Principle
Cholesterol esterase hydrolysis cholesterol esters into free cholesterol and fatty acids. In the second reaction cholesterol oxidase converts cholesterol to cholest-4-en-3-one and hydrogen peroxide. In presence of peroxidase, hydrogen peroxide oxidatively couples with4-aminoantipyrine and phenol to produce red quinoneimine dye which has absorbance maximum at 510 nm (500 530). The intensity of the red colour is proportional to the amount of total cholesterol in the specimen.
Procedure
Reaction type ……………….. End-Point (2 Step)
Reaction time ………………..5 + 5 mins.
Wavelength ………………..546 nm.
Flow Cell temperature ………………..37°C
Zero setting with ………………..Distilled water
Sample volume ………………..Q.01 ml (10 μt)
Reagent volume ………………..0.750 ml + 0.250 ml
Linearity ………………..450 mg/dl
Quality Control
To ensure adequate quality control, it is recommended that each batch should include a normal and an abnormal commercial reference control serum. t should be realized that the use of quality control material checks both instrument and reagent functions together. Factors which might affect the performance of this test include proper instrument function, temperature Control, cleanliness of glassware and accuracy of pipetting.
Other Details
The reagent kit should be stored at 2- 89°C and is stable till the expiry date indicated on the label. R and R, reagents are stable till expiry at 2- 8°C. The working solution (4 R 1 R.) is stable for 30 days at 2-8°C.
Pankaj Lakhe –
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