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Method of lipid assay and reagent for use therein

a technology which is applied in the field of lipid assay and reagent for use therein, can solve the problems of direct methods which have been put into practice, interference of aggregates on assay precision, damage to measuring equipment,

Inactive Publication Date: 2005-06-30
SEKISUI MEDICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This approach allows for precise and efficient lipid component analysis in specific lipoproteins, enhancing assay specificity and minimizing equipment damage, while reducing costs and complexity.

Problems solved by technology

Some of these direct methods are used in practice in daily clinical tests due to their simple procedures as compared with the precipitation method involving complicated procedures.
However, even the direct methods which have been put into practice have problems.
For example, the method of adding a precipitation agent to the reagent has problems: interference of the aggregates on assay precision and a damage to measuring equipment such as clogged flow passages with the products produced from reaction of the precipitation agent with an alkaline detergent for washing measuring equipment.
As for a method of using a modification enzyme, there are problems such as process control in enzyme modification procedures (quality control) and cost increase.

Method used

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  • Method of lipid assay and reagent for use therein
  • Method of lipid assay and reagent for use therein
  • Method of lipid assay and reagent for use therein

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0049] The effect of the present invention was confirmed using HDL and LDL fractions prepared by ultracentrifugation as the samples. The reagent with the following formulation was used for assaying cholesterols in the HDL and LDL fractions.

(First reagent)PIPES buffer solution (pH 6.5) 50 mmol / L4-Aminoantipyrine0.5 mmol / L(Second reagent)PIPES buffer solution (pH 6.5) 50 mmol / LCholesterol esterase  1 unit / mLCholesterol oxidase  1 unit / mLDisulfobutylmetatoluidine1.0 mmol / LPeroxidase  5 unit / mLOrganic silicon compound1%(all manufactured by NipponUnicar Co., Ltd.)

[0050] Hitachi 7170 automatic analyzer was used. 240 μL of the first reagent was added to 2.4 μL of the sample. Five minutes later, 80 μL of the second reagent was added. Absorbance at a wavelength of 600 nm was measured immediately before the addition of the second reagent and 5 minutes thereafter. The difference of the two measurements was regarded as the absorbance of the sample. As a control, a reagent which contains 1% Tr...

example 2

[0053] The effect of the present invention was confirmed using 20 serum samples containing lipoproteins. Reagents with the following formulation were used for assaying.

(First reagent)Bis-Tris buffer solution (pH 6.0)  50 mmol / LCholesterol oxidase  1 unit / mLPeroxidase1.25 unit / mLDisulfobutylmetatoluidine 0.5 mmol / LFlufenamic acid 150 μmol / L(Second reagent)Bis-Tris buffer solution (pH 6)  50 mmol / LCholesterol esterase 1.5 unit / mL(Asahi Kasei Corporation)4-Aminoantipyrine 1.0 mmol / LEmulgen B-66 1.5%Organic silicon compound0.001%(NUC-Silicon L720 manufactured byNippon Unicar Co., Ltd.)

[0054] Hitachi 7170 automatic analyzer was used. 240 μL of the first reagent was added to 2.4 μL of the sample. Five minutes later, 80 μL of the second reagent was added. Absorbance at 600 nm wavelength was measured immediately before the addition of the second reagent and 5 minutes after the addition of the second reagent, to determine the HDL cholesterol concentration from the difference in the absorba...

example 3

[0057] The effect of the present invention was confirmed in the same condition as in Example 2 except that 26 serum samples containing lipoproteins were used as the samples and NET-SG-60A (Example 3A) or NET-SG-60C (Example 3B) (both at a concentration of 0.05%, manufactured by Nihon Surfactant Kogyo KK.) was used as the organic silicon compound of the present invention in the second reagent. The results are shown in Table 3 and FIG. 2.

TABLE 3Unit: mg / dLMethod of thepresent inventionPrecipitationExampleExampleSamplemethodControl3A3B 144.547.846.747.0 249.351.150.951.1 348.851.151.050.9 469.772.472.773.6 567.266.967.967.5 666.469.169.169.1 740.342.242.242.2 828.632.430.830.5 945.347.946.646.61046.848.248.148.21141.744.143.343.01241.145.042.743.61355.659.258.659.21452.656.255.356.11562.262.263.062.31632.735.934.835.01748.248.348.549.21847.449.249.649.01941.145.145.045.02053.354.854.254.32144.046.045.845.92275.378.279.580.12359.461.760.561.22437.139.739.339.62550.655.053.053.52636.94...

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Abstract

A method of lipid assay characterized by assaying the lipids contained in a blood component in the presence of an organic silicon compound. The method can cause specific conditions for direct methods while satisfying requirements such as no influence on precision of assay, no burden on assay apparatus, and easy availability.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method of separately assaying lipid components in specific lipoprotein fractions efficiently by a simple procedure using a small amount of sample. The present invention also relates to a reagent used for the method. DESCRIPTION OF BACKGROUND ART [0002] Cholesterols, triglycerides, and phospholipids which are major lipids in the living body are combined with apoproteins to form lipoproteins in blood. The lipoproteins are grouped into chylomicron, very low density lipoprotein (VLDL), low density lipoprotein (LDL), high density lipoprotein (HDL), and the like according to their physical properties. Among these lipoproteins, it is known that LDL is a causative factor for arteriosclerosis, whereas HDL exhibits an anti-arteriosclerosis effect. [0003] Total cholesterols and total triglycerides in blood have been assayed for the purpose of preventing ischemic heart diseases or evaluating the effects of treatments for such dise...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): G01N33/92
CPCG01N33/92C12Q1/60C12Q1/44G01N2800/044C12Q1/61C07F7/18
Inventor YAMAMOTO, MITSUAKIYAMAMOTO, SHOKONAKAMURA, MITSUHIROSAITO, KAZUNORI
Owner SEKISUI MEDICAL CO LTD
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