Biosystems Alfa Reagent
PRINCIPLE OF THE METHOD
α-Amylase catalyzes the hydrolysis of 2-chloro-4-nitrophenyl-malto-trioside (CNP-G3) to
2-chloro-4-nitrophenol (CNP). The catalytic concentration is determined from the rate of
2-chloro-4-nitrophenol formation, measured at 405 nm1,2,3
A. Reagent: MES 50 mmol/L, calcium chloride 5 mmol/L, sodium chloride 300 mmol/L, sodium
thiocyanate 450 mmol/L, CNP-G3 2.25 mmol/L, pH 6.1.
Store at 2-8ºC.
Reagent is stable until the expiry date shown on the label when stored tightly closed and if
contaminations are prevented during their use.
Indications of deterioration:
Reagent: Presence of particulate material, turbidity, absorbance of the blank over 0.500 at
405 nm (1 cm cuvette).
Reagent (A) is provided ready to use.
− Analyzer, spectrophotometer or photometer with cell holder thermostatable at 25, 30 or 37ºC
and able to read at 405 nm.
− Cuvettes with 1 cm light path.
Serum, plasma or urine collected by standard procedures.
α-Amylase in serum or plasma is stable for 1 month at 2-8ºC. Use heparin as anticoagulant.
α-Amylase in urine is stable for 1 month at 2-8ºC if pH is adjusted to approximately 7 before
1. Bring the Reagent and the instrument to reaction temperature.
2. Pipette into a cuvette: (Notes 1,2)
Serum or plasma Urine
37ºC 25ºC,30ºC 37ºC 25ºC,30ºC
Reagent (A) 1.0 mL 1.0 mL 1.0 mL 1.0 mL
Sample 20 µL 50 µL 10 µL 20 µL
3. Mix and insert the cuvette into the photometer. Start the stopwatch.
4. Record initial absorbance and at 1 minute intervals thereafter for 3 minutes.
5. Calculate the difference between consecutive absorbances, and the average absorbance
difference per minute (∆A/min).