Enzymatic method and stabilized solutions for determining total cholesterol in human serum

An aqueous stabilized enzyme solution for use in the determination of total cholesterol which comprises dissolved in an aqueous buffer solution having a pH range of about 4 to 9, a surfactant present in an amount up to about 2.5 percent by volume of solution and an effective amount of sodium cholate, the cholesterol esterase, cholesterol oxidase, peroxidase, 4-aminoantipyrine, phenol and a polyhydroxy organic compound in an amount of about 7.5 percent by volume of solution to an amount insufficient to materially reduce enzyme activity of said cholesterol esterase, cholesterol oxidase and peroxidase.

A two-solution total cholesterol assay kit formed of an aqueous enzyme concentrate solution and a chromogen diluent solution, in which:

• (a) said aqueous enzyme concentrate solution comprises an aqueous buffer solution having a pH range of about 4 to 9, a surfactant and an effective amount of a salt of cholic acid, cholesterol esterase, cholesterol oxidase, 4-aminoantipyrine, and a polyhydroxy organic compound present in an amount of about 7.5 percent by volume of the enzyme concentrate solution to an amount insufficient to reduce enzyme activity of said cholesterol esterase, cholesterol oxidase and peroxidase in combined aqueous enzyme concentrate solution and chromogen diluent solutions;
• and
• (b) said chromogen diluent solution comprises dissolved in an aqueous solution having a pH of about 4 to 9, a surfactant and an effective amount of phenol, the total amount of surfactant present being up to 2.5 percent by volume of the combined aqueous enzyme concentrate solution and chromogen diluent solution.

A stabilized aqueous enzyme solution for use in the determination of total cholesterol which comprises dissolved in an aqueous solution having a pH of about 4 to 9, a surfactant present in an amount up to about 2.5 percent by volume of solution, a salt of cholic acid present in an amount up to about 15 grams per liter of solution, cholesterol oxidase present in an amount of from about 300 to 1,000 IU/l of solution, cholesterol esterase present in an amount up to about 1,000 IU/liter of solution, and a polyhydroxy organic compound present in concentration of from about 7.5 to about 50 percent by volume of solution.

An aqueous stabilized enzyme solution for use in the determination of total cholesterol which comprises dissolved in an aqueous buffer solution having a pH range of about 4 to 9, a surfactant present in an amount up to about 2.5 percent by volume of solution, sodium cholate present in an amount up to about 15 grams per liter of solution, cholesterol esterase present in an amount up to about 1,000 IU per liter of solution, cholesterol oxidase present in an amount of from about 300 to 1,000 IU per liter of solution, peroxidase, present in an amount of from about 150 to 300 KU per liter of solution, 4-aminoantipyrine present in an amount of from about 150 to about 450 mg per liter of solution, an effective amount of phenol is present and a polyhydroxy organic compound in an amount of about 7.5 to about 50 percent by volume of solution.

A two-solution total cholesterol assay kit formed of an aqueous enzyme concentrate solution and a chromogen diluent solution, in which:

• (a) said aqueous enzyme concentrate solution comprises an aqueous buffer solution having a pH range of about 4 to 9, a surfactant and a salt of cholic acid present in an amount up to 15 grams per liter of enzyme concentrate solution, cholesterol esterase present in an amount up to about 1,000 IU per liter of solution, cholesterol oxidase present in an amount of from about 300 to 1,000 IU per liter of solution, 4-aminoantipyrine present in an amount of from about 150 to about 450 mg per liter of solution, peroxidase present in an amount of from about 150 to 300 IU per liter of solution, and a polyhydroxy organic compound present in an amount of about 7.5 percent to about 50 percent by volume of the enzyme concentrate solution;
• and
• (b) said chromogen diluent solution comprises dissolved in an aqueous buffer solution and having a pH of about 4 to 9, a surfactant and an effective amount of phenol, the total amount of surfactant present being up to 2.5 percent by volume of the combined aqueous enzyme concentrate solution and chromogen diluent solution.

A kit including a stabilized enzyme concentrate solution and chromogen diluent solution for use in assaying total cholesterol in a sample comprising:

• (a) a stabilized enzyme concentrate solution comprising an aqueous buffer solution providing a pH of about 4 to about 9 and containing an effective amount of each of sodium cholate, cholesterol esterase, cholesterol oxidase, peroxidase, 4-aminoantipyrine, a surfactant, and a a polyhydroxy compound selected from the group consisting of glycerol, ethylene glycol, sorbitol and propylene glycol;
• and
• (b) a chromogen diluent solution comprising an aqueous solution containing an effective amount of phenol and a surfactant, the total of said surfactant provided being in an amount up to about 2.5 percent by volume of the total volume of the stabilized enzyme concentrate and chromogen diluent solution and said polyhydroxy organic compound being provided in an amount insufficient to materially reduce enzyme activity of said cholesterol esterase, cholesterol oxidase and peroxidase.

A kit for use in assaying total cholesterol in a sample comprising a stabilized aqueous enzyme concentrate solution which is diluted with water in a ratio of about one part enzyme solution to about four parts water prior to conducting an asay, the concentrate solution comprising about 50 percent by volume of an aqueous buffer solution providing a pH of about 6.60, about 50 percent by volume glycerol, 10 mM/l.+-.2 percent of sodium cholate, 1,000 IU/l.+-.5 percent of cholesterol esterase, about 1.5 percent by volume surfactant, 500 IU/l.+-.5 percent cholesterol oxidase, 150,000 IU/l.+-.5 percent peroxidase, 1.5 mM/l.+-.20 percent of 4-aminoantipyrine and 30 mM/l.+-.2 percent of phenol.

A kit for use in the oxygen consumption assay of total cholesterol in a sample comprising a stabilized aqueous enzyme solution comprising about 92.5 percent by volume of an aqueous buffer solution providing a pH of about 6.6, 10 mM/l.+-.2 percent of sodium cholate, about 1000 IU/l.+-.5 percent cholesterol esterase, 7.5 percent by volume glycerol, about 0.225 percent by volume surfactant, and about 1000 IU/l.+-.5 percent cholesterol oxidase.

A kit for use in the oxygen consumption assay of total cholesterol in a sample comprising an aqueous stabilized enzyme concentrate solution which can be diluted with water in a ratio of one part enzyme concentrate to four parts water, the stabilized enzyme concentrate solution comprising about 50 percent by volume of an aqueous buffer solution providing a pH of about 6.6, about 50 percent by volume glycerol 10 MM/l.+-.2 percent sodium cholate, and about 5333 IU/l.+-.5 percent cholesterol esterase, about 1.5 percent by volume surfactant, and about 6,666 IU/l cholesterol oxidase.