PRINCIPLE

Alkaline Phosphatase in serum catalyzes the hydrolysis of p-nitrophenyl phosphate to p-nitrophenol and phosphate.

The rate of formation of p-Nitrophenol is measured as an increase in absorbance, which is proportional to ALP activity in the sample.

REACTION

P-Nitrophenylphosphate —(ALP)—→ p-Nitrophenol + Phosphate

PROCEDURE

Pipette into a clean dry test tube labeled as Test (T):

Mix well and read the initial absorbance A1 after 1 minute and record the absorbance after every 1, 2, 3 minutes.

Calculate the mean absorbance change per minute (ΔA/min).

CALCULATION

ALP Activity in U/L = ΔA/min × 2764

NORMAL VALUE

• Children (0–15 yrs): 104–390 IU/L

• Adults (>15 yrs): 25–140 IU/L

Note: Each laboratory should establish its own normal range.

CLINICAL SIGNIFICANCE

Alkaline Phosphatase (ALP) is an enzyme of the Hydrolase class of enzymes and acts in an alkaline medium. It is found in high concentrations in the liver, biliary tract epithelium and bones.
Normal levels are age-dependent and increase during bone development.

Increased levels are associated mainly with liver and bone disease. Moderate increases are seen in Hodgkin’s disease and congestive heart failure.

LIMITATIONS & PRECAUTIONS

1. Storage conditions as mentioned on the kit to be adhered.

2. Do not freeze or expose the reagents to higher temperature as it may affect kit performance.

3. Before the assay, bring the reagent to room temperature.

4. Avoid contamination of the reagent during assay process.

5. Use clean glassware free from dust or debris.

6. Reagent/sample ratio as mentioned must be strictly observed as any change will affect the result.

7. Do not use the reagent if found hazy or cloudy.

8. GENERAL SYSTEM PARAMETERS

9. • Reaction type: Kinetic reaction (Increasing)

   • Wave length: 405 nm

   • Temperature: 37 °C

   • Delay: 60 sec

   • Interval: 30 sec

   • No. of readings: 5

   • Sample volume: 20 µL

   • Reagent volume: 1000 µL

   • Factor: 2764

   • Zero setting: Deionized water