Alkaline phosphatase activity on osteoblast - (Mar/10/2013 )

Hello

I have cultured human fetal osteoblast (hFOB 1.19) and I need to check for the alkaline phosphatase activity (ALP) for that cells.
I use the plant extract as for the treatment for differentiation and proliferation of the osteoblast.

I never did ALP test before. Does everybody can help regarding the protocol for ALP assay?
I will use ALP kit from RANDOX.

Thank you for help.

Bone ALP Activity : sample is pretreated by incubating 100 μl of serum for 30 min. at 37 °C with 10 μl of a 20 g/L Triton x-100 aqueous dH2O solution. After this pretreatment, to prevent biliary alkaline phosphatase isoenzyme co-precipitation, add at sample 100 μl of an aqueous dH2O solution 5 g/L Lectin from Triticum vulgaris (Wheat germ – Sigma cod. L9640), dissolving 2 mg highly purified essentially salt free, lyophilized powder in 0.4 ml dH2O and after recostitution it is stable for at least a month at 2-8 °C. After the second incubation, the serum mixture is centrifuged at 2000 g for 10 min. Without disturbing the precipitate, aspirate the supernatant, determine its ALP activity by means DGKC kinetic method at 37 °C, and multiplied the result by 2.1 to correct sample dilution. Bone ALP activity is calculated by subtracting this corrected value from the total ALP activity. For calculating the total analytical recovery of isoenzyme activities is useful resuspending the precipitate in 200 μl of isotonic saline and determined its ALP activity. Combining the activities of the precipitate and supernatant and adjusting for sample dilution gave a value for the total recovery of isoenzyme activity. Normal value : male 20-60 year old 27-96 U/L; Female 20-60 year old  32-102 U/L; Children 0-14 year old 120-544 U/L; Pregnacy 36-100 U/L .

You can apply this method of fractional precipitation for to research, with the Randox kit, bone alkaline phosphatase. If you desire to have a protocol for to make a Home-made kit useful for alkaline phosphatase determination let me know and I will send you the protocol.

Modified reagent to measure BALP activity in serum   

Precipitating reagent : dissolve in acetate buffer (CH3COONa/CH3COOH) 5 mM at pH 4.6, preparated  dissolving 0.41 g of anhydrous CH3COONa (or 0.68 g of CH3COONa•3H2O) in 800 ml of distilled water. About 0.29 ml of glacial acetic acid is added to adjust pH. Check  and, if is necessary, adjust pH to 4.6. Then dissolve 20 g of Triton X-100 and 0.1 g NaN3. Mix gently, avoiding foam formation and bring to 1 liter volume with distilled water. Mix such as above and dissolve 4.5 g/L of weat germ lectin from Triticum vulgaris. This reagent is ready for use, and stable for 1 year when stored from +2 to +8 °C in amber glass bottle.

Procedure

Dispense in microcentrifuge tube (Eppendorf) 100 μl serum, non hemolysed that is stable for 6 hrs at RT while for several months when stored at -20 °C, with 100 μl of precipitating reagent. Mix, and allows stay for 30 min. at RT. Centrifuge at 4000 rpm for 15 min. at RT, and transfer supernatant, for residual alkaline phosphatase (ALP_R) determination, with DEA-pNPP method at 37°C, according to DKCG. At the same time, measure total ALP on untreated sample.  

Bone alkaline phosphatase (BALP) is expressed in U/L and calculate using the following correcting function :

BALP = (1.118 x ALP_T) – (2.35 x ALP_R)

where : ALP_T = total ALP activity and ALP_R = residual supernatant activity.