Alkaline Phosphatase

Reagent Set

Intended Use Materials Provided

For the quantitative determination of Alkaline Phosphatase in human serum. Alkaline Phosphatase reagent.

Method History Materials Required but not Provided

Alkaline phosphatase in serum is determined by measuring the rate of 1. Accurate pipetting devices
hydrolysis of various phosphate esters under specified conditions. ρ- 2. Test tubes/rack
Nitrophenyl Phosphate is one such phosphate ester and was introduced as a 3. Timer
substrate by Fujita in 1939.1 Bessey, Lowry, and Brock published an 4. Spectrophotometer able to read at 405 nm (UV).
endpoint procedure in 19462 while Bowers and McComb reported a kinetic 5. Heating bath/block (37°C).
procedure in 1966.3 In 1974, the Committee on Enzymes of the
Scandinavian Society of Clinical Chemistry and Clinical Physiology adopted Procedure (Automated)
a modification of the above procedure as the recommended procedure.4 The Refer to specific instrument application instructions.
present method is based on the above two methods and that of Wilkinson, et
al.5 Procedure (Manual)
1. Reconstitute reagent according to instructions.
Principle 2. Pipette 1.0 ml of reagent into appropriate tubes and prewarm at 37°C for five
Alk Phos minutes.
ρ-NPP + H2O ----------------------------
ρ-Nitrophenol + H3PO4 3. Zero spectrophotometer with water at 405nm.
4. Add 0.025ml (25ul) of sample to reagent, mix and incubate at 37°C for one
ρ-Nitrophenyl phosphate is hydrolyzed to ρ-nitrophenol and inorganic minute.
phosphate. The rate at which the ρ-NPP is hydrolyzed, measured at 405 5. After one minute read and record the absorbance. Return tube to 37°C.
nm, is directly proportional to the alkaline phosphatase activity. Repeat readings every minute for the next two minutes.*
6. Calculate the average absorbance difference per minute (∆abs./min.)
Reagents 7. The ∆abs./min. multiplied by the factor 2187 (see Calculations) will yield
(Concentrations refer to reconstituted reagent). results in IU/L.
Alkaline Phosphatase Reagent: ρ-Nitrophenylphosphate 10.0mM, 8. Samples with values above 800 IU/L should be diluted 1:1 with saline, re-
Magnesium Ions 1.0mM, Buffer (pH 10.1±0.1), activator and binder. assayed and the results multiplied by two.
*NOTE: If the spectrophotometer being used is equipped with a temperature
Reagent Preparation controlled cuvette, the reaction mixture may be left in the cuvette while the
Reconstitute vial with the volume of distilled water stated on the vial label. absorbance readings are taken.
Swirl to dissolve.
Limitations
Reagent Storage This methodology measures total Alkaline Phosphatase irrespective of tissue or
Store reagent set at 2-8°C. Reconstituted reagent is stable for sixty days organ of origin. Further tests may be necessary to assist in differential diagnosis.
when stored at 2-8°C in amber glass bottle, and seven days at room
temperature. Calibration
The procedure is standardized by means of the millimolar absorptivity of ρ-
Reagent Deterioration Nitrophenol (18.75 at 405nm) under the specified conditions. Results are based
Do not use reagent when: on the change in absorbance per unit of time; all parameters must be known and
1. The dry powder cakes because of moisture penetration. controlled.
2. The reconstituted reagent has an optical density greater than 1.0 at
405nm. Calculation
One international Unit (IU/L) is defined as the amount of enzyme that catalyzes the
Precautions transformation of one micromole of substrate per minute under specified
1. This reagent is for in vitro diagnostic use only. conditions.
2. Avoid ingestion of all materials as toxicity has not been determined.
(IU/L) = ∆Abs./Min. x 1000 x 1.025 = ∆Abs./min. x 2187
Specimen Collection and Storage 18.75 x 1 x .025
1. Use non-hemolyzed serum (plasma should not be used since Where ∆Abs./Min. = Average absorbance change per minute
anticoagulants inhibit alkaline phosphatase activity).6,7 1000 = Conversion of IU/ml to IU/L
2. Serum samples should be stored at 2-8°C and run within two days.8 1.025 = Total reaction volume (ml)
18.75 = Millimolar absorptivity of ρ-Nitrophenol
. 025 = Sample Volume (ml)
Interferences 1 = Light path in cm
A number of drugs and substances affect alkaline phosphatase activity. See
Young, et al.6
Example: If your ∆ Abs/min. = 0.06
Then 0.06 x 2187 = 131IU/L

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 Alkaline Phosphatase
Reagent Set

NOTE: If test parameters are altered the factor has to be recalculated using
the above formula.

SI Units: To convert to SI Units (nkat/L) multiply IU/L by 16.67.

Quality Control
Serum controls with known normal and abnormal values should be run
routinely to monitor the validity of the reaction.

Suggested Values
Adults 35-123 IU/L at 37°C.
Children have a higher normal value. It is strongly suggested that each
laboratory establish its own normal range.

Performance
1. Linearity: 800 IU/L
2. Comparison: Studies between the present method and a similar
method yielded a correlation coefficient of 0.999 and a regression
equation of y=0.98x-2.5.
3. Precision:

Within Run Run to Run

Mean S.D. C.V.% Mean S.D. C.V.%
66 0.5 0.8 69 1.7 2.5
147 0.7 0.5 151 1.6 1.1

References
1. Fujita, H., J. Biochem., (Japan) 30:69 (1969).
2. Bessey, O.A., Lowry, O.H., Brock, M.J., J. Biol. Chem. 164:321 (1964).
3. Bowers, G.N., Jr., McComb, R.B., Clin. Chem. 12:70 (1966).
4. The Committee on Enzymes of the Scandinavian Society for Clinical
Chemistry and Clinical Physiology, Scand. J. Clin. Lab. Invest. 32:291
(1974).
5. Wilkinson, J.H., et al. Clin. Chem. 15:487 (1969).
6. Young, D.S., et al, Clin. Chem. 15:487 (1969).
7. Demetriou, J.A., Drewes, P.A., Gin, J.B., Clinical Chemistry: Principles
and Technics, 2nd Ed., Hagerstown (MD), Harper & Row, p.927 (1974).
8. Rej, R., Clin. Chem. 23:1903 (1977).

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