Print

DISCRETPAK™ REAGENTS - Cholesterol

Intended Use

For IN VITRO diagnostic use in the quantitative determination of Cholesterol in serum using manual or automated applications.

Method Principle

Cholesterol esters present in the serum sample are hydrolyzed into free Cholesterol and fatty acids by pancreatic Cholesterol Esterase (CE). The free Cholesterol formed and in the presence of oxygen, is then oxidized to Cholest-4-en-3-one by Cholesterol Oxidase (CO) with concomitant production of Hydrogen Peroxide. The Hydrogen Peroxide produced is quantitatively determined by coupling 4-aminoantipyrine with p-Hydroxybenzoic acid where a quinoneimine dye with maximum absorption at 500 nm is produced. The intensity of the dye color thus produced is directly proportional to the concentration of the total Cholesterol in the serum sample. The reaction scheme below illustrates the reactions that occur in this method.

CE
           Cholesterol esters ────────> Cholesterol + fatty acids

CO
               Cholesterol + O2 ────────> Cholest-4-en-3-one + H2O2

                 Peroxidase
2H2O2 + 4AA + p-Hydroxybenzoic acid ────────> Quinoneimine dye + 4H2O


Preparation Of Working Reagent

Catachem Cholesterol reagent is packaged in ready-to-use form. No preparation is required.

Storage and Stability

Store the Cholesterol Reagent at 2-8°C. When stored as directed, the reagent is stable until the expiration date stated on the label. When stored as directed, the Working Cholesterol Reagent is stable for 12 months at 2-8°C and 12 weeks at room temperature.

Method Performance Characteristics

Sensitivity: 0.0010-0.0020 absorbance units per mg/dL.
Linear Range: 0-600 mg/dL.
Precision:  Within-run and day-to-day precision is summarized below.

Cholesterol

Within-Run Precision

Total Precision

MEAN

SD

CV

SD

CV

mEq/L

mg/dL

%

mg/dL

%

56

0.71

1.2

0.71

1.30

261

1.95

0.75

6.30

2.40

456

2.97

0.65

3.00

0.70


Correlation

A comparison of this method using an automated analyzer and a reference method based on the CO and CE reaction resulted in the following regression statistics:

Correlation Data

Parameter

Data Observed

N

155

Range

56 - 456

Regression

Y = 1.003x + 4.3

Correlation

  r = 0.997

Sy,x

5.7