H-1117 Budapest,
CHOLESTEROL PAP
Budafoki street 111-113 STABLE LIQUID REAGENT
Tel.: +36-1-205-3617 Cat. No.: 47061,47061S 47062,47062S 47063
120 ml 600 ml 20x20 ml
Tel./Fax: +36-1-205-3616
Reagent kit for the quantitative determination of total cholesterol concentration in
serum. Enzymatic colorimetric method (PAP).
The biosynthesis of Cholesterol predominantly takes place in the liver and in intestinal
mucosa, but almost all cells synthesize it. It is a constituent of many membranes, it is Calculation using calibration
also essential in the synthesis of bile acids and steroid hormones. It circulates in blood as
Asample
cholesterol ester bound to beta lipoproteins. The measurement of the level of Cholesterol as
well as Triglycerides and Lipoproteins is important in examining the metabolism of lipids.
xC standard = C sample
Changes in the level of Cholesterol mainly reflect disorders of liver function. Cholesterol level
Astandard
is increased in obstructive jaundice, diabetes mellitus and hypothyroidism. The level is A = Absorbance,
decreased in some cases of hyperthyroidism and certain forms of anaemia. Identification of the C = Concentration
different density fractions (HDL, LDL, VLDL) as well as total Cholesterol plays a role in
the diagnosis. Quality control
Principle A quality control program is recommended for all clinical laboratories. The analysis of
The Cholesterol esters of the sample are hydrolysed by Cholesterol esterhydrolase (ChEH). 4- control material in both the normal and abnormal ranges with each assay is recommended
Cholesten-3-one and H2O2 are then formed from the released free Cholesterol by Cholesterol for monitoring the performance of the procedure. Each laboratory should establish corrective
oxidase (ChOD).A measurable Red quinonimine derivative which absorbance light measures to be taken if values fall outside the limits.
at 505 nm is formed from Hydrogenperoxide (H2O2) and 4-Aminoantipyrine in the
presence of Phenol and peroxidase (POD). PERFORMANCES DATA
Cholestero
l → Cholesterol + Fatty acids
esterase The following data were obtained using the Olympus 400 analyzer (37°C).
Cholesterol ester+H2O
Linearity
Cholesterol+O2 Cholestero
l → 4-Cholesten-3-one+ H2O2
oxidase
Up to 20.0 mmol/l (773 mg/dl).
2H2O2+Phenol+4-Aminoantipyrine → Red quinone+4H O
peroxidase
2
Sensitivity
Reference values It is recommended that each laboratory establishes its own range of sensitivity as this is
Serum cholesterol: 2.8-5.2 mmol/l (109-202 mg/dl) limited by the sensitivity of the spectrophotometer used. Under manual conditions however,
It is recommended that each laboratory should assign its own normal range. a change of 0.001 Abs is equivalent to 0.015 mmol/l (0,58 mg/dl) Cholesterol concentration
at 492 nm.
Reagents
1. Reagent (R1) Precision
Pipes buffer,pH=6.90 50 mmol/l
Phenol 24 mmol/l
Sodium cholate 0.5 mmol/l Reproducibility
4-Aminoantipyrine 0.5 mmol/l Average concentration
Cholesterol esterase 180 U/l SD CV%
(mmol/l)
Cholesterol oxidase 200 U/l
Peroxidase 1000 U/l Sample I 2.5 0.044 1.79
Sample II. 5.4 0.093 1.72
2. Cholesterol standard
Ready for use.For details please check the insert.
Available only in Cat. No.: 47062S and 47061S Repeatability
Average concentration
Precautions SD CV%
Discard cloudy reagent. Avoid contamination by using clean laboratory materials (pipettes, (mmol/l)
plastic vials….). The reagents contain 0.1 % sodium azide. To avoid the possible build-up of Sample I 3.6 0.034 1.02
azide compounds, flush waste-pipes with water after the disposal of undiluted reagent.
Sample II. 11.8 0.094 0.80
PROCEDURE Correlation
Comparative studies were done to compare our reagent with another commercial Cholesterol
Preparation and stability of working reagents PAP reagent.
The reagent is ready for use. The results from these studies are detailed below.
If the absorbance of working reagent is higher than 0.28 at 492 nm the reagent can not Correlation coefficient: r =0.9994
be used. Linear regression: y (mmol/l)= 1.006x-0.105
(x= other commercial reagent, y= own reagent).
Samples
Serum free of haemolysis. Specificity
Bilirubin 855 µmol/l (50 mg/dl), lipid 450 mg/dl, glucose 55.5 mmol/l (1000 mg/dl) and
Assay conditions ascorbic acid 0.6 mmol/l (10 mg/dl) don’t interfere with the assay up to the given levels.
Wavelength: 505 (480-520) nm
Temperature: 37°C NOTE
Cuvette: 1 cm light path Do not use reagents after the expiry date stated on each reagent container label. Do not use
Read against: reagent blank products, test solutions and reagents described above for any purpose other than described
Method: endpoint (increasing) herein.
Pipette into cuvette
For in vitro diagnostic use only.
Blank Standard Sample
Reagent 1 ml 1 ml 1 ml The following symbols are used on labels
Distilled water 10µl
Standard 10µl
For in vitro diagnostic use
Sample 10µl
Mix and read the absorbance (A) after a 5-minute incubation. Use by (last day of the month)
Calibration: (37°C, Cholesterol oxidase method)
S1: Distilled water Temperature limitation
S2: Cholesterol standard Cat. No.: 50611N or
Roche C.F.A.S. (Calibrator for automated system) Batch Code
Randox Calibration Serum Level I or
Randox Calibration Serum Level II Code
Calibration frequency:
Two point calibration is recommended
- after reagent lot change, Bibliography
- as required following quality control procedures.
Allain C.C and al., Clin.Chem.,20, (1974), 470.
Cholesterol PAP stable liquid reagent Date of revision: 2013-02 70-EN-2013-10
