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  1. Top
  2. Life Science
  3. Metabolism Research
  4. ELISA / Assay Kits (Metabolism)
  5. Alkaline phosphatase (ALP) Assay Kits
  6. LabAssay (TM) ALP

LabAssay (TM) ALP

for Cellbiology
Manufacturer :
FUJIFILM Wako Pure Chemical Corporation
Storage Condition :
Keep at 2-10 degrees C.
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Product Number
Package Size
Price
Inventory
Distributor
297-93501
Barcode No
4548995103048
500Tests
List Price
JPY 26,000

In stock in Japan

Please check here for notes on products and prices.

Document

SDS
Product Specification Sheet
Package Insert
Spectral Data
Certificate of Analysis
Calibration Certificate
Analytical Charts

Kit component

For 500 tests

Substrate Tablet 10 tablets
Buffer Solution 50 mL/1 bottle
Stop Solution 50 mL/1 bottle
Standard Solution 5 mL/1 bottle

Product Overview

Alkaline Phosphatase (ALP) is distributed in a variety of tissues such as liver, bone, and small intestine in animals. The change of the enzyme activity in tissues is an important hallmark for physiological phenomena as osteogenesis and so on. This kit is for Alkaline Phosphatase assay in a simultaneous multi-sample assay format with a microplate using p-Nitrophenylphosphate as a substrate.

[Note] LabAssay™ series are reagents for research purposes. They cannot be used for diagnostic purposes.

Kit Performance

Analysis sample Human Serum
Mouse Serum
Rat Serum
Dog Serum
Cat Serum
Culture medium (D-MEM)*
Calibration curve range 0.0625-0.5 mmol/L *p-nitrophenol
Sample volume 20 μL
Measurement duration Approx. 20 min
Wavelength 405 nm
*It is tested by using the samples that standard substances have been added to the culture medium.
Measurement availability depends on the culture medium, cell type and culture conditions. Please perform a spiked recovery test or dilution linearity test in advance using the culture medium to be used in your experiment.

Example of Calibration Curve

Example of Calibration Curve

Assay Principle

p-Nitrophenylphosphate is hydrolyzed into p-Nitrophenol and Phosphoric Acid in the carbonate buffer (pH 9.8) in the presence of Alkaline Phosphatase in sample. Released p-Nitrophenol showing yellow color is optically measured at 405 nm wavelength as the enzyme activity.

Procedure

Perform the assay in the wells according to the following table scheme.

Test Standard Blank
Working Assay Solution 100 μL 100 μL 100 μL
Sample Sample 20 μL Dilution series 20 μL Distilled water 20 μL
Shake for 1 minute by plate mixer and incubate for 15 minutes at 37 ℃
Stop Solution 80 μL 80 μL 80 μL
Shake for 1 minute by plate mixer and measure the absorbance at 405 nm of wavelength using microplate reader.

Data

Repeatability (within run precision)

Repeatability was conducted by measuring sixfold assays of serum samples from human, mouse, rat at two concentrations with this product.

n＼ID Human serum Mouse serum Rat serum
ID1 (mmol/L) ID2 (mmol/L) ID1 (mmol/L) ID2 (mmol/L) ID1 (mmol/L) ID2 (mmol/L)
1 0.768 0.383 0.503 0.269 0.568 0.325
2 0.761 0.368 0.490 0.268 0.580 0.324
3 0.746 0.367 0.508 0.263 0.571 0.311
4 0.753 0.378 0.485 0.280 0.568 0.332
5 0.739 0.370 0.522 0.264 0.578 0.332
6 0.738 0.386 0.507 0.281 0.580 0.333
mean 0.751 0.375 0.503 0.271 0.574 0.326
SD 0.0121 0.00814 0.0134 0.00783 0.00581 0.00838
CV(%) 1.6 2.2 2.7 2.9 1.0 2.6

[Result]
The CV (%) of human serum was 1.6-2.2%, mouse serum was 2.7-2.9% and rat serum was 1.0-2.6%, indicating good repeatability.

Reproducibility (between run precision)

Reproducibility was conducted by measuring serum samples from human, mouse, rat at three concentrations each for four days with this product.

Day＼ID Human serum Mouse serum Rat serum
ID3 (mmol/L) ID4 (mmol/L) ID5 (mmol/L) ID3 (mmol/L) ID4 (mmol/L) ID5 (mmol/L) ID3 (mmol/L) ID4 (mmol/L) ID5 (mmol/L)
1 0.735 0.494 0.359 0.489 0.380 0.254 0.565 0.436 0.325
2 0.734 0.489 0.367 0.490 0.369 0.240 0.544 0.433 0.318
3 0.733 0.504 0.367 0.505 0.387 0.263 0.601 0.478 0.352
4 0.717 0.490 0.350 0.489 0.372 0.234 0.571 0.435 0.323
mean 0.730 0.494 0.361 0.493 0.377 0.248 0.570 0.446 0.330
SD 0.00854 0.00685 0.00810 0.00785 0.00812 0.0132 0.0235 0.0217 0.0153
CV(%) 1.2 1.4 2.2 1.6 2.2 5.3 4.1 4.9 4.6

[Result]
The CV (%) of human serum was 1.2-2.2%, mouse serum was 1.6-5.3% and rat serum was 4.1-4.9%, indicating good reproducibility.

Dilution Linearity Test

Serum from human, mouse and rat, as well as D-MEM containing 10% FBS spiked with the standard, were serially diluted twofold with saline and measured in duplicate to assess linearity.

human serum
mouse serum
rat serum
D-MEM/10% FBS medium

[Result]
All measurement results showed good linearity within the measurement range.

Spike and Recovery Test

Standard solutions at four concentrations were added to serum from human, mouse and rat. Next, spike recovery tests were conducted, with each measurement performed in duplicate.

Human sample

Spiked amount
(mmol/L)		 Measurement value
(mmol/L)		 Recovery volume
(mmol/L)		 Recovery rate
(%)
Serum - 0.429 - -
0.0970 0.527 0.0980 101
0.197 0.587 0.158 80.2
0.297 0.672 0.243 81.8
0.507 0.866 0.437 86.2
Average 87.3

Mouse sample

Spiked amount
(mmol/L)		 Measurement value
(mmol/L)		 Recovery volume
(mmol/L)		 Recovery rate
(%)
Serum - 0.284 - -
0.0600 0.343 0.0590 98.3
0.127 0.399 0.115 90.6
0.251 0.507 0.223 88.8
0.500 0.753 0.469 93.8
Average 92.9

Rat sample

Spiked amount
(mmol/L)		 Measurement value
(mmol/L)		 Recovery volume
(mmol/L)		 Recovery rate
(%)
Serum - 0.374 - -
0.0600 0.439 0.0650 108
0.127 0.483 0.109 85.8
0.251 0.598 0.224 89.2
0.500 0.855 0.481 96.2
Average 94.9

[Result]
Good recovery rates were confirmed.

Measurement Using Actual Samples

Human sample

ID＼n Measurement value
(mmol/L)		 mean SD CV
(%)
1 2
Serum ID1 0.558 0.555 0.557 0.00212 0.4

Rat sample (S.D. rat)

ID＼n Measurement value
(mmol/L)		 mean SD CV
(%)
1 2
Serum ID1 0.541 0.534 0.538 0.00495 0.9
ID2 0.434 0.448 0.441 0.00990 2.2
ID3 0.357 0.357 0.357 0.00 0.0
ID4 0.461 0.458 0.460 0.00212 0.5
ID5 0.308 0.293 0.301 0.0106 3.5

Mouse sample (CD-1(ICR))

ID＼n Measurement value
(mmol/L)		 mean SD CV
(%)
1 2
Serum ID1 0.320 0.301 0.311 0.0134 4.3
ID2 0.452 0.454 0.453 0.00141 0.3
ID3 0.505 0.484 0.495 0.0148 3.0
ID4 0.433 0.451 0.442 0.0127 2.9
ID5 0.372 0.375 0.374 0.00212 0.6

Dog sample (Beagle)

ID＼n Measurement value
(mmol/L)		 mean SD CV
(%)
1 2
Serum ID1 0.395 0.385 0.390 0.00707 1.8
ID2 0.138 0.138 0.138 0.00 0.0
ID3 0.314 0.303 0.309 0.00778 2.5
ID4 0.323 0.299 0.311 0.0170 5.5
ID5 0.323 0.326 0.325 0.00212 0.7
ID6 0.297 0.290 0.294 0.00495 1.7
ID7 0.535 0.535 0.535 0.00 0.0
ID8 0.297 0.295 0.296 0.00141 0.5

Cat sample (Short hair)

ID＼n Measurement value
(mmol/L)		 mean SD CV
(%)
1 2
Serum ID1 0.204 0.196 0.200 0.00566 2.8
ID2 0.158 0.165 0.162 0.00495 3.1
ID3 0.221 0.213 0.217 0.00566 2.6
ID4 0.229 0.234 0.232 0.00354 1.5
ID5 0.297 0.295 0.296 0.00141 0.5
ID6 0.155 0.149 0.152 0.00424 2.8
ID7 0.398 0.388 0.393 0.00707 1.8
ID8 0.343 0.333 0.338 0.00707 2.1

FAQ

About calibration curve

Are there any precautions to consider when generating the calibration curve?
Even when distilled water is used as the sample, a background absorbance of approximately 0.3 OD is observed. Therefore, when measuring samples, we recommend subtracting the background OD value prior to generating the calibration curve. If the measured absorbance values differ significantly from the calibration curve shown, please review the following points.
  • Ensure that no undissolved substrate tablet remains (allow the tablet to dissolve completely).
  • Check the condition of the substrate solution (store refrigerated and protected from light; use within 2 weeks).
  • Strictly adhere to the specified enzyme reaction temperature (37°C) and reaction time (15 minutes).

About sample

What should I do with samples that exceed the measurable range?
For samples exceeding the upper limit of the assay range, dilute the sample with distilled water or an appropriate buffer prior to measurement. Do not use phosphate-based buffers. Multiply the result by the dilution factor to obtain the final value.
Is there any influence from substances that interfere with measurement (bilirubin, etc.)?
Hemolysis and bilirubin have almost no effect on the measured values.

About kit usage

What instruments, and equipment are required for the assay using this kit?
The instruments and equipment required for the use of this kit are listed below.
  • 96 well microplate (transparent type)
  • Micropipette
  • Pippette
  • Test tube
  • Plate mixer*
  • Incubator maintaining at 37°C*
  • Microplate reader with 405 nm wavelength filter
(* if the microplate reader is not equipped.)
What are the storage conditions and shelf-life guidelines for each reagent?
Working Assay Solution
Dissolve a Substrate Tablet into 5 mL of Buffer for Substrate Tablet provided.
It can be stored for two weeks at 2-10°C in the dark after reconstitution.

Stop Solution
Ready-to-use. It can be stored at 2-10°C.

Dilution series of Standard Solution
Store unused standard products at 2–10°C.

Overview / Applications

Property

Manufacturer Information

Alias

  • LabAssay
    633-51021 (Previous code)
    LABALP-M1 (Previous code)
    291-58601 (Previous code)

For research use or further manufacturing use only. Not for use in diagnostic procedures.

Product content may differ from the actual image due to minor specification changes etc.

If the revision of product standards and packaging standards has been made, there is a case where the actual product specifications and images are different.

The prices are list prices in Japan.Please contact your local distributor for your retail price in your region.

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