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JP-7856752-B2 - Reagent compositions and kits

JP7856752B2JP 7856752 B2JP7856752 B2JP 7856752B2JP-7856752-B2

Inventors

  • 伊藤 康樹

Assignees

  • デンカ株式会社

Dates

Publication Date
20260511
Application Date
20230410
Priority Date
20220411

Claims (10)

  1. A step of reacting the sample with the first reagent composition, After the step of reacting the sample with the first reagent composition, the process involves reacting the sample with a second reagent composition for quantifying small dense LDL cholesterol (sdLDL-C) to quantify the cholesterol remaining in the lipoprotein, A reagent composition used as the first reagent composition for a method of quantifying the sdLDL-C in the sample, comprising: Contains surfactants, The surfactant is at least one nonionic surfactant selected from the group consisting of polyoxyethylene benzylphenyl ether derivatives and polyoxyethylene styrene-phenyl ether derivatives. It possesses cholesterol esterase activity, cholesterol oxidase activity, and sphingomyelinase activity, A hydrogen donor and a coupler, or a hydrogen donor, but not the other. The hydrogen donor is one or more compounds selected from the group consisting of N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline, N-ethyl-N-(3-sulfopropyl)-3-methylaniline, N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline, N-(3-sulfopropyl)aniline, N-(3-sulfopropyl)-3-methoxy-5-aniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-4-fluoro-3,5-dimethoxyaniline, and N-ethyl-N-(3-methylphenyl)-N'-succinylethylenediamine. The coupler is one or more compounds selected from the group consisting of 4-aminoantipyrine, vanillin diamine sulfonic acid, methylbenzthiazolinone hydrazone, and sulfonated methylbenzthiazolinone hydrazone. The contact angle between the reagent composition and the polyethylene terephthalate (PET) substrate, as measured by the following method 1, is 63.0° or more and 67.0° or less. The second reagent composition comprises a buffer, an enzyme, a preservative, and a surfactant, and contains the hydrogen donor and the other of the coupler, but does not include one of the latter . (Method 1) (1) PET substrate: PET (amorphous polyester) resin sheet, transparent, 2 mm thick (2) Pretreatment: Wipe the surface of an unused PET substrate with a 70% ethanol aqueous solution. Perform the measurement within 5 minutes after wiping. (3) Measurement method and conditions: Droplet method, θ/2 method, temperature: 15-25°C, syringe: Teflon coated 18G, dropping method, dropping volume: 2 μL, measurement taken 1 second after dropping. (4) Calculation of contact angle: Measure five times and calculate the average of the five measurements.
  2. The reagent composition according to claim 1, wherein the viscosity of the reagent composition at 5°C, as measured by the following method 2-1, is 2.5 mPa·s or less. (Method 2-1) (1) Apparatus: EMS viscometer (Electro Magnetically Spinning Viscometer) (2) Measurement method and conditions: Measurement method: Electromagnetic spinning method, Motor rotation speed: 1000 rpm, Holding time: 600 seconds, Measurement temperature: 5°C, Sequence loop: 1 time, Sample: 500 μL (3) Calculation of viscosity: Five measurements are taken and the average value of the five measurements is calculated.
  3. The reagent composition according to claim 1 or 2, wherein the viscosity of the reagent composition at 37°C, as measured by the following method 2-2, is 1.05 mPa·s or less. (Method 2-2) (1) Apparatus: EMS viscometer (Electro Magnetically Spinning Viscometer) (2) Measurement method and conditions: Measurement method: Electromagnetic spinning method, Motor rotation speed: 1000 rpm, Holding time: 600 seconds, Measurement temperature: 37°C, Sequence loop: 1 time, Sample: 500 μL (3) Calculation of viscosity: Five measurements are taken and the average value of the five measurements is calculated.
  4. The reagent composition according to claim 1 or 2, wherein the nonionic surfactant comprises polyoxyethylene monostyrene-modified phenyl ether.
  5. The reagent composition according to claim 4, wherein the degree of polymerization n of polyoxyethylene in the polyoxyethylene monostyrene-phenyl ether is 5 or more and 80 or less.
  6. The reagent composition according to claim 4, wherein the content of the polyoxyethylene monostyrene-phenyl ether in the reagent composition is 0.05% (w/v) or more and 0.6% (w/v) or less relative to the entire reagent composition.
  7. The reagent composition according to claim 1 or 2, wherein the reagent composition used as the first reagent composition further has ascorbic acid oxidase activity.
  8. The reagent composition according to claim 1 or 2, wherein the reagent composition further possesses at least one activity selected from the group consisting of peroxidase activity and catalase activity.
  9. A first reagent composition comprising the reagent composition according to claim 1 or 2, A second reagent composition for quantifying the aforementioned sdLDL-C, A kit used for quantifying the sdLDL-C in the sample, including the above.
  10. The kit according to claim 9, wherein the second reagent composition has peroxidase activity.

Description

This invention relates to reagent compositions and kits. One technique for measuring LDL cholesterol is described in Patent Document 1 (Japanese Patent Publication No. 2000-325097). This document describes a method for measuring lipoprotein cholesterol, which includes a first step of selectively enzymatically reacting HDL cholesterol by adding an enzyme and a first surfactant to a sample containing lipoprotein, a second step of selectively enzymatically reacting LDL cholesterol by adding a second surfactant, and measuring HDL cholesterol and/or LDL cholesterol by measuring the compounds consumed or produced by the reaction with the enzyme in the first or second step. This method does not require a lipoprotein flocculant that increases the turbidity of the reaction solution, there are no restrictions on the enzyme used, and there is no need to add another enzyme in the step of reacting LDL cholesterol. It is a simple and inexpensive way to quantify LDL cholesterol, and if necessary, it can accurately and inexpensively measure HDL cholesterol without forming lipoprotein aggregates that interfere with optical measurement. Therefore, it is considered particularly useful in the field of clinical testing for arteriosclerosis and other conditions. Japanese Patent Publication No. 2000-325097 This figure shows the evaluation results of the accuracy of sdLDL-C measurement values after sample dilution.This figure shows the evaluation results of the reaction specificity (correlation with the reference method) of the measured values of sdLDL-C.This figure shows the relationship between the reaction specificity (correlation coefficient in correlation with the reference method) of sdLDL-C measurements and the contact angle.This figure shows the evaluation results of the dilution linearity of the measured values of sdLDL-C.This figure shows the evaluation results of the dilution linearity of the measured values of sdLDL-C. The embodiments will be described below. In this embodiment, the composition of the measurement reagent, etc., may contain each component individually or in combination of two or more. In this specification, the numerical range "x to y" represents "x or more and y or less", and includes both the lower limit x and the upper limit y. First, let's explain lipoproteins. Lipoproteins are broadly divided into Very Low Density Lipoprotein (VLDL), Low Density Lipoprotein (LDL), and High Density Lipoprotein (HDL). LDL is further divided into small dense LDL (sdLDL) and other subfractions. sdLDL is sometimes called small particle LDL, small LDL (SLDL), dense LDL, or small, dense LDL, while other LDL is sometimes called large LDL (LLDL) or light LDL. These lipoprotein fractions and subfractions can be distinguished by particle size or specific gravity. The diameter of lipoprotein particles varies among reporters, but for example, VLDL is 30 nm to 80 nm (or 30 nm to 75 nm), LDL is 22 nm to 28 nm (or 19 nm to 30 nm), and HDL is 7 nm to 10 nm. Regarding the specific gravity of lipoproteins, for example, VLDL is 1.006 or less, LDL is 1.019 to 1.063, and HDL is 1.063 to 1.21. Among lipoproteins, the LDL particle diameter can be measured, for example, by gradient gel electrophoresis (GGE) (JAMA, 260, pp. 1917-21, 1988) or NMR (HANDBOOK OF LIPOPROTEIN TESTING 2nd Edition, edited by Nader Rifai et al., pp. 609-623; AACC PRESS: The Fats of Life Summer 2002, LVDD 15 YEAR ANNIVERSARY ISSUE, Volume AVI No. 3, pp. 15-16). Furthermore, specific gravity can be determined, for example, based on analysis by ultracentrifugation (Atherosclerosis, 106, pp. 241-253, 1994; Atherosclerosis, 83, pp. 59, 1990). In this embodiment, the sdLDL to be measured generally refers to a subfraction of the LDL fraction with a diameter of approximately 22.0 to approximately 25.5 nm, or a subfraction with a specific gravity of 1.040 to 1.063. LDL is divided into subfractions based on size because smaller particles of LDL are more likely to induce arteriosclerosis and are considered more malignant than other types of LDL, making it necessary to separately measure the smaller particles. Within LDL, the diameter and specific gravity distributions are continuous, and it is not possible to clearly distinguish which specific gravity levels indicate particularly high malignancy. Therefore, the specific gravity values of 1.040 to 1.063 mentioned above are not established characteristics of sdLDL, but rather represent the higher specific gravity values obtained by dividing the widely used and established LDL specific gravity range of 1.019 to 1.063 at the midpoint. For example, regarding the specific gravity of sdLDL, another report divides it into fractions of 1.044 to 1.060 (Atherosclerosis: 106 241-253 1994). While there are slight differences among reporters regarding the specific gravity range for sdLDL, in all cases, the presence of sdLDL within that range is associated with clinical malignancy. In this specification, sdLDL specifically refers to LDL with