Veterinary Clinical Biochemistry and Laboratory Diagnosis

PROCEDURE

Specimen

•  Serum / plasma

Reagents (Agappe Diagnostics kit)

• Buffered alkaline – µ – Ketoglutarate substrate – pH 7.4 
• DNPH Reagent
• Sodium hydroxide (4N) – stock
• Working pyruvate standard, 2 mM

Preparation of working solution

• Dilute 1 ml Sodium hydroxide (4N – stock to 10ml with purified water).
• Other reagents are ready to use.

Procedure [reagents in the kit vary from manufacturer to

                   manufacturer so the procedure in  the kit used may be followed.]

• Mark 6 test tubes 1, 2, 3, 4, 5 for standards and ‘T’ for test and proceed as follows:

Tube No.	1	2	3	4	5	Test
Enzyme activity (Units/ml)	0	28	57	97	150	Find out from standard graph
Buffered substrate (ml)	0.50	0.45	0.40	0.35	0.30	0.50
Pyruvate standard 2 mM (ml)	-	0.05	0.10	0.15	0.20	Serum – 0.10 ml
Distilled water (ml)	0.10	0.10	0.10	0.10	0.10	0.10
Mix well and incubate at 37°C for 30 min
DNPH reagent (ml)	0.50	0.50	0.50	0.50	0.50	0.50
Mix well and allow to stand at R.T for 20 minutes
Working NaOH (ml)	5.0	5.0	5.0	5.0	5.0	5.0
Mix well; After 10 minutes measure the O.D at 505 nm
OD values at 505 nm

Standard graph

• Plot standard graph by taking the enzyme units on X-axis and OD values in the Y-axis.

Calculation

• For test
  • From the standard curve obtained in the graph, extrapolate the test OD value to the corresponding enzyme activity in the X-axis.