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Alkaline Phosphatase Kit

This document provides instructions for determining alkaline phosphatase (ALP) activity in serum using a kinetic method with p-nitrophenylphosphate (PNPP) as the substrate. ALP is an enzyme found in the liver, biliary tract, and bones. The assay involves mixing the serum sample with a working reagent containing PNPP buffer and measuring the increase in absorbance at 405nm over time, which is directly proportional to ALP activity in the sample. The change in absorbance per minute is used to calculate the ALP level based on defined parameters. Normal ALP levels vary with age but are elevated in liver and bone diseases.

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Dinesh Sreedharan
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1K views2 pages

Alkaline Phosphatase Kit

This document provides instructions for determining alkaline phosphatase (ALP) activity in serum using a kinetic method with p-nitrophenylphosphate (PNPP) as the substrate. ALP is an enzyme found in the liver, biliary tract, and bones. The assay involves mixing the serum sample with a working reagent containing PNPP buffer and measuring the increase in absorbance at 405nm over time, which is directly proportional to ALP activity in the sample. The change in absorbance per minute is used to calculate the ALP level based on defined parameters. Normal ALP levels vary with age but are elevated in liver and bone diseases.

Uploaded by

Dinesh Sreedharan
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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ALKALINE PHOSPHATASE

(PNPP Kinetic Method)


For the determination of Alkaline Phosphatase activity in Serum
(For In vitro Diagnostic Use Only)

SUMMARY:
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 in the 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 Hodgkins disease and congestive heart failure.

PRINCIPLE: ALP
p-Nitrophenylphosphate+H2O p - Nitrophenol +phosphate

CONTENTS:

PACK SIZE SUBSRATE REAGENT(A1) BUFFER REAGENT(A2) STANDARD (S)

5x5ml 5x5ml 30ml 5ml

6x10ml 6x10ml 60ml 5ml

50x5ml 50x5ml 50x5ml 5ml

STORAGE AND STABILITY:


Contents are stable at 2-8C0 till the expiry mentioned on the labels.

REAGENT PREPARATION:

WORKING REAGENT: Dissolve (A1) substrate vial using equivalent volume of (A2) Buffer. This working reagent is
stable for at least 45 days when stored at 2-8°C.THE SUBSTRATE IS LIGHT & TEMPERATURE SENSITIVE. TAKE
ADEQUATE CARE, ESPECIALLY AFTER RECONSTITUTION.

SAMPLE:
Serum Free from hemolysis. ALP is reported to be stable in serum for 3 days at 2-8°C.

GENERAL SYSTEM PARAMETERS:


Reaction Mode Kinetic Sample Volume 20 µl
Wavelength 405 nm Reagent volume 1000 µl
Blank Distilled Water Factor 2754
Temperature 37˚C Reaction Slope Increasing
Delay Time 60 sec Linearity 1000 IU/L
Read Time 180 sec Units IU/L
ASSAY PROCEDURE:

Wavelength/ Filter : 405 nm


Temperature : 37˚C
Light Path : 1 cm
Pipette into a clean dry test tube labeled as Test (T):

Addition Sequence T(µl)


Working Reagent(WR) 1000 µl
Incubate at the assay temperature for 1 min and add
Sample 20 µl
Mix well and read the initial absorbance A0 after 1 minute & repeat the absorbance reading after every 1,2 & 3
minutes. Calculate the mean absorbance change per minute (∆A / min)

CALCULATIONS:
ALP Activity in IU/L = ∆A / min x 2754 x TF

TEMPERATURE CONVERSION FACTORS:

Assay Temperature Factor TF

25⁰C 1.7

30⁰C 1.34

37⁰C 1.00

NORMAL REFERENCE RANGES:


Serum (Adults) : 80-290 IU/L at 370C
(Children) : 245-770 IU/L at 370C

LINEARITY:

The procedure is linear up to 1000 IU/L at 37°C. If the absorbance change (∆A/min.) exceeds 0.250, use only the
value of the first two minutes to calculate the result, or dilute the sample 1+9 with normal saline (NaCl 0.9%) and
repeat the assay (Results x 10).

NOTE:

Samples having a very high activity show a very high initial absorbance. If this is suspected then dilute the
sample and repeat the assay. Adherence to the reaction time should be meticulously followed. Substrate Reagent
and Working Reagent should not be exposed to light or left at R.T.

REFERENCES:

1. Bowers, G.N.McCommb, R.B.(1972) Clin. Chem. 18:97


2. Recommendations of German society for clinical Chemistry, (1972)
3. Z. Clin. Chem. Bio. 10:182

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