Veterinary Clinical Biochemistry and Laboratory Diagnosis

PROCEDURE

Sample

• Serum / plasma

Reagents:[The reagents in the kit vary from manufacturer to manufacturer, so the procedure in  the kit used may be followed.] 

• Cholesterol Reagent
• Working cholesterol standard, 200 mg%
• Precipitating reagent

Procedure: (Agappe Diagnostics kit)

For total cholesterol

• Mark three test tubes as Blank, Standard and test and proceed as follows.

S. No.	Reagents	Blank (B)	Standard (S)	Test (T)
1.	Cholesterol Reagent (ml)	3.0 ml	3.0	3.0
2.	Cholesterol Standard (µl)	-	15	-
3.	Serum (μl)	-	-	15

• Mix well and keep the tubes immediately in the boiling water bath exactly for 90 seconds (1 ½ minutes).
• Cool them immediately to R.T. under running tap water. Measure the O.D. of the tubes at 560 or using a yellow green filter.

For HDL Cholesterol

• Step I: HDL Cholesterol separation

Pipette into centrifuge tube	Quantity
Sample	200 μl
Precipitating reagent	2 ml

• Mix well keep at R.T. for 10 minutes.
• Then centrifuge at 200 rpm for 15 minutes to obtain a clear supernatant.
• Step II: HDL- Cholesterol estimation
  • Mark three test tubes, Blanks, Standard and test and proceed as follows.

  Reagents	Blank (B)	Standard (S)	Test (T)
  Cholesterol Reagent	3.0 ml	3.0 ml	3.0 ml
  Standard	-	15 μl	-
  Serum	-	-	120 μl

  • Mix well and keep the tubes immediately in the boiling water bath exactly for 90 seconds (1 ½ minutes).
  • Cool them immediately to R.T. under running tap water. Measure the O.D of all the tubes at 560 nm using a yellow green filter.

Calculations