Hdl Cholesterol Sensor
A method for the determination of the amount of cholesterol in high density lipoproteins in a high density lipoprotein containing sample, the method comprising reacting the sample with a PEG-ylated protein to selectively complex non-HDL lipoproteins in the sample with the PEG-ylated protein, or with a PEG-ylated enzyme capable of selective reaction with high density lipoproteins, and subsequently measuring the amount of cholesterol in the high density lipoproteins, for example using an electrochemical technique.
1 . A method for the determination of the amount of cholesterol in high density lipoproteins in a high density lipoprotein containing sample, said method comprising reacting the sample with (a) a PEG-ylated protein and subsequently measuring the amount of cholesterol in the high density lipoproteins.
2 . A method according to claim 1 , wherein the amount of cholesterol in the high density lipoproteins is measured by reacting the sample with (b) a cholesterol ester hydrolysing reagent and (c) cholesterol oxidase or cholesterol dehydrogenase and determining the amount of cholesterol which has reacted with the cholesterol oxidase or cholesterol dehydrogenase.
3 . A method according to claim 1 , wherein the sample is additionally reacted with (f) a surfactant.
4 . A method according to claim 1 , wherein the PEG-ylated protein is a PEG-ylated polyaminoacid, gelatine, lysozyme, lactalbumin or serum albumin.
5 . A method according to claim 4 , wherein the PEG-ylated protein is PEG-ylated bovine serum albumin.
6 . A method according to claim 1 , wherein the amount of cholesterol in the high density lipoproteins is measured by an electrochemical technique.
7 . A method according to claim 6 , wherein the method comprises reacting the sample with
(a) a PEG-ylated protein;
(b) a cholesterol ester hydrolysing reagent;
(c) cholesterol oxidase or cholesterol dehydrogenase;
(d) a coenzyme;
(g) a redox agent capable of being oxidised or reduced to form a product; and optionally
(h) a surfactant,
and electrochemically detecting the amount of product formed.
8 . An electrochemical method for the determination of the amount of cholesterol in high density lipoproteins in a high density lipoprotein containing sample, said method comprising reacting the sample with
(b) a cholesterol ester hydrolysing reagent;
(c) cholesterol oxidase or cholesterol dehydrogenase;
(d) a coenzyme;
(e) a redox agent capable of being oxidised or reduced to form a product; and optionally
(f) a surfactant,
(h) a complexing reagent capable of forming a complex with low density and very low density lipoproteins; and/or
(i) a PEG-ylated protein;
and electrochemically detecting the amount of product formed, wherein the cholesterol ester hydrolysing reagent and/or cholesterol oxidase or cholesterol dehydrogenase is modified with polyethylene glycol.
9 . A method according to claim 8 , wherein the complexing agent (h) comprises a polyanion selected from phosphotungstic acid and salts thereof, dextran sulphuric acid and salts thereof, polyethylene glycol and heparin and salts thereof and optionally also comprises a divalent metal salt.
10 . A method according to claim 8 , wherein the complexing reagent (h) comprises an antibody capable of binding to apoB-containing lipoproteins.
11 . A method according to claim 8 , one of claims 8 to 10 , wherein the cholesterol ester hydrolysing reagent (b) is an optionally PEG-ylated cholesterol esterase or lipase.
12 . A method according to claim 11 wherein component (b) is an optionally PEG-ylated cholesterol esterase or lipase and component (c) is optionally PEG-ylated cholesterol dehydrogenase.
13 . A method according to claim 8 , wherein the sample is additionally reacted with (g) a reductase.
14 . (canceled)
15 . A method according to claim 8 , wherein the coenzyme (d) is NAD + or an analogue thereof.
16 . (canceled)
17 . A method according to claim 8 , wherein the sample is reacted simultaneously with the cholesterol ester hydrolysing reagent, cholesterol oxidase or cholesterol dehydrogenase and, if used, the PEG-ylated protein or complexing reagent.
18 . (canceled)
19 . A reagent mixture for use in a method for the determination of the amount of cholesterol in high density lipoproteins in a high density lipoprotein containing sample, the reagent mixture comprising
(a) a PEG-ylated protein;
(b) a cholesterol ester hydrolysing reagent;
(c) cholesterol oxidase or cholesterol dehydrogenase; and optionally
(f) a surfactant.
20 . A reagent mixture according to claim 19 , which additionally comprises (d) a coenzyme, (e) a redox agent capable of being oxidised or reduced to form a product; and optionally (g) a reductase.
21 . A reagent mixture for use in an electrochemical method for the determination of the amount of cholesterol in high density lipoproteins in a high density lipoprotein containing sample, the reagent mixture comprising
(b) a cholesterol ester hydrolysing reagent;
(c) cholesterol oxidase or cholesterol dehydrogenase;
(d) a coenzyme;
(e) a redox agent capable of being oxidised or reduced to form a product; and optionally
(h) a complexing reagent capable of forming a complex with low density and very low density lipoproteins; and/or
(i) a PEG-ylated protein;
wherein the cholesterol ester hydrolysing reagent and/or the cholesterol oxidase or cholesterol dehydrogenase is modified with polyethylene glycol.
22 . A reagent mixture according to claim 21 , which additionally comprises (f) a surfactant and/or (g) a reductase.
23 . A kit for the determination of the amount of cholesterol in high density lipoproteins in a high density lipoprotein containing sample, the kit comprising (a) a PEG-ylated protein capable of selectively complexing with non-HDL lipoproteins, (b) a cholesterol ester hydrolysing reagent, (c) cholesterol oxidase or cholesterol dehydrogenase, optionally (f) a surfactant, and means for measuring the amount of cholesterol which reacts with the cholesterol oxidase or cholesterol dehydrogenase.
24 . A kit for the determination of the amount of cholesterol in high density lipoproteins in a high density lipoprotein containing sample, the kit comprising
an electrochemical cell having a working electrode, a reference or pseudo reference electrode and optionally a separate counter electrode;
the reagents defined in claim;
a power supply for applying a potential across the cell; and
a measuring instrument for measuring the resulting electrochemical response.
25 . A kit according to claim 24 , wherein the reagents are present as a single reagent mixture.
26 . A kit according to claim 25 , wherein the reagent mixture is in dried form.
27 . A kit according to claim 24 , wherein the working electrode is a microelectrode having at least one dimension of less than 50 μm.
28 . A kit according to claim 24 , wherein the electrochemical cell is in the form of a receptacle or partial receptacle, the working electrode is in a wall of the receptacle or partial receptacle and the reagents are at least partly contained within the receptacle or partial receptacle.
29 . A kit according to claim 24 comprising a strip having at least one receptacle or partial receptacle formed therein, the receptacle or partial receptacle having a first open part in a first surface of the strip to enable a sample to enter the receptacle or partial receptacle,
wherein the working electrode of the electrochemical cell is in a wall of the receptacle or partial receptacle, and
wherein the reference or pseudo reference electrode of the electrochemical cell comprises a reference or pseudo reference electrode layer formed on at least a part of the first surface of the strip, and
wherein the reagents are at least partly contained within the receptacle or partial receptacle.
30 . A kit for the determination of the amount of cholesterol in high density lipoproteins in a high density lipoprotein containing sample, the kit comprising
an electrochemical cell having a working electrode, a reference or pseudo reference electrode and optionally a separate counter electrode;
the reagents defined in claim 21 ;
a power supply for applying a potential across the cell; and
a measuring instrument for measuring the resulting electrochemical response.
31 . A method of operating a kit as defined in claim 24 , said method comprising
(i) contacting (1) the reagents and (2) a high density lipoprotein containing sample, with each other and with the electrodes;
(ii) applying a potential across the electrochemical cell; and
(iii) electrochemically detecting the amount of product formed by measuring the resulting electrochemical response.
32 . (canceled)