Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving cholesterol
Reexamination Certificate
2001-03-19
2004-09-21
Gitomer, Ralph (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving cholesterol
C435S019000, C435S026000
Reexamination Certificate
active
06794157
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a method for the quantitative determination of cholesterol in low density lipoproteins (LDL) (hereinafter referred to as LDL cholesterol), which is important in the field of clinical diagnosis, and a reagent for use in the method. The present invention also relates to a method for the continuous fractional determination of cholesterol in high density lipoproteins (HDL) (hereinafter referred to as HDL cholesterol) and LDL cholesterol, which are also important in the field of clinical diagnosis, and a reagent kit for use therein. The present invention further relates to a method for the continuous fractional determination of HDL cholesterol, LDL cholesterol and total cholesterol [the term is used to mean total cholesterol in HDL, LDL, very low density lipoproteins (hereinafter referred to as VLDL) and chylomicron (hereinafter referred to as CM)], which are important in the field of clinical diagnosis, as well as a reagent kit to be used therefor.
BACKGROUND ART
In general, HDL is called good cholesterol since HDL functions to remove cholesterol accumulated on arterial walls and transport cholesterol to liver. On the other hand, LDL is generally termed bad cholesterol because of its action to transport cholesterol to peripheral tissues including arterial walls. In the field of clinical investigations, the levels of HDL cholesterol, LDL cholesterol and total cholesterol are useful indices for total judgement of lipid-related diseases such as arteriosclerosis, etc.
These cholesterol levels are separately determined using reagents exclusively specific to each type of cholesterol so that an autoanalyzer is designed so as to be suitable for individual determination of these cholesterol levels. It has been desired to further improve the specificity of a reagent to each cholesterol. Besides, no simple and automated method for the continuous fractional determination of HDL cholesterol, LDL cholesterol, total cholesterol, etc in the same detection system is known.
DISCLOSURE OF THE INVENTION
An object of the present invention is to provide a method for the quantitative determination of LDL cholesterol and a determination reagent for use in such a method.
Another object of the present invention is to provide a method for the continuous fractional determination of HDL cholesterol and LDL cholesterol in the same sample and a reagent kit for use therein.
More specifically, the present invention relates to (1) through (27) below.
(1) A method for quantitatively determining LDL cholesterol in a biological sample, which comprises
performing the reaction of cholesterol in the presence of:
a) a biological sample,
b) cholesterol esterase and cholesterol oxidase or cholesterol dehydrogenase (hereinafter collectively referred to as CH enzymes), and
c) a reagent enabling the CH enzymes of b) to act only on LDL cholesterol, and
measuring the amount of the hydrogen peroxide or reduced coenzyme formed by the reaction to quantitatively determine the concentration of LDL cholesterol.
(2) The method according to (1), wherein the reagent enabling CH enzymes to act only on LDL cholesterol is a reagent containing at least a polyoxyethylene derivative and a polyoxyethylene-polyoxypropylene copolymer.
(3) The method according to (2), wherein the polyoxyethylene derivative is a polyoxyethylene alkyl ether or a polyoxyethylene alkylaryl ether.
(4) The method according to (2) or (3), wherein the polyoxyethylene-polyoxypropylene copolymer is a surfactant represented by general formula (I):
HO—(C
2
H
4
O)
a
—(C
3
H
6
O)
b
—(C
2
H
4
O)
c
—H (I)
(wherein a, b and c, which may be the same or different, each represents an integer of 1 to
200).
(5) A method for the continuous fractional determination of HDL cholesterol and LDL cholesterol in a biological sample, which comprises
subjecting cholesterol to the first reaction in the presence of:
a) a biological sample,
b) CH enzymes, and
c) a reagent enabling the CH enzymes of b) to act only on HDL cholesterol, and
measuring the amount of the hydrogen peroxide or reduced coenzyme formed by the first reaction to quantitatively determine the concentration of HDL cholesterol,
then adding
d) a reagent enabling the CH enzymes of b) to act only on LDL cholesterol,
subjecting cholesterol to the second reaction, and
measuring the amount of the hydrogen peroxide or reduced coenzyme formed by the second reaction to quantitatively determine the concentration of LDL cholesterol.
(6) A method for the continuous fractional determination of HDL cholesterol and LDL cholesterol in a biological sample, which comprises
subjecting cholesterol to the first reaction in the presence of:
a) a biological sample,
b) CH enzymes, and
c) a reagent enabling the CH enzymes of b) to act only on HDL cholesterol, and
measuring the amount of the hydrogen peroxide or reduced coenzyme formed by the first reaction to quantitatively determine the concentration of HDL cholesterol,
then adding
d) CH enzymes, and
e) a reagent enabling the CH enzymes of d) to act only on LDL cholesterol,
subjecting cholesterol to the second reaction, and
measuring the amount of the hydrogen peroxide or reduced coenzyme formed by the second reaction to quantitatively determine the concentration of LDL cholesterol.
(7) The method according to (5) or (6), wherein the reagent enabling CH enzymes to act only on LDL cholesterol is a reagent containing at least a polyoxyethylene derivative and a polyoxyethylene-polyoxypropylene copolymer.
(8) The method according to (7), wherein the polyoxyethylene derivative is a polyoxyethylene alkyl ether or a polyoxyethylene alkylaryl ether.
(9) The method according to (7) or (8), wherein the polyoxyethylene-polyoxypropylene copolymer is a surfactant represented by general formula (I):
HO—(C
2
H
4
O)
a
—(C
3
H
6
O)
b
—(C
2
H
4
O)
c
—H (I)
(wherein a, b and c, which may be the same or different, each represents an integer of 1 to
200).
(10) A method for the continuous fractional determination of HDL cholesterol and total cholesterol in a biological sample, which comprises
subjecting cholesterol to the first reaction in the presence of:
a) a biological sample,
b) CH enzymes, and
c) a reagent enabling CH enzymes of b) to act only on HDL cholesterol, and
measuring the amount of the hydrogen peroxide or reduced coenzyme formed by the first reaction to quantitatively determine the concentration of HDL cholesterol,
then adding
d) a reagent enabling the CH enzymes of b) to act on cholesterol in all lipoproteins,
subjecting cholesterol to the second reaction, and
measuring the amount of the hydrogen peroxide or reduced coenzyme formed by the second reaction to quantitatively determine the concentration of total cholesterol.
(11) A method for the continuous fractional determination of HDL cholesterol and total cholesterol in a biological sample, which comprises
subjecting cholesterol to the first reaction in the presence of:
a) a biological sample,
b) CH enzymes, and
c) a reagent enabling the CH enzymes of b) to act only on HDL cholesterol, and
measuring the amount of the hydrogen peroxide or reduced coenzyme formed by the first reaction to quantitatively determine the concentration of HDL cholesterol,
then adding
d) CH enzymes, and
e) a reagent enabling the CH enzymes of d) to act on cholesterol in all lipoproteins,
subjecting cholesterol to the second reaction, and
measuring the amount of the hydrogen peroxide or reduced coenzyme formed by the second reaction to quantitatively determine the total cholesterol.
(12) The method according to any one of (5) through (11), wherein the reagent enabling CH enzymes to act only on cholesterol in HDL is a reagent for aggregating lipoproteins other than HDL.
(13) The method according to (12), wherein the reagent for aggregating lipoproteins other than HDL further contains a nonionic surfactant that does not solubilize the aggregated lipoproteins.
(14) The method according to (12) or (13), wherein the reagent for aggregating lipoproteins other than HDL is a reagent comprising heparin or a sa
Fitzpatrick ,Cella, Harper & Scinto
Kyowa Medex Co., Ltd.
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