Enzymatic reagent system for total cholesterol assay using oxygen-rate method
First Claim
1. A cholesterol assay enzymatic reagent for rate determination of cholesterol in a sample to be assayed of the type comprising cholesterol oxidase, and a buffering agent in an amount to produce a solution having a pH of between about 5.5 and about 8, characterized in that said reagent further comprises means for neutralizing substantially all oxygen consumption inhibiting effects of inhibiting effects present in said sample to be assayed, said means consisting of alkyldimethylbenzylammonium salt in an amount sufficient to neutralize substantially all oxygen consumption inhibiting effects of inhibiting agents present in said sample to be assayed.
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Abstract
A novel enzymatic reagent system is provided for assaying for total cholesterol in a liquid containing cholesterol, cholesterol esters, oxygen and substances which appear to inhibit the reactions between the enzymes and cholesterol and/or cholesterol esters. The novel reagent system comprises an aqueous solution of a buffering agent, cholesterol oxidase, cholesterol esterase, and a cationic surfactant in an amount sufficient to neutralize the effect of the inhibiting agents.
15 Citations
27 Claims
- 1. A cholesterol assay enzymatic reagent for rate determination of cholesterol in a sample to be assayed of the type comprising cholesterol oxidase, and a buffering agent in an amount to produce a solution having a pH of between about 5.5 and about 8, characterized in that said reagent further comprises means for neutralizing substantially all oxygen consumption inhibiting effects of inhibiting effects present in said sample to be assayed, said means consisting of alkyldimethylbenzylammonium salt in an amount sufficient to neutralize substantially all oxygen consumption inhibiting effects of inhibiting agents present in said sample to be assayed.
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3. A cholesterol assay enzymatic reagent for rate determination of cholesterol in a sample to be assayed comprising:
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(a) from about 0.2 to about 5 International Units of cholesterol oxidase per milliliter of reagent; (b) from about 0 to about 5 International Units of cholesterol esterase per milliliter of reagent; (c) from about 0.01 to about 0.4 percent by weight of said reagent of an alkyldimethylbenzylammonium salt; and (d) a buffering agent in an amount to produce a solution having a pH of between about 5.5 and about 8. - View Dependent Claims (4, 5)
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6. A cholesterol assay enzymatic reagent for rate determination of cholesterol in a sample to be assayed comprising:
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(a) a buffering agent in an amount to produce a solution having a pH of about 6; (b) about 1.2 International Units of cholesterol oxidase per milliliter of reagent; (c) about 0.014 International Units of cholesterol esterase per milliliter of reagent; (d) about 0.08 percent by weight of said reagent of a bile salt; (e) about 0.05 percent by weight of said reagent of isooctylphenoxypolyethoxyethanol; and (f) about 0.1 percent by weight of said reagent of an alkyldimethylbenzylammonium salt. - View Dependent Claims (7)
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8. In a cholesterol assay enzymatic reagent kit capable of being employed for rate determination of cholesterol in a sample to be assayed of the type comprising:
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(a) a first reagent comprising; (i) cholesterol esterase and (ii) cholesterol oxidase; and (b) a second reagent comprising; (i) a buffer solution having a pH of between about 5.5 to about 8; said reagents adapted to be employed together;
the improvement being that said second reagent further comprises means for neutralizing substantially all oxygen consumption inhibiting effects of inhibiting agents present in said sample to be assayed, said means consisting of an alkyldimethylbenzylammonium salt in an amount sufficient to neutralize substantially all oxygen consumption inhibiting effects of inhibiting agents present in said sample to be assayed.
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9. In a cholesterol assay enzymatic reagent kit capable of being employed for rate determination of cholesterol in a sample to be assayed of the type comprising:
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(a) a first reagent comprising cholesterol esterase; (b) a second reagent comprising cholesterol oxidase; and (c) a third reagent comprising a buffering solution having a pH of from about 5.5 to about 8; said reagents adapted to be employed together;
the improvement being that said third reagent further comprises a means for neutralizing substantially all oxygen consumption inhibiting effects of inhibiting agents present in said sample to be assayed, said means consisting of an alkyldimethylbenzylammonium salt in an amount sufficient to neutralize substantially all oxygen consumption inhibiting of inhibiting agents present in said sample to be assayed.
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10. A cholesterol assay enzymatic reagent kit capable of being employed for rate determination of cholesterol in a sample to be assayed comprising, base upon a combined solution of a first reagent comprising cholesterol esterase;
- a second reagent comprising cholesterol oxidase; and
a third reagent comprising a buffer solution having a pH of about 6, a bile salt, and a non-ionic surfactant, said reagents adapted to be employed together;(a) an aqueous solution of potassium orthophosphate in an amount to produce a solution having a pH of about 6; (b) about 0.08 percent by weight of said combined solution of sodium cholate; (c) about 0.05 percent by weight of said combined solution of isooctylphenoxypolyethanol; (d) about 0.1 percent by weight of said combined solution of an alkyldimethylbenzylammonium salt; (e) about 1.2 International Units of cholesterol oxidase per milliliter of said combined solution; (f) about 0.014 International Units of cholesterol esterase per milliliter of said combined solution. - View Dependent Claims (11)
- a second reagent comprising cholesterol oxidase; and
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12. In a rate method for determining the cholesterol concentration in a cholesterol containing sample comprising the steps of:
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(a) oxidizing the cholesterol present in the sample in an oxygen saturated aqueous solution by means of a cholesterol assay enzymatic reagent of the type comprising; (i) cholesterol oxidase and; (ii) a buffering agent in an amount to produce a solution having a pH of between 5.5 and about 8;
in the presence of a sensor which serves to monitor a property or characteristic of oxygen in said solution related to the oxygen concentration thereof;(b) generating a first electrical signal related to said oxygen concentration; (c) differentiating said first electrical signal to produce an output signal proportional to the instantaneous time rate of change of oxygen concentration; and (d) measuring said output signal to determine said cholesterol concentration;
the improvement comprising neutralizing substantially all oxygen consumption inhibiting effects of inhibiting agents in said sample to be assayed by including in said cholesterol assay enzymatic reagent a cationic surfactant in an amount sufficient to neutralize substantially all oxygen consumption inhibiting effects of inhibiting agents present in said sample to be assayed. - View Dependent Claims (13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23)
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24. In a method of determining the cholesterol concentration in a cholesterol containing sample comprising the steps of:
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(a) oxidizing the cholesterol present in the sample in an oxygen saturated aqueous solution by means of a cholesterol assay enzymatic reagent of the type comprising; (i) cholesterol oxidase; and (ii) a buffering agent in an amount to produce a solution having a pH of between about 5.5 and about 8 in the presence of a sensor which serves to monitor a property or characteristic of oxygen in said solution related to the oxygen concentration thereof; (b) generating a first electrical signal related to said oxygen concentration; (c) differentiating said first electrical signal to produce an output signal proportional to the instantaneous time rate of change of oxygen concentration; and (d) measuring said output signal to determine said cholesterol concentration; the improvement comprising neutralizing substantially all oxygen consumption inhibiting effects of inhibiting agents in said sample to be assayed by including in said cholesterol assay enzymatic reagent from about 0.01 to about 0.4 percent by weight of said reagent of a cationic surfactant. - View Dependent Claims (25, 26, 27)
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Specification