LabAssay (TM) ALP

Introduction

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.

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.

• Performance

  Sample	Plasma, Serum, Cell culture medium
  Target animals	Human, Mouse, Rat, Cat, Dog
  Standard curve range	0.0625 - 0.5 mmol/L
  Measurement time	Approx. 20 min.
  Amount of sample	20 μL
  Measurement wavelength	405 nm

• Standard Curve

  

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

Spike-and-Recovery Test

Spike and Recovery Test of ALP, a known amount of standard is spiked into in human,mouse,rat serum sample n=2.

• Human serum

  Spike (mmol/L)	Mean (mmol/L)	Recovery (mmol/L)	Recovery(%)
  -	0.429	-	-
  0.097	0.527	0.098	101.0
  0.197	0.587	0.158	80.2
  0.297	0.672	0.243	81.8
  0.507	0.866	0.437	86.2

• Mouse serum

  Spike (mmol/L)	Mean (mmol/L)	Recovery (mmol/L)	Recovery(%)
  -	0.284	-	-
  0.060	0.343	0.059	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

• Rat Serum

  Spike (mmol/L)	Mean (mmol/L)	Recovery (mmol/L)	Recovery(%)
  -	0.374	-	-
  0.060	0.439	0.065	108.3
  0.127	0.483	0.109	85.8
  0.251	0.598	0.224	89.2
  0.500	0.855	0.481	96.2

Linearity-of-Dilution Test

• Human serum
  
  
• Mouse serum
  
  
• Rat Serum
  
  

Example of measurement

• 20% Diluted Normal　Mouse serum, CD-1(ICR), male
  No.	Measured(mmol/L)		Mean(mmol/L)	SD	CV(%)
  1	0.320	0.301	0.310	0.013	4.2
  2	0.452	0.454	0.453	0.001	0.2
  3	0.505	0.484	0.495	0.015	3.0
  4	0.433	0.451	0.442	0.013	2.9
  5	0.372	0.375	0.373	0.002	0.5

• Normal　Mouse plasma K2EDTA, CD-1(ICR), male
  No.	Measured(mmol/L)		Mean(mmol/L)	SD	CV(%)
  1	0.081	0.079	0.080	0.001	1.5
  2	0.213	0.202	0.208	0.008	3.8
  3	0.099	0.098	0.098	0.000	0.1
  4	0.056	0.061	0.059	0.004	6.5
  5	0.110	0.109	0.109	0.001	0.6

• 10% Diluted Normal Rat serum, Sprague Dawley, male
  No.	Measured(mmol/L)		Mean(mmol/L)	SD	CV(%)
  1	0.541	0.534	0.538	0.005	1.0
  2	0.434	0.448	0.441	0.010	2.2
  3	0.357	0.357	0.357	0.000	0.0
  4	0.461	0.458	0.460	0.002	0.5
  5	0.308	0.293	0.301	0.011	3.6

• Normal Rat plasma K2EDTA, Sprague Dawley, male
  No.	Measured(mmol/L)		Mean(mmol/L)	SD	CV(%)
  1	0.170	0.172	0.171	0.001	0.7
  2	0.145	0.134	0.139	0.007	5.3
  3	0.262	0.257	0.260	0.004	1.4
  4	0.209	0.202	0.205	0.006	2.7
  5	0.174	0.171	0.172	0.002	1.2

• Normal Canine serum, Beagle, male
  No.	Measured(mmol/L)		Mean (mmol/L)	SD	CV(%)	Age(years)
  1	0.395	0.385	0.390	0.005	1.2	4
  2	0.138	0.138	0.138	0.000	0.2	6
  3	0.314	0.303	0.309	0.005	1.7	6
  4	0.323	0.299	0.311	0.012	3.8	11
  5	0.323	0.326	0.325	0.002	0.5	12
  6	0.297	0.290	0.293	0.004	1.2	6
  7	0.535	0.535	0.535	0.000	0.0	10
  8	0.297	0.295	0.296	0.001	0.5	4

• Normal Canine plasma EDTA, Beagle, male
  No.	Measured(mmol/L)		Mean (mmol/L)	SD	CV(%)	Age(years)
  1	0.025	0.031	0.028*	0.003	10.7	4
  2	0.030	0.033	0.032*	0.002	4.8	6
  3	0.030	0.036	0.033*	0.003	8.6	10
  4	0.056	0.069	0.062*	0.006	10.2	12
  5	0.058	0.061	0.059*	0.002	2.8	6
  6	0.026	0.028	0.027*	0.001	3.8	11
  7	0.021	0.022	0.022*	0.001	3.1	10
  8	0.065	0.070	0.067	0.003	3.7	12

• Normal Feline serum, Short hair, male
  No.	Measured(mmol/L)		Mean (mmol/L)	SD	CV(%)	Age(years)
  1	0.204	0.196	0.200	0.004	2.1	3
  2	0.158	0.165	0.162	0.004	2.2	3
  3	0.221	0.213	0.217	0.004	1.8	6
  4	0.229	0.234	0.232	0.003	1.2	4
  5	0.297	0.295	0.296	0.001	0.3	6
  6	0.155	0.149	0.152	0.003	2.0	6
  7	0.398	0.388	0.393	0.005	1.2	1
  8	0.343	0.333	0.338	0.005	1.4	1

• Normal Feline plasma EDTA, Short hair, male
  No.	Measured(mmol/L)		Mean (mmol/L)	SD	CV(%)	Age(years)
  1	0.227	0.227	0.227	0.000	0.0	0.5
  2	0.050	0.047	0.048*	0.002	3.4	1
  3	0.086	0.088	0.087	0.001	1.0	3
  4	0.107	0.103	0.105	0.002	2.1	0.5
  5	0.138	0.136	0.137	0.001	0.7	3
  6	0.062	0.058	0.060*	0.002	3.3	3
  7	0.031	0.028	0.030*	0.001	4.0	0.5
  8	0.057	0.056	0.056*	0.000	0.6	1

References

1. Ito, S. et al. : J. Pharmacol. Exp. Ther., 333, 341(2010). *Extraction liqiud of mouse kidney
2. Matsuyama, A. et al. : Clin. Exp. Pharmacol. Physiol., 45, 75(2018). *MC3T3-E1cell, C2C12Ccell
3. Chiba, T. et al. : J. Atheroscler. Thromb, 23, 1099(2016). *Mouse plasma
4. Kohno, Y. et al. : Stem Cell Res. Ther., 8, 115(2017). *Mesenchymal stem cell