme, ALP (alkaline phosphatase) and isozyme, CK (creatine kinase) and isozyme, amylase (Amy) and isozyme, lipase, γ -GTP (γ-glutamyltranspeptidase), cholinesterase (ChE) and the like. As electrolytes and metals, sodium (Na), potassium (K), chloride (Cl), calcium (Ca), phosphorus (P) [inorganic phosphorus (IP)], iron (Fe), magnesium (Mg), etc. mentioned. Proteins include total protein (TP), serum protein fraction (PF) and the like. Nitrogen-containing components include urea nitrogen (BUN), creatinine (Cr), uric acid (UA), bilirubin (Bil), and ammonia. Lipids include cholesterol, HDL-cholesterol (HDL-C, also called high-density lipoprotein cholesterol), LDL-cholesterol (LDL-C, also called low-density lipoprotein cholesterol), triglycerides (TG), and the like. be done. Sugar-related substances include blood sugar (BS, GLU) and glycated hemoglobin (HbA1c). Such reagents for performing biochemical tests are called biochemical test reagents, and are sold as liquid or freeze-dried products. Generally used in liquid form. A liquid one may also be used by appropriately diluting it with a solvent such as water before use.

The biochemical test reagent using the present invention can be a biochemical test reagent used for any of the above test items. It is more preferably used as a reagent, more preferably used as a high-density lipoprotein cholesterol measuring reagent and/or a low-density lipoprotein cholesterol measuring reagent, and particularly used as a high-density lipoprotein cholesterol measuring reagent. preferable. Biochemical test reagents for measuring lipids generally often contain components (such as enzymes) that have fat-soluble moieties. According to the present invention, measurement errors are reduced even in biochemical test reagents for measuring lipids that may contain components having fat-soluble parts that are likely to cause measurement errors by being adsorbed to metal reagent probes. It is possible to

A biochemical analyzer is a device that uses biochemical test reagents, and is not particularly limited, but includes a container for storing samples and reagents, a reaction cell for storing samples and reagents, and a reaction cell for storing samples and reagents. A dispensing mechanism that automatically injects into the reaction cell, an automatic stirring mechanism that mixes the sample and reagents in the reaction cell, a mechanism that measures the spectroscopic spectrum of the sample during or after the reaction, and aspirates and discharges the reaction solution after spectroscopic measurement. It is composed of an automatic cleaning mechanism that cleans the reaction cell. These biochemical automatic analyzers are also capable of measuring a plurality of different measurement items together. When measuring a plurality of different measurement items, the process of setting containers containing different reagents respectively, sucking and discharging from the containers containing these different reagents with the same probe, and distributing them to the reaction cells is repeated. Analysis may be performed. The present invention is preferably carried out in such a manner that different reagents are dispensed with the same metallic reagent probe.

Among the mechanisms for aspirating reagents and automatically dispensing them into reaction cells, the nozzle portion is particularly called a reagent probe. The reagent probe is an interface with the apparatus that comes into direct contact with the reagent, such as for sucking and discharging the reagent, and is generally a tubular structure. As the material of the reagent probe, metal is usually used in many cases. Among others, stainless steel is often used as the material of the reagent probe, but it is not particularly limited. Although the present invention is practiced in biochemical tests using reagent probes made of metal, the reagent probe need not be entirely made of metal, and at least a portion of the surface that may come into contact with the reagent is made of metal. If it is In certain embodiments, the present invention may be suitably practiced in biochemical assays using reagent probes in which at least a portion of the surface that may come into contact with the reagent is made of stainless steel. In addition, the inner wall of the reagent probe may be coated to prevent contamination, and the material thereof may be polytetrafluoroethylene, but is not particularly limited.

In addition, reagent probes are used in various environments. For example, the usage environment is not particularly limited, but the coating may peel off over time, and the liquid may easily be adsorbed on the surface of the metal reagent probe such as the inner wall. Furthermore, due to improper washing, proteins such as enzymes and polymer compounds such as polystyrene particles may remain on the surface of the reagent probe such as the inner wall. Also, normally it is desirable to replace with a new one once every 1-2 years due to regular maintenance, but in developing countries, for reasons such as cost savings and lack of engineers who can perform maintenance. Without regular replacement, the coating may peel off, exposing the metal inside, or the metal parts may be rusted.

Contamination in the present specification is not particularly limited, but after aspirating and discharging the first reagent with a metal (for example, stainless steel) reagent probe, a second separate reagent with the same reagent probe , the first reagent adsorbed to the surface (including the inner surface) of the reagent probe is absorbed into the surface of the reagent probe, the reaction cell, or the reagent bottle in the first reagent and the second reagent. It refers to the state in which the first reagent is mixed. It is known that measurement errors occur when the first reagent and the second reagent are mixed in this manner.

"Reduction of measurement error" or "avoidance of contamination" in the present invention means that, for example, after the reagent probe aspirates and discharges the first reagent containing the contents of the present invention, two A state in which the measured value obtained with the second different reagent is less than ±5% from the value that should have been originally obtained when another reagent is aspirated and discharged. preferably less than ±4%, more preferably less than ±3%, even more preferably less than ±2%, even more preferably less than ±1% from the value that should have been originally obtained; Particularly preferably, it is less than ±0.5%. A measurement error from a value that should have been obtained originally can be measured in the same manner as in the test examples described later.

The type of nonionic surfactant used in the present invention is not particularly limited. As the nonionic surfactant, for example, the following commercially available products can also be suitably used.
Examples of polyoxyethylene octylphenyl ethers include Triton X-100 and Triton X-114 (“Triton” is a registered trademark. The same shall apply hereinafter).
For example, polyoxyethylene sorbitan monolaurate includes Tween 20 ("Tween" is a registered trademark. The same shall apply hereinafter).
For example, polyoxyethylene sorbitan monooleate includes Tween 80.
For example, polyoxyethylene alkyl ethers include polyoxyethylene lauryl ether, polyoxyethylene cetyl ether, polyoxyethylene stearyl ether, polyoxyethylene oleyl ether, polyoxyethylene myrister ether, or polyoxyethylene octyldodecyl ether. . Furthermore, as polyoxyethylene lauryl ether, Kao Emulgen 103 (“Emulgen” is a registered trademark. The same shall apply hereinafter.), Emulgen 104P, Emulgen 105, Emulgen 106, Emulgen 108, Emulgen 109P, Emulgen 120, Emulgen 123P, Emulgen 130K, and Emulgen 147. or Emulgen 150, polyoxyethylene cetyl ethers such as Emulgen 210P or Emulgen 220, polyoxyethylene stearyl ethers such as Emulgen 306P, Emulgen 320P or Emulgen 350, polyoxyethylene oleyl ethers such as Emulgen 404, Emulgen 408, Emulgen 409PV, Emulgen 420, or Emulgen 430; polyoxyethylene myrister ethers such as Emulgen 4085; polyoxyethylene octyldodecyl ethers such as Emulgen 2020G-HA or Emulgen 2025G; Oxyethylene alkyl ethers include Emulgen 705, Emulgen 707, Emulgen 709, Emulgen 1108, Emulgen 1118S-70, Emulgen 1135S-70, or Emulgen 1150S-60.
For example, polyoxyethylene distyrenated phenyl ethers include Emulgen A60, Emulgen A90 or Emulgen A500.
For example, polyoxyethylene tribenzylphenyl ether includes Emulgen B66.

Among them, the nonionic surfactants are Triton X-114 (polyoxyethylene octylphenyl ether, HLB 12.3), Tween 20 (polyoxyethylene sorbitan monolaurate, HLB 16.7), Tween 80 (polyoxyethylene sorbitan monolaurate). oleate, HLB 15.0), Emulgen 420 (polyoxyethylene oleyl ether, HLB 13.6), Emulgen A60 (polyoxyethylene distyrenated phenyl ether, HLB 12.8), Emulgen A90 (polyoxyethylene distyrenated phenyl ether , HLB 14.5), and Emulgen B66 (polyoxyethylene tribenzylphenyl ether, HLB 13.2).

The concentration of the nonionic surfactant in the solution (in the solution after dissolution/dilution if dissolved/diluted before use) is not particularly limited, but the upper limit of the range is the solution is preferably 20 g/L, more preferably 10 g/L, and more preferably 5 g/L, relative to the total amount of The lower end of the range is preferably used at 0.01 g/L, more preferably 0.05 g/L, and even more preferably 0.1 g/L of the total amount of the solution. When two or more nonionic surfactants are used to reduce measurement errors, the total amount of these nonionic surfactants is preferably within the above concentration range.

Moreover, in the present invention, two or more types of nonionic surfactants may be mixed and contained in the solution, and the mixing ratio is not particularly limited. Further, in the present invention, in addition to nonionic surfactants, other surfactants (anionic surfactants, cationic surfactants, amphoteric surfactants, etc.) impair the effects of the present invention. may be used together unless

In addition, the method of reducing measurement error or avoiding contamination of the present invention can be applied to various biochemical test reagents. Examples of biochemical test reagents include AST (GOT), ALT (GPT), LDH (lactate dehydrogenase) and isozyme, ALP (alkaline phosphatase) and isozyme, CK (creatine kinase) and isozyme, and amylase (Amy). and isozymes, lipase, γ-GTP (γ-glutamyl transpeptidase), various enzymes such as cholinesterase (ChE), sodium (Na), potassium (K), chloride (Cl), calcium (Ca), phosphorus (P) [ inorganic phosphorus (IP)], electrolytes and metals such as iron (Fe) and magnesium (Mg), proteins such as total protein (TP), serum protein fraction (PF), urea nitrogen (BUN), creatinine (Cr), Nitrogen-containing components such as uric acid (UA), bilirubin (Bil), ammonia, lipids such as cholesterol, HDL-cholesterol (HDL-C), LDL-cholesterol (LDL-C), triglycerides (TG), blood sugar (BS, GLU), glycated hemoglobin (HbA1c) and other sugar-related substances. Specifically, in the measurement error reduction method or the contamination avoidance method of the present invention, other components required for the biochemical test reagent may coexist. For example, the components constituting the neutral fat measurement reagent include nonionic surfactants as well as other components such as other surfactants, lipase, glycerol kinase, ATP, magnesium, peroxidase, and chromogens. All of these may coexist, or, in addition to the nonionic surfactant, necessary components may be appropriately selected from these other components and coexisted.

The present invention may also be a biochemical test reagent comprising any of the components described above or a composition composed thereof. Various biochemical test reagents are already established in the art. Therefore, according to a known method, the method for reducing measurement error or the method for avoiding contamination of the present invention, the composition with reduced measurement error or the composition that avoids contamination is applied to biochemical test reagents, and in various samples can measure the amount or concentration of the biological component. Its composition is not particularly limited as long as it contains a nonionic surfactant. In addition, the compositions of the present invention (including the forms contained in kits) may be aqueous solutions or lyophilized. Aqueous solutions can be used by appropriately diluting them with a solvent such as water before use. Lyophilized products are dissolved before use and finally used in liquid form.

Moreover, the biochemical test reagent to which the present invention is applied contains an enzyme. Accurate analysis is possible by containing the enzyme. A functional protein such as an antibody may further contain a compound for more accurate analysis. For example, in the field of biochemical testing, detection systems using enzymes are widely used, and in such cases, enzymes such as oxidase (oxidase), dehydrogenase (dehydrogenase), esterase, and peroxidase are used. Furthermore, these detection systems contain other enzymes and coloring agents. Detect by dye. Also, in a detection system using dehydrogenase (dehydrogenase), a coenzyme and a reduced dye are used.

The present invention is embodied in biochemical test reagents containing enzymes. Although the type of enzyme used in the present invention is not particularly limited, enzymes that act on fat-soluble components are preferred. Enzymes that act on fat-soluble components generally often have a fat-soluble portion. ADVANTAGE OF THE INVENTION According to this invention, even when using the enzyme which has a fat-soluble component which is easy to adsorb|suck to a metallic reagent probe, adsorption|suction to a probe can be suppressed effectively and a measurement error can be reduced. From such a point of view, the enzymes used in the present invention preferably include cholesterol oxidase (herein sometimes abbreviated as COO), cholesterol esterase (herein sometimes abbreviated as COE), Examples include lipoprotein lipase, more preferably cholesterol oxidase and cholesterol esterase, and still more preferably cholesterol oxidase. In one embodiment, the biochemical test reagent of the present invention preferably contains a combination of cholesterol oxidase and cholesterol esterase.

The source of enzymes used in such biochemical test reagents is not particularly limited. For example, in the cholesterol measuring reagent, cholesterol esterase and cholesterol oxidase are used as enzymes essential for measurement, and as the cholesterol esterase, for example, substances derived from microorganisms such as Schizophyllum and Pseudomonas can be used. As cholesterol oxidase, for example, substances derived from microorganisms such as Streptomyces and Pseudomonas can be used. It also includes those produced by gene recombination technology in which these genes are incorporated into microorganisms such as Escherichia coli, or those whose properties have been modified by gene modification or the like. These are commercially available.

The enzyme concentration in the biochemical test reagent used in the present invention is not particularly limited as long as the effects of the present invention can be exhibited. For example, the concentration of the enzyme in the solution (if it is dissolved/diluted before use, in the solution after dissolution/dilution; the same shall apply hereinafter) is not particularly limited, but the upper limit of the range is , preferably about 30 U/mL, more preferably about 25 U/mL. The lower limit of the range is preferably about 0.01 U/mL, more preferably about 0.1 U/mL, relative to the total amount of the solution. When cholesterol oxidase or cholesterol esterase is used as the enzyme, the preferred concentration of each enzyme is as follows.
(a) When cholesterol oxidase is used: the upper limit of the range is preferably about 5 U/mL, more preferably about 3 U/mL, relative to the total amount in the solution, and the lower limit of the range is relative to the total amount of the solution On the other hand, it is preferably about 0.3 U/mL, more preferably about 0.75 U/mL, still more preferably about 1.5 U/mL. It has been recognized that the higher the concentration of cholesterol oxidase, the more likely it is to adsorb to the metal reagent probe, which tends to cause measurement errors. According to the present invention, the measurement error can be effectively reduced even within the concentration range described above.
(b) When cholesterol esterase is used: The upper limit of the range is preferably about 3 U/mL, more preferably about 1 U/mL, and the lower limit of the range is preferably 0.3 U relative to the total amount of the solution. /mL, more preferably about 0.5 U/mL.

The present invention is practiced when using two or more different reagents for different test items. For example, AST (GOT), ALT (GPT), LDH (lactate dehydrogenase) and isozyme, ALP (alkaline phosphatase) and isozyme, CK (creatine kinase) and isozyme, amylase (Amy) and isozyme, lipase, γ-GTP (γ-glutamyltranspeptidase), cholinesterase (ChE), sodium (Na), potassium (K), chloride (Cl), calcium (Ca), phosphorus (P) [inorganic phosphorus (IP)], iron (Fe), Magnesium (Mg), total protein (TP), serum protein fraction (PF), urea nitrogen (BUN), creatinine (Cr), uric acid (UA), bilirubin (Bil), ammonia, cholesterol, HDL cholesterol (HDL-C , high-density lipoprotein-cholesterol), LDL-cholesterol (LDL-C, also known as low-density lipoprotein-cholesterol), triglycerides (TG), blood sugar (BS, GLU), glycated hemoglobin (HbA1c) Examples include, but are not limited to, two or more different reagents for two or more measurement items selected from a group. Preferably, one of the two or more measurement items is a high-density lipoprotein cholesterol (HDL-C) measurement reagent, and the other is a measurement reagent for measurement items other than HDL-C. In one preferred embodiment, the present invention can be practiced in a biochemical test using a high-density lipoprotein-cholesterol assay reagent and a triglyceride assay reagent. In another preferred embodiment, a biochemical test using a high density lipoprotein cholesterol measuring reagent and a uric acid measuring reagent can be performed. In the preferred embodiment described above, in addition to the combination of the high-density lipoprotein cholesterol measuring reagent and the triglyceride measuring reagent or the uric acid measuring reagent, reagents for measuring other test items may be used in combination. .

In addition, when a biological sample is to be analyzed, an enzyme such as ascorbate oxidase that is not directly involved in the detection system or a chelating agent such as EDTA should be added in order to avoid the effects of interfering substances contained in the biological sample. There is In addition, preservatives may be added to the extent that they do not adversely affect the reaction of the main enzyme. Examples of preservatives include azides, chelating agents, antibiotics, and antibacterial agents. Various metal ions such as Ca and Mg may coexist for enzyme stabilization.

The method for reducing measurement error or the method for avoiding contamination of the present invention is not particularly limited as long as the effect of the present invention is exhibited, but the pH of the composition is preferably in the range of 5 to 10, and the range of pH 6.0 to 8.0 is preferable. Especially preferred. As shown in the results of test examples described later, the pH of the solution of the biochemical test reagent used in the present invention tends to reduce measurement errors more effectively by increasing the pH. Therefore, by setting the pH of the solution of the biochemical test reagent of the present invention within the above range, the measurement error can be reduced more effectively. Moreover, as a buffer solution used so as to achieve the above pH range, a conventionally known buffer solution having a buffering capacity within the above pH range can be appropriately used. Such buffers include, for example, phosphoric acid, boric acid, tris(hydroxymethyl)aminomethane, imidazole, glycylglycine, MES, Bis-Tris, ADA, ACES, Bis-Tris propane, PIPES, MOPSO, MOPS , BES, HEPES, TES, DIPSO, TAPSO, POPSO, HEPPS (also known as EPPS), HEPPSO, Tricine, Bicine, TAPS and the like. Although not particularly limited, the biochemical test reagent used in the present invention preferably uses Good's buffer, ADA buffer, MOPS buffer, MES buffer, Bis-Tris buffer, PIPES buffer, ACES buffer, Using MOPSO buffer, BES buffer, TES buffer, HEPES buffer, DIPSO buffer, TAPSO buffer, POPSO buffer, HEPPSO buffer, HEPPS buffer, Tricine buffer, Bicine buffer, TAPS buffer is more preferred.

EXAMPLES Hereinafter, the present invention will be specifically described with reference to Examples. In addition, this invention is not specifically limited by an Example.

[Comparative example and reference example]
A high-density lipoprotein (HDL) cholesterol measuring reagent having the following composition was prepared, and a contamination test with the triglyceride measuring reagent was carried out.
(1) Preparation of reagent <Composition A>
First reagent 80.0 mM ADA buffer (pH 6.50)
1.00 U/mL cholesterol esterase (COE-301 manufactured by Toyobo)
3.00 U/mL cholesterol oxidase (Toyobo COO-321)
2.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L N-ethyl-N-sulfopropyl-m-anisidine second reagent 100.0 mM MOPS buffer (pH 7.50)
7.00 U/mL lipoprotein lipase (manufactured by Toyobo)
8.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L 4-Aminoantipyrine 9.000 g/L Polyoxyethylene lauryl ether

<Composition B-1>
First reagent 80.0 mM ADA buffer (pH 6.50)
1.00 U/mL cholesterol esterase (COE-301 manufactured by Toyobo)
3.00 U/mL cholesterol oxidase (Toyobo COO-321)
2.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L N-ethyl-N-sulfopropyl-m-anisidine 0.900 g/L cholic acid second reagent 100.0 mM MOPS buffer (pH 7.50)
7.00 U/mL lipoprotein lipase (manufactured by Toyobo)
8.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L 4-Aminoantipyrine 9.000 g/L Polyoxyethylene lauryl ether

<Composition B-2>
First reagent 80.0 mM ADA buffer (pH 6.50)
0.50 U/mL cholesterol esterase (COE-301 manufactured by Toyobo)
3.00 U/mL cholesterol oxidase (Toyobo COO-321)
2.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L N-ethyl-N-sulfopropyl-m-anisidine 0.900 g/L cholic acid second reagent 100.0 mM MOPS buffer (pH 7.50)
7.00 U/mL lipoprotein lipase (manufactured by Toyobo)
8.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L 4-Aminoantipyrine 9.000 g/L Polyoxyethylene lauryl ether

<Composition B-3>
First reagent 80.0 mM ADA buffer (pH 6.50)
3.00 U/mL cholesterol oxidase (Toyobo COO-321)
2.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L N-ethyl-N-sulfopropyl-m-anisidine 0.900 g/L cholic acid second reagent 100.0 mM MOPS buffer (pH 7.50)
7.00 U/mL lipoprotein lipase (manufactured by Toyobo)
8.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L 4-Aminoantipyrine 9.000 g/L Polyoxyethylene lauryl ether

<Composition C>
First reagent 80.0 mM ADA buffer (pH 6.50)
1.00 U/mL cholesterol esterase (COE-301 manufactured by Toyobo)
2.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L N-ethyl-N-sulfopropyl-m-anisidine 0.900 g/L cholic acid second reagent 100.0 mM MOPS buffer (pH 7.50)
7.00 U/mL lipoprotein lipase (manufactured by Toyobo)
8.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L 4-Aminoantipyrine 9.000 g/L Polyoxyethylene lauryl ether

(2) Test method <Contamination test>
Using a Hitachi High-Technologies 7180 type automatic analyzer (using a stainless steel reagent probe) as a biochemical analyzer, first, Toyobo neutral fat measurement reagent Diacolor Liquid TG-S (R1: R2/2: 1 mixture) After measuring the serum sample 5 times, measure the above sample with each of the above HDL cholesterol measuring reagents, and again with Toyobo's neutral fat measuring reagent Diacolor Liquid TG-S (R1: R2/2: 1 mixture). The above sample was measured using This series of operations was carried out continuously. Here, since the first reagent and the second reagent in each measurement reagent are aspirated by different reagent probes, there is no risk of contamination occurring between the first reagent and the second reagent. In addition, since the reaction cell used for each measurement is different, the test was conducted under conditions where there is no risk of contamination due to adsorption and residual reagent components in the reaction cell. Before the analysis, calibration was performed using a standard sample, and the absorbance was converted to triglyceride concentration from the obtained calibration curve and output as a measured value.
<Method for judging results>
In the contamination test, whether or not the reagent for measuring HDL cholesterol measured at the sixth time remained in the reagent probe and affected the measured value of the reagent for measuring triglycerides measured at the seventh time was evaluated by "influence". If the degree of influence is less than 5%, it is judged that there is no contamination, and if the degree of influence is 5% or more, it is judged that there is contamination. The degree of influence was calculated using the following formula.
Influence (%) = (7th measured value - 1st to 5th measured value) / 1st to 5th measured value x 100

(3) Analysis Results Table 1 shows the results. According to the results in Table 1, in Comparative Examples 1 to 4, the degree of influence was ≧5%, and contamination with the reagent probe was observed regardless of the presence or absence of cholic acid, which is an anionic surfactant, and the concentration of cholesterol esterase. confirmed. On the other hand, in Reference Example 1, contamination with reagent probes did not occur. This suggested that cholesterol oxidase remained on the reagent probe and adversely affected the subsequently aspirated reagent.
Separately, when the reagent probe used in the 6th measurement and the reagent probe used in the 7th measurement were exchanged for evaluation, an improvement in the measurement error was observed. From this, it was clarified that the cause of the contamination that causes the measurement error is the adsorption and residue on the reagent probe.

[Example 1]
A high-density lipoprotein (HDL) cholesterol measuring reagent having the following composition was prepared, and a contamination test with the triglyceride measuring reagent was carried out.
(1) Preparation of reagent <Composition D-1>
First reagent 80.0 mM ADA buffer (pH 6.50)
1.00 U/mL cholesterol esterase (COE-301 manufactured by Toyobo)
3.00 U/mL cholesterol oxidase (Toyobo COO-321)
2.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L N-ethyl-N-sulfopropyl-m-anisidine 0.900 g/L cholic acid 1.000 g/L Tween20
Second reagent 100.0 mM MOPS buffer (pH 7.50)
7.00 U/mL lipoprotein lipase (manufactured by Toyobo)
8.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L 4-Aminoantipyrine 9.000 g/L Polyoxyethylene lauryl ether

<Composition D-2>
First reagent 80.0 mM ADA buffer (pH 6.50)
1.00 U/mL cholesterol esterase (COE-301 manufactured by Toyobo)
3.00 U/mL cholesterol oxidase (Toyobo COO-321)
2.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L N-ethyl-N-sulfopropyl-m-anisidine 0.900 g/L cholic acid 5.000 g/L Tween20
Second reagent 100.0 mM MOPS buffer (pH 7.50)
7.00 U/mL lipoprotein lipase (manufactured by Toyobo)
8.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L 4-Aminoantipyrine 9.000 g/L Polyoxyethylene lauryl ether

<Composition E-1>
First reagent 80.0 mM ADA buffer (pH 6.50)
1.00 U/mL cholesterol esterase (COE-301 manufactured by Toyobo)
3.00 U/mL cholesterol oxidase (Toyobo COO-321)
2.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L N-ethyl-N-sulfopropyl-m-anisidine 0.900 g/L cholic acid 1.000 g/L Tween80
Second reagent 100.0 mM MOPS buffer (pH 7.50)
7.00 U/mL lipoprotein lipase (manufactured by Toyobo)
8.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L 4-Aminoantipyrine 9.000 g/L Polyoxyethylene lauryl ether

<Composition E-2>
First reagent 80.0 mM ADA buffer (pH 6.50)
1.00 U/mL cholesterol esterase (COE-301 manufactured by Toyobo)
3.00 U/mL cholesterol oxidase (Toyobo COO-321)
2.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L N-ethyl-N-sulfopropyl-m-anisidine 0.900 g/L cholic acid 5.000 g/L Tween80
Second reagent 100.0 mM MOPS buffer (pH 7.50)
7.00 U/mL lipoprotein lipase (manufactured by Toyobo)
8.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L 4-Aminoantipyrine 9.000 g/L Polyoxyethylene lauryl ether

<Composition F-1>
First reagent 80.0 mM ADA buffer (pH 6.50)
1.00 U/mL cholesterol esterase (COE-301 manufactured by Toyobo)
3.00 U/mL cholesterol oxidase (Toyobo COO-321)
2.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L N-ethyl-N-sulfopropyl-m-anisidine 0.900 g/L Cholic acid 1.000 g/L Triton X-114
Second reagent 100.0 mM MOPS buffer (pH 7.50)
7.00 U/mL lipoprotein lipase (manufactured by Toyobo)
8.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L 4-Aminoantipyrine 9.000 g/L Polyoxyethylene lauryl ether

<Composition F-2>
First reagent 80.0 mM ADA buffer (pH 6.50)
1.00 U/mL cholesterol esterase (COE-301 manufactured by Toyobo)
3.00 U/mL cholesterol oxidase (Toyobo COO-321)
2.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L N-ethyl-N-sulfopropyl-m-anisidine 0.900 g/L cholic acid 5.000 g/L Triton X-114
Second reagent 100.0 mM MOPS buffer (pH 7.50)
7.00 U/mL lipoprotein lipase (manufactured by Toyobo)
8.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L 4-Aminoantipyrine 9.000 g/L Polyoxyethylene lauryl ether

(2) Test method <Contamination test>
Using a Hitachi High-Technologies 7180 type automatic analyzer (using a stainless steel reagent probe) as a biochemical analyzer, first, Toyobo neutral fat measurement reagent Diacolor Liquid TG-S (R1: R2/2: 1 mixture) After measuring the serum sample 5 times, measure the above sample with each of the above HDL cholesterol measuring reagents, and again with Toyobo's neutral fat measuring reagent Diacolor Liquid TG-S (R1: R2/2: 1 mixture). The above sample was measured using This series of operations was carried out continuously. Here, since the first reagent and the second reagent in each measurement reagent are aspirated by different reagent probes, there is no risk of contamination occurring between the first reagent and the second reagent. In addition, since the reaction cell used for each measurement is different, the test was conducted under conditions where there is no risk of contamination due to adsorption and residual reagent components in the reaction cell. Before the analysis, calibration was performed using a standard sample, and the absorbance was converted to triglyceride concentration from the obtained calibration curve and output as a measured value.
<Method for judging results>
In the contamination test, whether or not the reagent for measuring HDL cholesterol measured at the sixth time remained in the reagent probe and affected the measured value of the reagent for measuring triglycerides measured at the seventh time was evaluated by "influence". If the degree of influence is less than 5%, it is judged that there is no contamination, and if the degree of influence is 5% or more, it is judged that there is contamination. The degree of influence was calculated using the following formula.
Influence (%) = (7th measured value - 1st to 5th measured value) / 1st to 5th measured value x 100

(3) Analysis Results Table 2 shows the results. According to the results in Table 2, the degree of influence was 6.1 to 8.9% in Comparative Examples 1 to 4 in Table 1, in which the solution containing cholesterol oxidase (first reagent) did not contain a nonionic surfactant. In contrast, in Examples 1-1 to 1-6, in which nonionic surfactants (Tween 20, Tween 80, and Triton X-114) were added, the degree of influence was less than 5%, and contamination with reagent probes was reduced. Nation did not occur.

[Example 2]
A high-density lipoprotein (HDL) cholesterol measuring reagent having the following composition was prepared, and a contamination test with the triglyceride measuring reagent was carried out.
(1) Preparation of reagent <Composition G-1>
First reagent 80.0 mM ADA buffer (pH 6.50)
1.00 U/mL cholesterol esterase (COE-301 manufactured by Toyobo)
3.00 U/mL cholesterol oxidase (Toyobo COO-321)
2.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L N-ethyl-N-sulfopropyl-m-anisidine 0.900 g/L Cholic acid 1.000 g/L Emulgen A60 (manufactured by Kao Corporation)
Second reagent 100.0 mM MOPS buffer (pH 7.50)
7.00 U/mL lipoprotein lipase (manufactured by Toyobo)
8.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L 4-Aminoantipyrine 9.000 g/L Polyoxyethylene lauryl ether

<Composition G-2>
First reagent 80.0 mM ADA buffer (pH 6.50)
1.00 U/mL cholesterol esterase (COE-301 manufactured by Toyobo)
3.00 U/mL cholesterol oxidase (Toyobo COO-321)
2.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L N-ethyl-N-sulfopropyl-m-anisidine 0.900 g/L Cholic acid 5.000 g/L Emulgen A60 (manufactured by Kao Corporation)
Second reagent 100.0 mM MOPS buffer (pH 7.50)
7.00 U/mL lipoprotein lipase (manufactured by Toyobo)
8.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L 4-Aminoantipyrine 9.000 g/L Polyoxyethylene lauryl ether

<Composition H-1>
First reagent 80.0 mM ADA buffer (pH 6.50)
1.00 U/mL cholesterol esterase (COE-301 manufactured by Toyobo)
3.00 U/mL cholesterol oxidase (Toyobo COO-321)
2.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L N-ethyl-N-sulfopropyl-m-anisidine 0.900 g/L Cholic acid 1.000 g/L Emulgen A90 (manufactured by Kao Corporation)
Second reagent 100.0 mM MOPS buffer (pH 7.50)
7.00 U/mL lipoprotein lipase (manufactured by Toyobo)
8.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L 4-Aminoantipyrine 9.000 g/L Polyoxyethylene lauryl ether

<Composition H-2>
First reagent 80.0 mM ADA buffer (pH 6.50)
1.00 U/mL cholesterol esterase (COE-301 manufactured by Toyobo)
3.00 U/mL cholesterol oxidase (Toyobo COO-321)
2.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L N-ethyl-N-sulfopropyl-m-anisidine 0.900 g/L Cholic acid 5.000 g/L Emulgen A90 (manufactured by Kao Corporation)
Second reagent 100.0 mM MOPS buffer (pH 7.50)
7.00 U/mL lipoprotein lipase (manufactured by Toyobo)
8.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L 4-Aminoantipyrine 9.000 g/L Polyoxyethylene lauryl ether

<Composition I-1>
First reagent 80.0 mM ADA buffer (pH 6.50)
1.00 U/mL cholesterol esterase (COE-301 manufactured by Toyobo)
3.00 U/mL cholesterol oxidase (Toyobo COO-321)
2.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L N-ethyl-N-sulfopropyl-m-anisidine 0.900 g/L Cholic acid 1.000 g/L Emulgen B66 (manufactured by Kao Corporation)
Second reagent 100.0 mM MOPS buffer (pH 7.50)
7.00 U/mL lipoprotein lipase (manufactured by Toyobo)
8.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L 4-Aminoantipyrine 9.000 g/L Polyoxyethylene lauryl ether

<Composition I-2>
First reagent 80.0 mM ADA buffer (pH 6.50)
1.00 U/mL cholesterol esterase (COE-301 manufactured by Toyobo)
3.00 U/mL cholesterol oxidase (Toyobo COO-321)
2.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L N-ethyl-N-sulfopropyl-m-anisidine 0.900 g/L Cholic acid 5.000 g/L Emulgen B66 (manufactured by Kao Corporation)
Second reagent 100.0 mM MOPS buffer (pH 7.50)
7.00 U/mL lipoprotein lipase (manufactured by Toyobo)
8.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L 4-Aminoantipyrine 9.000 g/L Polyoxyethylene lauryl ether

<Composition J>
First reagent 80.0 mM ADA buffer (pH 6.50)
1.00 U/mL cholesterol esterase (COE-301 manufactured by Toyobo)
3.00 U/mL cholesterol oxidase (Toyobo COO-321)
2.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L N-ethyl-N-sulfopropyl-m-anisidine 0.900 g/L Cholic acid 1.500 g/L Emulgen A60 (manufactured by Kao Corporation)
0.500 g/L Emulgen A90 (manufactured by Kao Corporation)
0.500 g/L Emulgen 420 (manufactured by Kao Corporation)
Second reagent 100.0 mM MOPS buffer (pH 7.50)
7.00 U/mL lipoprotein lipase (manufactured by Toyobo)
8.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L 4-Aminoantipyrine 9.000 g/L Polyoxyethylene lauryl ether

(2) Test method <Contamination test>
Using a Hitachi High-Technologies 7180 type automatic analyzer (using a stainless steel reagent probe) as a biochemical analyzer, first, Toyobo neutral fat measurement reagent Diacolor Liquid TG-S (R1: R2/2: 1 mixture) After measuring the serum sample 5 times, measure the above sample with each of the above HDL cholesterol measuring reagents, and again with Toyobo's neutral fat measuring reagent Diacolor Liquid TG-S (R1: R2/2: 1 mixture). The above sample was measured using This series of operations was carried out continuously. Here, since the first reagent and the second reagent in each measurement reagent are aspirated by different reagent probes, there is no risk of contamination occurring between the first reagent and the second reagent. In addition, since the reaction cell used for each measurement is different, the test was conducted under conditions where there is no risk of contamination due to adsorption and residual reagent components in the reaction cell. Before the analysis, calibration was performed using a standard sample, and the absorbance was converted to triglyceride concentration from the obtained calibration curve and output as a measured value.
<Method for judging results>
In the contamination test, whether or not the reagent for measuring HDL cholesterol measured at the sixth time remained in the reagent probe and affected the measured value of the reagent for measuring triglycerides measured at the seventh time was evaluated by "influence". If the degree of influence is less than 5%, it is judged that there is no contamination, and if the degree of influence is 5% or more, it is judged that there is contamination. The degree of influence was calculated using the following formula.
Influence (%) = (7th measured value - 1st to 5th measured value) / 1st to 5th measured value x 100

(3) Analysis Results Table 3 shows the results. According to the results in Table 3, the degree of influence was 6.1 to 8.9% in Comparative Examples 1 to 4 in Table 1, in which the solution containing cholesterol oxidase (first reagent) did not contain a nonionic surfactant. On the other hand, in Examples 2-1 to 2-7 in which nonionic surfactants (Emulgen A60, Emulgen A90, and Emulgen B66) were added, the degree of influence was less than 5%, and contamination with the reagent probe was reduced. Nation did not occur.

[Example 3]
A high-density lipoprotein (HDL) cholesterol measuring reagent having the following composition was prepared, and a contamination test with the triglyceride measuring reagent was carried out.
(1) Preparation of reagent <Composition K-1>
First reagent 80.0 mM ADA buffer (pH 6.50)
1.00 U/mL cholesterol esterase (COE-301 manufactured by Toyobo)
3.00 U/mL cholesterol oxidase (Toyobo COO-321)
2.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L N-ethyl-N-sulfopropyl-m-anisidine 0.900 g/L Cholic acid 0.100 g/L Emulgen A90 (manufactured by Kao Corporation)
Second reagent 100.0 mM MOPS buffer (pH 7.50)
7.00 U/mL lipoprotein lipase (manufactured by Toyobo)
8.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L 4-Aminoantipyrine 9.000 g/L Polyoxyethylene lauryl ether

<Composition K-2>
First reagent 80.0 mM ADA buffer (pH 6.50)
1.00 U/mL cholesterol esterase (COE-301 manufactured by Toyobo)
3.00 U/mL cholesterol oxidase (Toyobo COO-321)
2.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L N-ethyl-N-sulfopropyl-m-anisidine 0.900 g/L Cholic acid 0.200 g/L Emulgen A90 (manufactured by Kao Corporation)
Second reagent 100.0 mM MOPS buffer (pH 7.50)
7.00 U/mL lipoprotein lipase (manufactured by Toyobo)
8.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L 4-Aminoantipyrine 9.000 g/L Polyoxyethylene lauryl ether

<Composition K-3>
First reagent 80.0 mM ADA buffer (pH 6.50)
1.00 U/mL cholesterol esterase (COE-301 manufactured by Toyobo)
3.00 U/mL cholesterol oxidase (Toyobo COO-321)
2.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L N-ethyl-N-sulfopropyl-m-anisidine 0.900 g/L Cholic acid 0.300 g/L Emulgen A90 (manufactured by Kao Corporation)
Second reagent 100.0 mM MOPS buffer (pH 7.50)
7.00 U/mL lipoprotein lipase (manufactured by Toyobo)
8.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L 4-Aminoantipyrine 9.000 g/L Polyoxyethylene lauryl ether

<Composition K-4>
First reagent 80.0 mM ADA buffer (pH 6.50)
1.00 U/mL cholesterol esterase (COE-301 manufactured by Toyobo)
3.00 U/mL cholesterol oxidase (Toyobo COO-321)
2.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L N-ethyl-N-sulfopropyl-m-anisidine 0.900 g/L Cholic acid 0.400 g/L Emulgen A90 (manufactured by Kao Corporation)
Second reagent 100.0 mM MOPS buffer (pH 7.50)
7.00 U/mL lipoprotein lipase (manufactured by Toyobo)
8.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L 4-Aminoantipyrine 9.000 g/L Polyoxyethylene lauryl ether

<Composition K-5>
First reagent 80.0 mM ADA buffer (pH 6.50)
1.00 U/mL cholesterol esterase (COE-301 manufactured by Toyobo)
3.00 U/mL cholesterol oxidase (Toyobo COO-321)
2.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L N-ethyl-N-sulfopropyl-m-anisidine 0.900 g/L Cholic acid 0.500 g/L Emulgen A90 (manufactured by Kao Corporation)
Second reagent 100.0 mM MOPS buffer (pH 7.50)
7.00 U/mL lipoprotein lipase (manufactured by Toyobo)
8.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L 4-Aminoantipyrine 9.000 g/L Polyoxyethylene lauryl ether

<Composition K-6>
First reagent 80.0 mM ADA buffer (pH 6.50)
1.00 U/mL cholesterol esterase (COE-301 manufactured by Toyobo)
3.00 U/mL cholesterol oxidase (Toyobo COO-321)
2.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L N-ethyl-N-sulfopropyl-m-anisidine 0.900 g/L Cholic acid 0.600 g/L Emulgen A90 (manufactured by Kao Corporation)
Second reagent 100.0 mM MOPS buffer (pH 7.50)
7.00 U/mL lipoprotein lipase (manufactured by Toyobo)
8.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L 4-Aminoantipyrine 9.000 g/L Polyoxyethylene lauryl ether

<Composition K-7>
First reagent 80.0 mM ADA buffer (pH 6.50)
1.00 U/mL cholesterol esterase (COE-301 manufactured by Toyobo)
3.00 U/mL cholesterol oxidase (Toyobo COO-321)
2.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L N-ethyl-N-sulfopropyl-m-anisidine 0.900 g/L Cholic acid 0.700 g/L Emulgen A90 (manufactured by Kao Corporation)
Second reagent 100.0 mM MOPS buffer (pH 7.50)
7.00 U/mL lipoprotein lipase (manufactured by Toyobo)
8.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L 4-Aminoantipyrine 9.000 g/L Polyoxyethylene lauryl ether

<Composition K-8>
First reagent 80.0 mM ADA buffer (pH 6.50)
1.00 U/mL cholesterol esterase (COE-301 manufactured by Toyobo)
3.00 U/mL cholesterol oxidase (Toyobo COO-321)
2.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L N-ethyl-N-sulfopropyl-m-anisidine 0.900 g/L Cholic acid 0.800 g/L Emulgen A90 (manufactured by Kao Corporation)
Second reagent 100.0 mM MOPS buffer (pH 7.50)
7.00 U/mL lipoprotein lipase (manufactured by Toyobo)
8.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L 4-Aminoantipyrine 9.000 g/L Polyoxyethylene lauryl ether

<Composition K-9>
First reagent 80.0 mM ADA buffer (pH 6.50)
1.00 U/mL cholesterol esterase (COE-301 manufactured by Toyobo)
3.00 U/mL cholesterol oxidase (Toyobo COO-321)
2.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L N-ethyl-N-sulfopropyl-m-anisidine 0.900 g/L Cholic acid 0.900 g/L Emulgen A90 (manufactured by Kao Corporation)
Second reagent 100.0 mM MOPS buffer (pH 7.50)
7.00 U/mL lipoprotein lipase (manufactured by Toyobo)
8.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L 4-Aminoantipyrine 9.000 g/L Polyoxyethylene lauryl ether

<Composition K-10>
First reagent 80.0 mM ADA buffer (pH 6.50)
1.00 U/mL cholesterol esterase (COE-301 manufactured by Toyobo)
3.00 U/mL cholesterol oxidase (Toyobo COO-321)
2.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L N-ethyl-N-sulfopropyl-m-anisidine 0.900 g/L Cholic acid 1.000 g/L Emulgen A90 (manufactured by Kao Corporation)
Second reagent 100.0 mM MOPS buffer (pH 7.50)
7.00 U/mL lipoprotein lipase (manufactured by Toyobo)
8.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L 4-Aminoantipyrine 9.000 g/L Polyoxyethylene lauryl ether

(2) Test method <Contamination test>
Using a Hitachi High-Technologies 7180 type automatic analyzer (using a stainless steel reagent probe) as a biochemical analyzer, first, Toyobo neutral fat measurement reagent Diacolor Liquid TG-S (R1: R2/2: 1 mixture) After measuring the serum sample 5 times, measure the above sample with each of the above HDL cholesterol measuring reagents, and again with Toyobo's neutral fat measuring reagent Diacolor Liquid TG-S (R1: R2/2: 1 mixture). The above sample was measured using This series of operations was carried out continuously. Here, since the first reagent and the second reagent in each measurement reagent are aspirated by different reagent probes, there is no risk of contamination occurring between the first reagent and the second reagent. In addition, since the reaction cell used for each measurement is different, the test was conducted under conditions where there is no risk of contamination due to adsorption and residual reagent components in the reaction cell. Before the analysis, calibration was performed using a standard sample, and the absorbance was converted to triglyceride concentration from the obtained calibration curve and output as a measured value.
<Method for judging results>
In the contamination test, whether or not the reagent for measuring HDL cholesterol measured at the sixth time remained in the reagent probe and affected the measured value of the reagent for measuring triglycerides measured at the seventh time was evaluated by "influence". If the degree of influence is less than 5%, it is judged that there is no contamination, and if the degree of influence is 5% or more, it is judged that there is contamination. The degree of influence was calculated using the following formula.
Influence (%) = (7th measured value - 1st to 5th measured value) / 1st to 5th measured value x 100

(3) Analysis Results Table 4 shows the results. According to the results in Table 4, the degree of influence was 6.1 to 8.9% in Comparative Examples 1 to 4 in Table 1, in which the solution containing cholesterol oxidase (first reagent) did not contain a nonionic surfactant. On the other hand, in Examples 3-1 to 3-10 in which 0.1 g / L or more of Emulgen A90, which is a nonionic surfactant, was added, the degree of influence was less than 5%, and contamination with the reagent probe did not happen.

[Example 4]
A high-density lipoprotein (HDL) cholesterol measuring reagent having the following composition was prepared, and a contamination test with the uric acid measuring reagent was carried out.
(1) Preparation of reagent <Composition L-1>
First reagent 80.0 mM ADA buffer (pH 7.00)
1.00 U/mL cholesterol esterase (COE-301 manufactured by Toyobo)
2.00 U/mL cholesterol oxidase (Toyobo COO-321)
2.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L N-ethyl-N-sulfopropyl-m-anisidine 0.900 g/L Cholic acid 0.5 g/L Emulgen A90 (manufactured by Kao Corporation)
0.5 g/L Emulgen B66 (manufactured by Kao Corporation)
Second reagent 100.0 mM MOPS buffer (pH 7.50)
7.00 U/mL lipoprotein lipase (manufactured by Toyobo)
8.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L 4-Aminoantipyrine 9.000 g/L Polyoxyethylene lauryl ether

<Composition L-2>
First reagent 80.0 mM ADA buffer (pH 7.40)
1.00 U/mL cholesterol esterase (COE-301 manufactured by Toyobo)
2.00 U/mL cholesterol oxidase (Toyobo COO-321)
2.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L N-ethyl-N-sulfopropyl-m-anisidine 0.900 g/L Cholic acid 0.5 g/L Emulgen A90 (manufactured by Kao Corporation)
0.5 g/L Emulgen B66 (manufactured by Kao Corporation)
Second reagent 100.0 mM MOPS buffer (pH 7.50)
7.00 U/mL lipoprotein lipase (manufactured by Toyobo)
8.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L 4-Aminoantipyrine 9.000 g/L Polyoxyethylene lauryl ether

<Composition M-1>
First reagent 80.0 mM ADA buffer (pH 6.50)
1.00 U/mL cholesterol esterase (COE-301 manufactured by Toyobo)
2.00 U/mL cholesterol oxidase (Toyobo COO-321)
2.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L N-ethyl-N-sulfopropyl-m-anisidine 0.900 g/L Cholic acid 3.5 g/L Emulgen A90 (manufactured by Kao Corporation)
1.5 g/L Emulgen B66 (manufactured by Kao Corporation)
Second reagent 100.0 mM MOPS buffer (pH 7.50)
7.00 U/mL lipoprotein lipase (manufactured by Toyobo)
8.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L 4-Aminoantipyrine 9.000 g/L Polyoxyethylene lauryl ether

<Composition M-2>
First reagent 80.0 mM ADA buffer (pH 6.50)
1.00 U/mL cholesterol esterase (COE-301 manufactured by Toyobo)
2.00 U/mL cholesterol oxidase (Toyobo COO-321)
2.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L N-ethyl-N-sulfopropyl-m-anisidine 0.900 g/L Cholic acid 4.0 g/L Emulgen A90 (manufactured by Kao Corporation)
1.0 g/L Emulgen B66 (manufactured by Kao Corporation)
Second reagent 100.0 mM MOPS buffer (pH 7.50)
7.00 U/mL lipoprotein lipase (manufactured by Toyobo)
8.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L 4-Aminoantipyrine 9.000 g/L Polyoxyethylene lauryl ether

<Composition M-3>
First reagent 80.0 mM ADA buffer (pH 6.50)
1.00 U/mL cholesterol esterase (COE-301 manufactured by Toyobo)
2.00 U/mL cholesterol oxidase (Toyobo COO-321)
2.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L N-ethyl-N-sulfopropyl-m-anisidine 0.900 g/L Cholic acid 4.5 g/L Emulgen A90 (manufactured by Kao Corporation)
0.5 g/L Emulgen B66 (manufactured by Kao Corporation)
Second reagent 100.0 mM MOPS buffer (pH 7.50)
7.00 U/mL lipoprotein lipase (manufactured by Toyobo)
8.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L 4-Aminoantipyrine 9.000 g/L Polyoxyethylene lauryl ether

<Composition M-4>
First reagent 80.0 mM ADA buffer (pH 6.50)
1.00 U/mL cholesterol esterase (COE-301 manufactured by Toyobo)
2.00 U/mL cholesterol oxidase (Toyobo COO-321)
2.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L N-ethyl-N-sulfopropyl-m-anisidine 0.900 g/L Cholic acid 5.0 g/L Emulgen A90 (manufactured by Kao Corporation)
0.5 g/L Emulgen B66 (manufactured by Kao Corporation)
Second reagent 100.0 mM MOPS buffer (pH 7.50)
7.00 U/mL lipoprotein lipase (manufactured by Toyobo)
8.00 U/mL peroxidase (manufactured by Toyobo)
0.200 g/L 4-Aminoantipyrine 9.000 g/L Polyoxyethylene lauryl ether

(2) Test method <Contamination test>
Using a MINDRAY BS-600 (using a stainless steel reagent probe) as a biochemical analyzer, a control serum sample manufactured by SPINREACT was first measured five times with a reagent for measuring uric acid manufactured by SPINREACT, and then measured with each reagent for measuring HDL cholesterol. The above sample was measured, and the above sample was measured again with the uric acid measuring reagent manufactured by SPINREACT. This series of operations was carried out continuously. This operation was performed as Tests 1 to 7, respectively. Here, since the first reagent and the second reagent in each measurement reagent are aspirated by different reagent probes, there is no risk of contamination occurring between the first reagent and the second reagent. In addition, since the reaction cell used for each measurement is different, the test was conducted under conditions where there is no risk of contamination due to adsorption and residual reagent components in the reaction cell. Before the analysis, calibration was performed using a standard sample, and the absorbance was converted to uric acid concentration from the obtained calibration curve and output as a measured value.
<Method for judging results>
In the contamination test, whether or not the HDL cholesterol measuring reagent measured at the sixth time remained in the reagent probe and affected the measured value of the uric acid measuring reagent measured at the seventh time was evaluated by the "degree of influence". If the degree of influence is less than 5%, it is judged that there is no contamination, and if the degree of influence is 5% or more, it is judged that there is contamination. The degree of influence was calculated using the following formula.
Influence (%) = (7th measured value - 1st to 5th measured value) / 1st to 5th measured value x 100

(3) Analysis Results Table 5 shows the results. According to the results in Table 5, in Comparative Example 2 in which the solution containing cholesterol oxidase (first reagent) did not contain a nonionic surfactant, the degree of influence was 7.6%, and contamination with the reagent probe was confirmed. . On the other hand, as in Examples 4-1 to 4-6, the addition of nonionic surfactants (Emulgen A90 and Emulgen B66) reduced the influence to less than 5%, and no contamination with reagent probes occurred. . Furthermore, increasing the pH reduced the effect, as shown in Examples 4-1 and 4-2. In addition, as in Examples 4-3 to 4-6, contamination in the metal reagent probe could be kept at a low level regardless of the blending ratio or blending amount of multiple nonionic surfactants. .

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a biochemical test reagent or the like that suppresses contamination with a metal reagent probe and reduces measurement errors.

Claims (17)

Occurs due to contamination caused by enzymes contained in biochemical test reagents adsorbing or remaining on the metal reagent probe in an automatic biochemical analyzer equipped with a mechanism for dispensing different reagents with the same metal reagent probe. A method for reducing measurement error , characterized by using at least one nonionic surfactant,
wherein the at least one nonionic surfactant is
(a) used in contact with a metallic reagent probe during operation in an automated biochemical analyzer using enzyme-containing biochemical test reagents, or
(b) A method of reducing measurement error, which is used by including a nonionic surfactant in the solution that contacts the metal reagent probe . The nonionic surfactant includes polyoxyethylene octylphenyl ether, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene alkyl ether, polyoxyethylene distyrenated phenyl ether, and polyoxyethylene 2. The method of reducing measurement error according to claim 1, wherein the surfactant is at least one nonionic surfactant selected from the group consisting of ethylene tribenzylphenyl ether. 3. The method of reducing measurement error according to claim 1, wherein at least part of said reagent probe is made of stainless steel. The method for reducing measurement error according to any one of claims 1 to 3, wherein the enzyme is cholesterol oxidase. The method for reducing measurement errors according to any one of claims 1 to 4, wherein the biochemical test reagent is a high-density lipoprotein cholesterol measurement reagent. The method for reducing measurement error according to any one of claims 1 to 5, wherein the biochemical test reagent has an enzyme concentration of 0.01 to 30 U/mL. The method for reducing measurement errors according to any one of claims 1 to 6, wherein the biochemical test reagent has a pH of 5 to 10. The nonionic surfactant is Triton (registered trademark) X-100, Triton (registered trademark) X-114, Tween (registered trademark) 20, Tween (registered trademark) 80, Emulgen (registered trademark) 404, Emulgen (registered trademark) Emulgen® 408, Emulgen® 409PV, Emulgen® 420, Emulgen® 430, Emulgen® A60, Emulgen® A90, Emulgen® A500, and Emulgen® ( The method according to any one of claims 1 to 7, wherein the surfactant is at least one nonionic surfactant selected from the group consisting of B66. Contamination caused by enzymes contained in biochemical test reagents adsorbing or remaining on the metal reagent probe when used in a biochemical automatic analyzer equipped with a mechanism for dispensing different reagents with the same metal reagent probe. A biochemical test reagent with reduced measurement error caused by contamination , containing at least one nonionic surfactant together with an enzyme, and used to contact a metallic reagent probe. , biochemical test reagents. The nonionic surfactant includes polyoxyethylene octylphenyl ether, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene alkyl ether, polyoxyethylene distyrenated phenyl ether, and polyoxyethylene The biochemical test reagent according to claim 9, which is at least one nonionic surfactant selected from the group consisting of ethylene tribenzylphenyl ether. 11. The reagent of claim 9 or 10, wherein at least part of said reagent probe is made of stainless steel. The reagent according to any one of claims 9 to 11, wherein said biochemical test reagent contains cholesterol oxidase as an enzyme. The reagent according to any one of claims 9 to 12, wherein said biochemical test is a cholesterol measurement test. The reagent according to any one of claims 9 to 13, wherein said biochemical test reagent is a high density lipoprotein cholesterol measuring reagent. The reagent according to any one of claims 9 to 14, wherein the biochemical test reagent has an enzyme concentration of 0.01 to 30 U/mL. The reagent according to any one of claims 9 to 15, wherein the biochemical test reagent has a pH of 5-10. The nonionic surfactant is Triton (registered trademark) X-100, Triton (registered trademark) X-114, Tween (registered trademark) 20, Tween (registered trademark) 80, Emulgen (registered trademark) 404, Emulgen (registered trademark) Emulgen® 408, Emulgen® 409PV, Emulgen® 420, Emulgen® 430, Emulgen® A60, Emulgen® A90, Emulgen® A500, and Emulgen® ( 17. The reagent according to any one of claims 9 to 16, which is at least one nonionic surfactant selected from the group consisting of B66.