LabAssay Ammonia Technical Information and Data

Assay Principle

The Deproteinizing Reagent deactivates the enzymes and removes the components that inhibit color reaction in a sample. After adding Deproteinizing Reagent to a sample and centrifuging, add Phenol, Sodium Pentacyanonitrosylferrate(III) Dihydrate, and Sodium Hypochlorite to the supernatant. The indophenol blue pigment is obtained by the following schematic reaction. The amount of ammonia contained in the sample is determined by measuring the absorbance of the blue color.

Performance

Sample	Blood, Serum, Plasma, Culture supernatant
Target animals	Mouse, Rat, Cat, Dog
Calibration curve range	100 - 400 μg / dL (μg / 100 mL)
Sample volume	70 μL
Measurement duration	Approx. 70 min.
Wavelength	630 nm

Standard Curve

Procedure

Assay in a microplate format
Perform the assay in the wells according to the following table scheme.

	Test	Standard	Blank
An experiment can be conducted in a microtube
Deproteinizing Reagent	Deproteinizing reagent：Sample = 4：1(v/v)	Deproteinizing reagent：Standard solution = 4：1(v/v)	70 μL
Sample	Deproteinizing reagent：Sample = 4：1(v/v)	Deproteinizing reagent：Standard solution = 4：1(v/v)	-
	Mix well. Centrifuge 5000×g, 4℃, for 15 min. Subsequent operations should be performed in the wells.	Mix well. Subsequent operations should be performed in the wells.
	Supernatant 70 μL	Mixed solution 70 μL	-
Chromogen Reagent A	70 μL	70 μL	70 μL
Chromogen Reagent B	35 μL	35 μL	35 μL
Chromogen Reagent C	70 μL	70 μL	70 μL
After adding each reagent to a microplate, mix well and incubate at 37 ℃, for 20 min. Mix well, then let stand at room temperature＊ for 30 min. Mix well, then measure the absorbance of the test sample and standard solution at 630 nm with the blank as the control.

*Room temperature: 20℃ - 25℃

Data

Spike-and-Recovery Test

Spike and Recovery Test of Ammoinia, a known amount of standard is spiked into in mous and rat sample n=2.

Mouse Sample

Spike (µg/dL)	Measured (µg/dL)	Recovery (µg/dL)	Recovery (%)
-	81.8	-	-
56.9	142	60.2	106
114	205	123	108
171	247	165	96.8

Rat Sample

Spike (µg/dL)	Measured (µg/dL)	Recovery (µg/dL)	Recovery (%)
-	77.3	-	-
56.9	137	59.7	105
114	188	111	97.5
171	236	159	93.1

Examples of measurement

Human serum/plasma, mouse (ICR) serum/plasma samples, and rat (SD) serum/plasma were measured with this product.

Mouse blood BALB/cAJc1 8W ♂

Sample No.	Measured (µg/dL)
1	118
2	124
3	95.6
4	116
5	95
mean	110
SD	13.6

(n=2)

Rat blood Jc1:SD 8W ♂

Sample No.	Measured (µg/dL)
1	99.3
2	91.6
3	90
4	93.1
5	97.7
mean	94.3
SD	4.01

(n=2)

Canine plasma EDTA

No.	Age (years)	Measured (μg/dL)		mean	SD	CV (%)
1	4	232	210	221	15	7.0
2	6	194	178	186	12	6.2
3	10	225	207	216	13	5.9
4	12	208	196	202	8.2	4.1
5	6	181	202	192	15	7.7
6	11	155	175	165	14	8.6
7	10	144	151	147	5	3.5
8	12	156	173	164	12	7.2

Canine serum

No.	Age (years)	Measured (μg/dL)		mean	SD	CV (%)
1	4	331	365	348	24	6.9
2	6	296	323	310	19	6.2
3	6	409	465	437	40	9.1
4	11	367	365	366	1.6	0.4
5	12	453	513	483	42	8.7
6	6	349	366	357	12	3.4
7	10	451	504	478	37	7.8
8	4	291	323	307	22	7.2

Feline plasma EDTA

No.	Age (years)	Measured (μg/dL)		mean	SD	CV (%)
1	0.5	191	204	197	9.4	4.8
2	1.0	109	109	109	0.14	0.13
3	3.0	113	119	116	4.1	3.5
4	0.5	138	156	147	13	8.6
5	3.0	133	137	135	2.9	2.2
6	3.0	103	114	108	7.6	7.0
7	0.5	129	137	133	5.8	4.4
8	1.0	170	169	170	0.84	0.50

Feline serum

No.	Age (years)	Measured (μg/dL)		mean	SD	CV (%)
1	3.0	381	366	374	11	2.8
2	3.0	370	378	374	5.7	1.5
3	6.0	381	342	361	28	7.8
4	4.0	357	329	343	20	5.8
5	6.0	249	245	247	2.9	1.2
6	6.0	315	299	307	11	3.7
7	1.0	403	388	395	10	2.6
8	1.0	198	193	196	4.2	2.1

No.	Age (years)	Measured (μg/dL)		mean	SD	CV (%)
1	4	232	210	221	15	7.0
2	6	194	178	186	12	6.2
3	10	225	207	216	13	5.9
4	12	208	196	202	8.2	4.1
5	6	181	202	192	15	7.7
6	11	155	175	165	14	8.6
7	10	144	151	147	5	3.5
8	12	156	173	164	12	7.2

No.	Age (years)	Measured (μg/dL)		mean	SD	CV (%)
1	4	331	365	348	24	6.9
2	6	296	323	310	19	6.2
3	6	409	465	437	40	9.1
4	11	367	365	366	1.6	0.4
5	12	453	513	483	42	8.7
6	6	349	366	357	12	3.4
7	10	451	504	478	37	7.8
8	4	291	323	307	22	7.2

No.	Age (years)	Measured (μg/dL)		mean	SD	CV (%)
1	3.0	381	366	374	11	2.8
2	3.0	370	378	374	5.7	1.5
3	6.0	381	342	361	28	7.8
4	4.0	357	329	343	20	5.8
5	6.0	249	245	247	2.9	1.2
6	6.0	315	299	307	11	3.7
7	1.0	403	388	395	10	2.6
8	1.0	198	193	196	4.2	2.1

No.	Age (years)	Measured (μg/dL)		mean	SD	CV (%)
1	4	232	210	221	15	7.0
2	6	194	178	186	12	6.2
3	10	225	207	216	13	5.9
4	12	208	196	202	8.2	4.1
5	6	181	202	192	15	7.7
6	11	155	175	165	14	8.6
7	10	144	151	147	5	3.5
8	12	156	173	164	12	7.2

No.	Age (years)	Measured (μg/dL)		mean	SD	CV (%)
1	4	331	365	348	24	6.9
2	6	296	323	310	19	6.2
3	6	409	465	437	40	9.1
4	11	367	365	366	1.6	0.4
5	12	453	513	483	42	8.7
6	6	349	366	357	12	3.4
7	10	451	504	478	37	7.8
8	4	291	323	307	22	7.2

No.	Age (years)	Measured (μg/dL)		mean	SD	CV (%)
1	3.0	381	366	374	11	2.8
2	3.0	370	378	374	5.7	1.5
3	6.0	381	342	361	28	7.8
4	4.0	357	329	343	20	5.8
5	6.0	249	245	247	2.9	1.2
6	6.0	315	299	307	11	3.7
7	1.0	403	388	395	10	2.6
8	1.0	198	193	196	4.2	2.1

No.	Age (years)	Measured (μg/dL)		mean	SD	CV (%)
1	4	232	210	221	15	7.0
2	6	194	178	186	12	6.2
3	10	225	207	216	13	5.9
4	12	208	196	202	8.2	4.1
5	6	181	202	192	15	7.7
6	11	155	175	165	14	8.6
7	10	144	151	147	5	3.5
8	12	156	173	164	12	7.2

No.	Age (years)	Measured (μg/dL)		mean	SD	CV (%)
1	4	331	365	348	24	6.9
2	6	296	323	310	19	6.2
3	6	409	465	437	40	9.1
4	11	367	365	366	1.6	0.4
5	12	453	513	483	42	8.7
6	6	349	366	357	12	3.4
7	10	451	504	478	37	7.8
8	4	291	323	307	22	7.2

No.	Age (years)	Measured (μg/dL)		mean	SD	CV (%)
1	3.0	381	366	374	11	2.8
2	3.0	370	378	374	5.7	1.5
3	6.0	381	342	361	28	7.8
4	4.0	357	329	343	20	5.8
5	6.0	249	245	247	2.9	1.2
6	6.0	315	299	307	11	3.7
7	1.0	403	388	395	10	2.6
8	1.0	198	193	196	4.2	2.1