JP3968374B2 - Liquid reagent for measurement of free fatty acid (NEFA) - Google Patents

Description

  The present invention includes coenzyme A (hereinafter referred to as CoA), acylcoenzyme A synthetase (hereinafter referred to as ACS), and ATP, which are liquid, can be stored for a long time, and can measure free fatty acid (hereinafter referred to as NEFA). A first liquid reagent, and a second liquid reagent containing acylcoenzyme A oxidase (hereinafter referred to as ACO), peroxidase, a hydrogen donor which is a chromogenic substrate for peroxidase, and N-ethylmaleimide (hereinafter referred to as NEM), and The present invention relates to a stabilization method in the production of NEFA, a NEFA measurement kit including them, and a NEFA measurement method using them.

  Conventional commercially available kits for automatic analysis reagents are supplied as a combination of a lyophilized product and a lysate, and have been dissolved in use and used for measurement. However, in the case of a freeze-dried product, in addition to the complexity of reagent preparation, there are problems that an erroneous measurement result is obtained due to mistakes during preparation, and that the effective period after dissolution is short (lack of stability). Therefore, in recent clinical examinations, the use of liquid reagents has become mainstream in order to avoid such problems.

  As a quantitative method of NEFA, first, in the first reagent, by the action of ACS in the presence of a divalent metal ion such as Mg ++ or Mn ++ and adenosine 5′-triphosphate (hereinafter referred to as ATP), CoA In the first step of producing acyl CoA from NEFA in the sample and in the second reagent, hydrogen peroxide produced by oxidizing acyl CoA by the action of ACO is converted into peroxidase and chromogenic substrate in the presence of NEM. A method comprising a second step of quantifying by combining is widely adopted.

However, since NEFA measuring reagents contain many unstable components, it has been considered difficult to liquefy. For example, once the conventionally known NEFA measuring first reagent containing CoA is dissolved, its effective period is about 5 to 15 days in refrigeration.
Furthermore, NEM becomes unstable in the region of higher pH than near neutral, and ACO is denatured by NEM. Therefore, once NEM and ACO coexist in a solution near neutral, both of them are rapidly stable. Will fall. Therefore, it is said that the coexisting solution of NEM and ACO is difficult to maintain the performance as a measurement reagent for a long period of time. Conventionally, NEM is stored in a strongly acidic solution, and ACO is lyophilized and stored. Thus, it is common to store NEM and ACO separately to ensure their stability.

  An object of the present invention is to provide a stable NEFA measurement liquid reagent capable of stabilizing a first reagent and a second reagent and maintaining measurement performance over a long period in a NEFA measurement method using an enzyme. Furthermore, an object of the present invention is to provide a simpler and more accurate NEFA measurement method using these reagents.

  As a result of intensive studies to achieve the above object, the present inventors have determined that the chelating agent and at least one compound selected from the group consisting of substituted α-cyclodextrin, mannitol and trehalose as the first reagent. It was found that the first reagent can be stabilized by allowing it to coexist with. In addition, the present inventors include ACO, peroxidase, a hydrogen donor that is a chromogenic substrate for peroxidase, and NEM, and a flavin adenine dinucleotide (hereinafter referred to as FAD) as a second reagent having a pH effective for stabilizing NEM. ), And 6,6′-dithiodinicotinic acid (hereinafter referred to as DTDN) and / or potassium ferrocyanide to further stabilize the second reagent, thereby completing the present invention. It came to.

That is, the present invention is capable of long-term storage that could not be achieved conventionally, and the following first liquid reagents (1) to (4) and second liquid reagents (5) to (8): I will provide a.
(1) A liquid reagent containing coenzyme A, acylcoenzyme A synthetase and ATP, and an amount of one or more chelating agents effective for stabilizing the reagent, and an amount effective for stabilizing the reagent A stabilized liquid reagent further comprising one or more compounds selected from the group consisting of α-cyclodextrin, mannitol and trehalose.
(2) The liquid reagent according to (1) above, which contains at least diethylenetriamine-N, N, N ′, N ″, N ″ -pentaacetic acid as a chelating agent.
(3) The liquid reagent according to the above (1) or (2), comprising at least a substituted α-cyclodextrin.
(4) The liquid according to (3) above, wherein the substituted α-cyclodextrin is 2,6-di-O-methyl-α-cyclodextrin and / or 6-O-α-D-maltosyl-α-cyclodextrin reagent.
(5) A liquid reagent containing acyl coenzyme A oxidase, peroxidase, a hydrogen donor which is a chromogenic substrate for peroxidase and N-ethylmaleimide, and having a pH effective for stabilizing N-ethylmaleimide, A stabilized liquid reagent further comprising an amount of FAD effective for stabilization, and an amount of 6,6'-dithiodinicotinic acid and / or potassium ferrocyanide effective for stabilizing said reagent.
(6) The liquid reagent according to (5) above, wherein the concentration of potassium ferrocyanide is 0.12 mg / 100 mL or more.
(7) The liquid reagent according to the above (5) or (6), wherein the concentration of 6,6′-dithiodinicotinic acid is 0.4 mmol / L or more.
(8) The liquid reagent according to any one of (5) to (7), further containing an anionic surfactant.

In addition, the present invention provides the following kit for measuring a free fatty acid, which can be stored for a long time which could not be achieved conventionally.
(9) A kit for measuring a free fatty acid, comprising the liquid reagent according to any one of (1) to (4) above and a reagent for quantifying acylcoenzyme A.
(10) The kit according to (9) above, wherein the reagent for quantifying acylcoenzyme A is the liquid reagent according to any of (5) to (8) above.
(11) A kit for measuring a free fatty acid, comprising a reagent for reaction for converting free fatty acid to acylcoenzyme A and the liquid reagent according to any one of (5) to (8) above.

The present invention also provides the following reagent stabilization method in reagent production.
(12) In a reagent solution containing coenzyme A, acylcoenzyme A synthetase and ATP, an amount of one or more chelating agents effective for stabilizing the reagent, and an amount of substituted α-effective for stabilizing the reagent One or more compounds selected from the group consisting of cyclodextrin, mannitol, and trehalose are added, and the reagent solution is stabilized to the extent that it can be used as a liquid reagent for measuring free fatty acids.
(13) The method according to (12) above, comprising at least diethylenetriamine-N, N, N ′, N ″, N ″ -pentaacetic acid as a chelating agent.
(14) The method according to (12) or (13) above, wherein at least a substituted α-cyclodextrin is added.
(15) The method according to (14) above, wherein the substituted α-cyclodextrin is 2,6-di-O-methyl-α-cyclodextrin and / or 6-O-α-D-maltosyl-α-cyclodextrin .
(16) A reagent solution containing acylcoenzyme A oxidase, peroxidase, a hydrogen donor that is a chromogenic substrate for peroxidase, and N-ethylmaleimide, an amount of FAD effective for stabilizing the reagent, and stabilization of the reagent An effective amount of 6,6′-dithiodinicotinic acid and / or potassium ferrocyanide is added and the solution is adjusted to a pH effective for stabilizing N-ethylmaleimide. A method of stabilizing to the extent that it can be used as a liquid reagent for measuring free fatty acids.
(17) The method according to (16) above, wherein the concentration of potassium ferrocyanide is 0.12 mg / 100 mL or more.
(18) The method according to (16) or (17) above, wherein the concentration of 6,6′-dithiodinicotinic acid is 0.4 mmol / L or more.
(19) The method according to any one of (16) to (18) above, further comprising adding an anionic surfactant.

Furthermore, the present invention provides the following methods for measuring free fatty acids.
(20) A sample characterized in that the liquid reagent according to any one of (1) to (4) above and a reagent for quantifying acylcoenzyme A are sequentially added to the sample, and the amount of acylcoenzyme A is measured. Method for measuring free fatty acids in the inside.
(21) The method according to (20) above, wherein the reagent for quantifying acylcoenzyme A is the liquid reagent of any one of (5) to (8) above.
(22) A reagent for reaction for converting free fatty acid to acylcoenzyme A and the liquid reagent according to any one of (5) to (8) above are sequentially added to the sample, and the color developed by the reaction of peroxidase is measured. A method for measuring free fatty acids in a sample.

  The first liquid reagent of the present invention further contains one or more compounds selected from the group consisting of substituted α-cyclodextrin, mannitol and trehalose in addition to CoA, ACS, ATP and one or more chelating agents. The first liquid reagent can also be stabilized with CoA, ACS, ATP and one or more chelating agents, but further includes one or more compounds selected from the group consisting of substituted α-cyclodextrin, mannitol and trehalose. By containing it, it is exceptionally stabilized. The concentration of CoA is not particularly limited as long as it is in a range suitable for the enzyme reaction in which the solution is used. As the chelating agent used in the present invention, any generally used chelate is used, and preferably, for example, diethylenetriamine-N, N, N ′, N ″, N ″ -pentaacetic acid ( Hereinafter referred to as DTPA), triethylenetetramine-N, N, N ′, N ″, N ′ ″, N ′ ″-hexaacetic acid and O, O′-bis (2-aminoethyl) ethylene glycol-N N, N ′, N′-tetraacetic acid and the like are used. As the chelating agent, one type of compound may be used alone, or two or more types may be used in combination. More preferably, the chelating agent used in the present invention contains at least DTPA. The concentration of the chelating agent is not particularly limited as long as it is an amount effective for stabilizing CoA, and varies depending on the type of chelating agent used. The density | concentration which does not precipitate with temperature is mentioned. When DTPA is used for the preparation of the first liquid reagent for NEFA measurement, the concentration of DTPA may be lower than that of a divalent metal ion added at the same time. More preferably, the use concentration of DTPA is 0.25 to 10 mmol / L.

  The concentration of the substituted α-cyclodextrin contained in the first liquid reagent is 0.1 to 10 g / 100 mL, preferably 1 to 5 g / 100 mL. Similarly, the concentration of mannitol is 0.1-10 g / 100 mL, preferably 1-5 g / 100 mL, and the concentration of trehalose is 0.1-10 g / 100 mL, preferably 1-5 g / 100 mL. Examples of the substituted α-cyclodextrin used in the first liquid reagent of the present invention include any α-cyclodextrin having one or more substituents. Substituents include hydrocarbon groups (eg, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl), aromatic groups (including heterocyclic groups, etc.), sugars (eg, monosaccharides, disaccharides, oligos) Examples include, but are not limited to, any natural or synthetic compound substituents such as sugars (which may be chained or branched), amino acids (including peptides) and lipids. Preferably, the substituted α-cyclodextrin is 2,6-di-O-methyl-α-cyclodextrin or 6-O-α-D-maltosyl-α-cyclodextrin. These substituted α-cyclodextrin, mannitol and trehalose may be present alone or in combination in the first liquid reagent. Here, the “effective amount for stabilization” in relation to the first liquid reagent means, for example, the performance as a liquid reagent even after storing the liquid reagent under severe conditions (37 ° C., 16 days) in a practical test. Means an effective amount to be able to. In addition, “stabilized” for the first liquid reagent means that the residual reagent has a residual reactivity of about 60% immediately after preparation, more preferably about 65% or more after the liquid reagent is stored under the above severe conditions in practical tests. It is said to be “remarkably stabilized” particularly when a reactive residual ratio of about 65% or more is maintained. This “effective amount for stabilization” should not be considered individually for each compound, but if the first liquid reagent contains different amounts of multiple compounds, the sum of the effects of each compound. If “stabilization” of the first liquid reagent is achieved, it is interpreted as “an amount effective for stabilization”.

  The pH of the first liquid reagent of the present invention is in a range suitable for the reaction of ACS, does not adversely affect CoA and other components, and comprises the chelating agent, substituted α-cyclodextrin, mannitol and trehalose There is no particular limitation as long as it is within a range suitable for stabilizing the reagent with one or more selected compounds. For example, the pH is 6.5 to 8.0, and each component is added to a buffer solution having a buffer capacity in this range, such as phosphate buffer solution, Tris buffer solution, citrate buffer solution, and Good's buffer solution. By dissolving, a first liquid reagent having a desired pH can be obtained.

  The concentration of ACS and ATP contained in the first liquid reagent of the present invention is not particularly limited as long as it is an amount sufficient to convert NEFA in the sample into acyl CoA. Further, since ACS is divalent cation-requiring, the first reagent for measuring NEFA of the present invention further contains divalent metal ions such as Mg ++ and Mn ++. The concentration of the divalent metal ion is not particularly limited as long as it is higher than the chelating agent added to the CoA-containing solution and ACS has a concentration sufficient to convert NEFA to acyl CoA.

  The second liquid reagent of the present invention contains ACO, peroxidase, a hydrogen donor which is a chromogenic substrate for peroxidase, and NEM, has a pH effective for stabilizing NEM, and is effective in stabilizing the reagent. And an amount of DTDN and / or potassium ferrocyanide effective to stabilize the reagent. The second liquid reagent includes ACO, peroxidase, a hydrogen donor that is a chromogenic substrate for peroxidase, and NEM, has a pH effective for stabilizing NEM, and can be stabilized by including FAD. , DTDN and / or potassium ferrocyanide is further added to stabilize the composition. The concentration of ACO is not particularly limited as long as it is in a range suitable for the enzyme reaction in which the solution is used, and is, for example, 1 to 200 U / mL.

  The peroxidase is not particularly limited as long as it is an enzyme that can oxidize a hydrogen donor that is a chromogenic substrate using hydrogen peroxide, and examples thereof include horseradish peroxidase (hereinafter referred to as HRP). The hydrogen donor is not particularly limited as long as it can be oxidized by peroxidase to produce a coloring substance together with an appropriate coupler such as 4-aminoantipyrine or 3-methyl-2-benzothiazoline. For example, N-ethyl-N-sulfo Propyl-m-anisidine (ADPS), N-ethyl-N-sulfopropyl-aniline (ALPS), N-methyl-N-sulfopropyl-aniline, N-butyl-N-sulfopropyl-aniline, N-ethyl-N -Sulfopropyl-3,5-dimethoxyaniline (DAPS), N-sulfopropyl-3,5-dimethoxyaniline (HDAPS), N-ethyl-N-sulfopropyl-3,5-dimethylaniline (MAPS), N- Ethyl-N-sulfopropyl-m-toluidine (TOPS), N-ethyl-N-sulfur Propyl-o-toluidine, N-ethyl-N-sulfopropyl-p-toluidine, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-anisidine (ADOS), N-ethyl-N- ( 2-hydroxy-3-sulfopropyl) aniline (ALOS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (DAOS), N- (2-hydroxy-3- Sulfopropyl) -3,5-dimethoxyaniline (HDAOS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethylaniline (MAOS), N-ethyl-N- (2- Hydroxy-3-sulfopropyl) -m-toluidine (TOOS), N-sulfopropylaniline (HALPS), p-chlorophenol, p-butyl Mophenol, 2,4-dichlorophenol, p-hydroxybenzoic acid, 3,3 ′, 5,5′-tetramethylbenzidine, N- (3-sulfopropyl) 3,3 ′, 5,5′-tetramethyl Examples include benzidine, N, N, N ′, N ″, N ″ -hexa (3-sulfopropyl) -4,4 ′, 4 ″ -triaminotriphenylmethane.

  The concentration of NEM is an amount effective for preventing color fading caused by peroxidase, and is not particularly limited as long as it does not adversely affect the reaction of ACO, and is, for example, 0.1 to 20 mmol / L. The pH of the second liquid reagent of the present invention is adjusted to a pH effective for stabilizing NEM within the stable pH range of ACO that has spread to the acidic side by incorporating the second liquid reagent stabilizer. Preferably, the pH of the second liquid reagent of the present invention is 5.5 to 6.8, more preferably 5.8 to 6.4, and phosphate buffer, Tris buffer, citrate buffer, Good ( A second liquid reagent having a desired pH can be obtained by dissolving each component in a buffer solution having a buffer capacity in this range, such as a Good's buffer solution. Here, “the pH effective for stabilizing NEM” means a pH that can ensure the stability of NEM to an extent sufficient to achieve stabilization of the second liquid reagent described later.

  The second liquid reagent of the present invention contains FAD as an essential stabilizer. The concentration of FAD used is not particularly limited as long as it is an amount effective for stabilizing the second liquid reagent, and examples thereof include a concentration of 10 μmol / L or more, more preferably 100 to 2000 μmol / L. Here, the “effective amount for stabilization” with respect to the second liquid reagent is, for example, in a liquid state even after storing the liquid reagent under severe conditions (37 ° C., 16 days, 22 days or 29 days) in a practical test. It means an amount effective to ensure the performance as a reagent. Further, “stabilized” with respect to the second liquid reagent means that in a practical test, after storing the liquid reagent at 37 ° C. for 16 days, it is about 70% immediately after preparation, preferably about 75%, more preferably about 80%. This means that the above-mentioned reactive residual rate is maintained. In particular, when the reactive residual rate is maintained at about 75% or more, it is said to have been “stably stabilized”. In addition, after storage at 37 ° C. for 22 days, it means that a reactive residual ratio of about 70%, preferably about 75%, more preferably about 80% or more immediately after preparation is maintained, in particular, about 75% or more. It is said to be “specially stabilized” when it retains the reactive residual rate. In addition, after storage for 29 days at 37 ° C., it means that a reactive residual ratio of about 60% immediately after preparation, preferably about 65% or more is maintained, and in particular, a reactive residual ratio of about 65% or more is maintained. In some cases, it is said to have been exceptionally stabilized. This “effective amount for stabilization” should not be considered as the effect of FAD alone, but with DTDN and / or potassium ferrocyanide (and optionally a surfactant) contained together in the second liquid reagent. If “stabilization” of the second liquid reagent is achieved by the sum of the effects, it is interpreted as “an amount effective for stabilization”.

  The second liquid reagent of the present invention further contains DTDN and / or potassium ferrocyanide as a stabilizer in addition to FAD. The use concentration of DTDN is not particularly limited as long as it is an amount effective for stabilizing the second liquid reagent, and is, for example, 0.4 mmol / L or more, more preferably 0.8 to 5 mmol / L. The concentration of potassium ferrocyanide used is not particularly limited as long as it is an amount effective for stabilizing the second liquid reagent, and is, for example, 0.12 mg / 100 mL or more, more preferably 0.35 to 2.8 mg / 100 mL. Here, the “effective amount for stabilization” is the same as that defined for the FAD, and these amounts should not be regarded as the effect of each compound alone, but include the FAD contained together in the second liquid reagent. In the case of containing both DTDN and potassium ferrocyanide, the sum of the effects of these and FAD, and in the case of further containing a surfactant, the sum of the effects of the surfactants. If stabilization of the two liquid reagents is achieved, each stabilizer is understood to be “an amount effective for stabilization”.

  Preferably, the second liquid reagent of the present invention contains both DTDN and potassium ferrocyanide as stabilizers in addition to FAD.

  In a preferred embodiment, the second liquid reagent further contains a surfactant. The stability of the second liquid reagent is further enhanced by the coexistence of the surfactant. The type of the surfactant is not particularly limited as long as it does not inhibit the reaction of ACO and peroxidase, but preferably an anionic surfactant, more preferably polyoxyethylene alkyl ether sulfate, polyoxyethylene alkyl phenyl ether sulfate, polyoxy These are ethylene dialkyl allyl ether sulfuric acid, polyoxyethylene alkenyl allyl ether sulfuric acid, and salts thereof. One of these may be used alone, or two or more may be used in combination. More specifically, polyoxyethylene alkyl ether sulfates [Example: Haitenol 18E (Daiichi Kogyo Seiyaku Co., Ltd.); Emar 20C and 20A, Lebenol WX (above, Kao Corporation)]], polyoxy Ethylene alkyl phenyl ether sulfate [Example: Haitenol N-17 (Daiichi Kogyo Seiyaku Co., Ltd.); Emar NC, Lebenol WZ (above, Kao Corporation)], polyoxyethylene dialkyl allyl ether sulfate [ Examples: Hightenol NE-15 (Daiichi Kogyo Seiyaku Co., Ltd.)], polyoxyethylene alkenyl allyl ether sulfate [Example: Hitenol NF-13 (Daiichi Kogyo Seiyaku Co., Ltd.)] and the like. . The concentration of the surfactant used is not particularly limited as long as it is an amount effective for stabilizing the second liquid reagent and does not adversely affect the reaction and measurement of ACO and peroxidase. Although different, for example, in the case of Haitenol 18E, it is 50 mg / 100 mL or more, preferably 100 to 1000 mg / 100 mL.

  By adding the surfactant, the solution can be prevented from becoming cloudy at the same time. For example, when Haitenol 18E is used, the turbidity prevention effect is achieved at 50 mg / 100 mL or more.

  The NEFA measurement kit of the present invention includes the first liquid reagent and / or the second liquid reagent of the present invention. When only one of the first liquid reagent and the second liquid reagent of the present invention is included, the NEFA measurement kit of the present invention is, for example, a reaction reagent for converting conventionally known NEFA to acyl CoA, or A kit can be combined with each of known reagents for quantifying acyl CoA. Any conventionally known reagent for converting NEFA to acyl-CoA may be any reagent that converts NEFA to acyl-CoA, but an ACS-containing reagent is preferred. The conventionally known reagent for quantifying acyl CoA may be any reagent capable of quantifying acyl CoA, but is preferably a reagent containing ACO. In this case, NEFA can be measured by measuring hydrogen peroxide generated by the ACO reaction. Hydrogen peroxide may be measured directly using a sensor that consumes oxygen, or chemiluminescence using oxalic acid diester, or chemiluminescence using luminol and peroxidase.

  The present invention also provides a method for stabilizing a reagent solution to such an extent that it can be used as a liquid reagent for NEFA measurement. The method of stabilizing the first liquid reagent includes a reagent solution containing CoA, ACS and ATP, an amount of one or more chelating agents effective for stabilization, and an amount of substituted α-cyclodextrin effective for stabilization, One or more compounds selected from the group consisting of mannitol and trehalose are added. These chelating agents and substituted α-cyclodextrin, mannitol and trehalose may be added separately in any order, or they may be once dissolved in another solution and then added to the reagent solution. . The addition amount is not particularly limited as long as these stabilizers stabilize the reagent solution as a whole. For example, the amount as described above with respect to the first liquid reagent of the present invention is preferably used.

  The method for stabilizing the second liquid reagent of the present invention comprises a reagent solution containing ACO, peroxidase, a hydrogen donor that is a chromogenic substrate for peroxidase, and NEM, an amount of FAD effective for stabilization, and effective for stabilization. Adding a sufficient amount of DTDN and / or potassium ferrocyanide and adjusting the solution to a pH effective for stabilizing the NEM. These FAD, DTDN and potassium ferrocyanide may be added separately in any order, or they may be once dissolved in another solution and then added to the reagent solution. The addition amount is not particularly limited as long as these stabilizers stabilize the reagent solution as a whole. For example, the amount as described above for the second liquid reagent of the present invention is preferably used. The pH may be adjusted before or after the addition.

  In the NEFA measurement method of the present invention, a reaction reagent for converting NEFA to acyl CoA, preferably the first liquid reagent for NEFA measurement of the present invention, is added to a sample to convert NEFA in the sample to acyl CoA. Then, a reagent for quantifying acyl CoA, preferably a reagent that oxidizes acyl CoA to produce hydrogen peroxide and a reagent that quantifies the generated hydrogen peroxide, more preferably the present invention The second liquid reagent for measuring NEFA is added to the reaction solution, and the produced acyl CoA (hydrogen peroxide) is quantified (provided that at least the first or second liquid for measuring NEFA of the present invention is used). Use one of the reagents). When the first or second liquid reagent for NEFA measurement according to the present invention is not used, a reagent for reaction for converting NEFA to acyl CoA and a reagent for quantifying acyl CoA are respectively known for NEFA measurement. It may be a first and a second liquid reagent.

  The sample to be subjected to the NEFA measurement of the present invention is any sample that is considered to contain NEFA. If the sample contains an inhibitor of NEFA measurement or a factor that reduces sensitivity, accuracy or reproducibility, the sample may be pretreated to remove such factor. This sample includes blood, serum, plasma, lymph, bile, urine, feces, saliva, body fluid (including semen, vaginal fluid, sweat, tear fluid, etc.), amniotic fluid, synovial fluid, gingival exudate, pleural fluid, brain Examples include, but are not limited to, biological samples such as chamber fluid and cerebrospinal fluid, liquid samples obtained by extraction from biological tissues, and arbitrary samples obtained from other than living organisms.

  The present invention will be described more specifically with reference to the following examples. However, these are merely examples and do not limit the scope of the present invention.

(Example 1 Stabilization of the first liquid reagent by addition of sugars and cyclodextrin)
A control serum (prepared by adding NEFA to Ceraclear LP (manufactured by Aswell)) having a NEFA value of about 2300 μEq / L was used as a sample for practical testing. As the first liquid reagent for test NEFA measurement, 60 mmol / L trishydroxymethylaminomethane (hereinafter referred to as Tris), 1.1 mmol / L 4-aminoantipyrine, 1.2 mmol / L adenosine 5′-triphosphate disodium trihydrate (Oriental Yeast Co., Ltd.), 1.2 mmol / L Coenzyme A trilithium (Oriental Yeast Co., Ltd.), 0.25 U / mL ACS (Asahi Kasei Co., Ltd.), 1 mmol / L DTPA (Dojin Chemical Co., Ltd.) ), 1.25 mmol / L magnesium chloride and 2 g / 100 mL of various sugars or cyclodextrins were prepared and stored at 37 ° C. for 16 days. As a NEFA measurement second liquid reagent, R2 of Nescoat NEFA-V2 (NEFA measurement automatic analysis kit; manufactured by Aswell Co., Ltd.) was used after being prepared according to the booklet. The reactivity of the 1st liquid reagent for a test which added various saccharides or cyclodextrin was measured as follows using the Hitachi 7170 automatic analyzer. Add 200 μl of the first liquid reagent to 3 μl of the sample, react for 5 minutes at 37 ° C., add 50 μl of R2 of Nescoat NEFA-V2, and further react at 37 ° C. for 5 minutes. The main wavelength is 546 nm and the sub wavelength is 700 nm. The change in absorbance at photometric points 16 to 34 was determined. On the day of preparation of the first liquid reagent for testing and after storage for 16 days at 37 ° C., the sample was measured by the above operation, and the reactivity of the first liquid reagent for testing on the day of preparation was set to 37%, assuming that the reactivity of the first liquid reagent for testing was 100%. The reaction rate (%) after storage for 16 days was determined. The results are shown in Table 1. From the results shown in Table 1, no added α-cyclodextrin such as 2,6-di-O-methyl-α-cyclodextrin, 6-O-α-D-maltosyl-α-cyclodextrin, mannitol and trehalose were added. It became clear that a high reactive residual ratio was maintained as compared with the control.

(Example 2 Stabilization of the second liquid reagent by addition of potassium ferrocyanide)
A control serum (prepared by adding NEFA to Ceraclear LP (manufactured by Aswell)) having a NEFA value of about 2300 μEq / L was used as a sample for practical testing. As the NEFA measurement first liquid reagent, R1 of Nescoat NEFA-V2 (NEFA measurement automatic analysis kit; manufactured by Aswell Co., Ltd.) was used after being prepared according to the booklet. NEFA measurement second liquid reagent for test, 10mmol / L phosphate buffer, 6mg / mL TOOS, 4mmol / L NEM, 2mmol / L FAD, 500mg / 100mL Hightenol 18E, 1U / mL HRP (Toyobo Co., Ltd.) ), 60 U / mL ACO (Asahi Kasei Co., Ltd.), and 0.12 to 2.80 mg / 100 mL potassium ferrocyanide were prepared and stored at 37 ° C. for 16 days. Add 200 μl of Nescoat NEFA-V2 R1 to 3 μl of sample, react for 5 minutes at 37 ° C., add 50 μl of NEFA measurement second liquid reagent for test, and further react at 37 ° C. for 5 minutes. The change in absorbance at measurement points 16 to 34 was determined at a wavelength of 700 nm. On the day of preparation of the second liquid reagent for test and after storage for 16 days at 37 ° C., the sample was measured by the above operation, and the reactivity of the second liquid reagent for test on the day of preparation was set to 100% and the temperature was adjusted to 37 ° C. The reaction rate (%) after storage for 16 days was determined. The results are shown in Table 2. From the results in Table 2, it was found that adding potassium ferrocyanide to the second liquid reagent improves stability in a dose-dependent manner.

(Example 3 Stabilization of the second liquid reagent by adding various compounds in the presence of potassium ferrocyanide)
A control serum (prepared by adding NEFA to Ceraclear LP (manufactured by Aswell)) having a NEFA value of about 2300 μEq / L was used as a sample for practical testing. As the NEFA measurement first liquid reagent, R1 of Nescoat NEFA-V2 (NEFA measurement automatic analysis kit; manufactured by Aswell Co., Ltd.) was used after being prepared according to the booklet. NEFA measurement second liquid reagent for test, 10mmol / L phosphate buffer, 6mg / mL TOOS, 4mmol / L NEM, 2mmol / L FAD, 500mg / 100mL Hightenol 18E, 2.80mg / 100mL potassium ferrocyanide, 1U / mL A pH 6.0 solution containing HRP (manufactured by Toyobo Co., Ltd.), 60 U / mL ACO (manufactured by Asahi Kasei Co., Ltd.), and 1 mmol / L of various compounds was prepared and stored at 37 ° C. for 22 days. The reactivity of the test NEFA measurement second liquid reagent was measured by the method described in Example 2. On the day of preparation of the second liquid reagent for test and after storage at 37 ° C. for 22 days, the sample was measured by the above operation, and the reactivity of the second liquid reagent for test on the day of preparation was set to 100% and the temperature was adjusted to 37 ° C. The reaction rate (%) after storage for 22 days was determined. The results are shown in Table 3. The results in Table 3 show that only the addition of DTDN improved the stability of the second liquid reagent. Therefore, the above-described excellent long-term stabilization effect of DTDN was further examined.

(Example 4 Dose-dependent stabilization effect of DTDN on the second liquid reagent in the presence of potassium ferrocyanide)
A control serum (prepared by adding NEFA to Ceraclear LP (manufactured by Aswell)) having a NEFA value of about 2300 μEq / L was used as a sample for practical testing. As the NEFA measurement first liquid reagent, R1 of Nescoat NEFA-V2 (NEFA measurement automatic analysis kit; manufactured by Aswell Co., Ltd.) was used after being prepared according to the booklet. NEFA measurement second liquid reagent for test, 10mmol / L phosphate buffer, 6mg / mL TOOS, 4mmol / L NEM, 2mmol / L FAD, 500mg / 100mL Hightenol 18E, 2.80mg / 100mL potassium ferrocyanide, 1U / mL A pH 6.0 solution containing HRP (Toyobo Co., Ltd.), 60 U / mL ACO (Asahi Kasei Co., Ltd.), and 0.2 to 5.0 mmol / L DTDN was prepared and stored at 37 ° C. for 29 days. The reactivity of the test NEFA measurement second liquid reagent was measured by the method described in Example 2. On the day of preparation of the second liquid reagent for test and after storage for 29 days at 37 ° C., the sample was measured by the above operation, and the reactivity of the second liquid reagent for test on the day of preparation was set to 100% and the temperature was adjusted to 37 ° C. The reaction rate (%) after storage for 29 days was determined. The results are shown in Table 4. The results in Table 4 revealed that DTDN stabilized the second liquid reagent in a dose-dependent manner. In particular, DTDN was found to show a significant stabilizing effect at a concentration of about 1.0 to 3.0 mmol / L in the second liquid reagent.

  As described above, as the first liquid reagent stabilizer, one or more chelating agents and one or more compounds selected from the group consisting of substituted α-cyclodextrin, mannitol and trehalose are added to the first liquid reagent. By doing so, the performance of the first liquid reagent in the NEFA measurement can be stabilized in a liquid state over a long period of time. Further, by adding FAD and DTDN and / or potassium ferrocyanide as the second liquid reagent stabilizer to the second liquid reagent, the performance of the second liquid reagent in the NEFA measurement can be stabilized in a liquid state over a long period of time. . Further, by using these first and second liquid reagents, it is possible to provide a NEFA measurement kit that is liquid and stable for a long period of time.

Claims (11)

A liquid reagent containing acyl coenzyme A oxidase, peroxidase, hydrogen donor which is a chromogenic substrate for peroxidase and N-ethylmaleimide and having a pH effective for stabilizing N-ethylmaleimide, effective amount of FAD, and further contains the amount effective for stabilization of the reagent, 6,6'-dithio-di nicotine San及beauty one or more compounds selected from the group consisting of potassium off Eroshian reduction, Stabilized liquid reagent.   The liquid reagent according to claim 1, wherein the concentration of potassium ferrocyanide is 0.12 mg / 100 mL or more.   The liquid reagent according to claim 1 or 2, wherein the concentration of 6,6'-dithiodinicotinic acid is 0.4 mmol / L or more.   The liquid reagent in any one of Claims 1-3 which further contains an anionic surfactant. A kit for measuring a free fatty acid comprising the following reagent (a) and the following reagent (b) :
(A) a liquid reagent comprising coenzyme A, acylcoenzyme A synthetase and ATP, in an amount effective for stabilizing the reagent, and a substitution of an amount effective for stabilizing the reagent a stabilized liquid reagent further comprising one or more compounds selected from the group consisting of α-cyclodextrin, mannitol and trehalose; and
(B) The liquid reagent in any one of Claims 1-4. A reagent solution containing acylcoenzyme A oxidase, peroxidase, a hydrogen donor that is a chromogenic substrate for peroxidase and N-ethylmaleimide, an amount of FAD effective for stabilizing the reagent, and an amount effective for stabilizing the reagent of 6,6'-dithio-di nicotine San及beauty adding one or more compounds selected from the group consisting of potassium off Eroshian reduction, and adjusting the solution to the effective pH for the stabilization of N- ethylmaleimide A method of stabilizing the reagent solution to such an extent that it can be used as a liquid reagent for measuring free fatty acids. The method of Claim 6 that the density | concentration of potassium ferrocyanide is 0.12 mg / 100mL or more. The method according to claim 6 or 7 , wherein the concentration of 6,6'-dithiodinicotinic acid is 0.4 mmol / L or more. The method according to any one of claims 6 to 8, wherein an anionic surfactant is further added. A method for measuring free fatty acid in a sample, comprising sequentially adding a liquid reagent (a) below and a reagent (b) below to a sample and measuring the amount of acylcoenzyme A:
(A) a liquid reagent comprising coenzyme A, acylcoenzyme A synthetase and ATP, in an amount effective for stabilizing the reagent, and a substitution of an amount effective for stabilizing the reagent A stabilized liquid reagent further comprising one or more compounds selected from the group consisting of α-cyclodextrin, mannitol and trehalose; and (b) the liquid reagent according to any one of claims 1 to 4. A method for measuring free fatty acid in a sample, which comprises sequentially adding a reagent (a) below and a reagent (b) below to a sample, and measuring color development due to peroxidase reaction :
(A) a liquid reagent comprising coenzyme A, acylcoenzyme A synthetase and ATP, in an amount effective for stabilizing the reagent, and a substitution of an amount effective for stabilizing the reagent a stabilized liquid reagent further comprising one or more compounds selected from the group consisting of α-cyclodextrin, mannitol and trehalose; and
(B) The liquid reagent in any one of Claims 1-4.