T0 ESTIMATE THE SERUM ALKALINE

PHOSPHATASE

REAGENTS
R1 ALP buffer

– DEA buffer
– Magnesium chloride

R2 ALP substrate

– 4-NPP
– Biocides

Mix 4ml of R1 with 1ml of R2, the mixture is known as working reagent.
Stable for 5 days at 20-25°C or for 15-30 days at 2-8°C.

PRINCIPLE
Alkaline phosphatase ALP catalyzes the hydrolysis of 4-nitrophenylnitrate 4-
NPP with the formation of 4-nitrophenol and inorganic phosphate, acting the
alkaline buffer as phosphate -group accepter.

The reaction is monitored kinetically at 405nm by the rate of formation of 4-

nitrophenol, proportional to the activity of Alkaline phosphatase in the
sample.

ALP,Mg⁺⁺

4-nitrophenylphosphate + H2O -------→ 4-nitrophenol +Pi

pH > 9
PROCEDURE

Wavelength: 405nm

Light path: 1cm

Temperature: 20-25 °C

1- Preincubate the working reagent, samples and controls to the reaction

temperature.
2- Set the photometer to 0 absorbance with distilled water.
3- Pipette into a cuvette

Working Reagent 2mL

Sample or Control 40µL

4- Mix gently by inversion. Insert cuvette into the cell holder and start
stopwatch.
5- Incubate for 1 minute and record initial absorbance reading.
6- Repeat the absorbance reading exactly after 1, 2 and 3 minutes.
7- Calculate the difference between absorbencies.
8- Calculate the mean of the results to obtain the average change in
absorbance (ΔA/min).

0 min A0

1 min A1 A1 – A0 ΔA1

2 min A2 A2 – A1 ΔA2

3 min A3 A3 – A2 ΔA3
ΔA1 + ΔA2 + ΔA3 / 3 = ΔA /min

CALCULATIONS

U/L = Δ/min x 2764

U/L x 0.01667 = ------ µkat/L

RESULT

INTERPRETATIONS