CN102645414B

CN102645414B - Reagent kit capable of determining urine inositol

Info

Publication number: CN102645414B
Application number: CN201210099629.7A
Authority: CN
Inventors: 李子樵; 朱雪青
Original assignee: SHANGHAI AILEX TECHNOLOGY Co Ltd
Current assignee: Ailex Technology Group Co ltd; Zhejiang Ailex Medical Co ltd
Priority date: 2012-04-06
Filing date: 2012-04-06
Publication date: 2014-04-02
Anticipated expiration: 2032-04-06
Also published as: CN102645414A

Abstract

The invention relates to a medicine reagent, in particular to a reagent kit capable of determining urine inositol. The reagent kit comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises buffer solution, 1.0 g/L of Thio-NAD+, 1.0 g/L of stabilizing agent and 0.5 g/L of preservative. The reagent R2 comprises buffer solution, 5-6 g/L of NADH, 50-100 KU/L of inositol dehydrogenase, 0.1-0.2 g/L of stabilizing agent and 0.5 g/L of preservative. Compared with the prior art, the reagent kit has the beneficial effects that the preparation is simple and convenient, no special instrument is required, the operation can be finished on a common fully-automatic or semi-automatic instrument in a clinical laboratory, the whole process only requires 10 minutes, the labor and the materials are greatly saved, and meanwhile, a large quantity of samples can be determined in the clinical.

Description

Measure the test kit of urine inositol

[technical field]

The present invention relates to a kind of pharmaceutical reagent, be specifically related to a kind of test kit of measuring urine inositol.

[background technology]

Inositol in human body is mainly present in (for the decades of times in blood) in cell, with the form of unbound state or phosphatidylinositols constituent, is distributed widely in human body.Phosphatidylinositols decomposes by approach such as receptor activations, generates inositol-3-phosphoric acid, the effect of performance " second messenger " in cell.In addition, under diabetic disease states (hyperglycemia), in especially neural cell, inositol content reduces (sorbyl alcohol increase), finally can cause the active decline of Na, K-ATPase, causes nerve block.

By measuring the urine mysoinositol content after glucose load, can under AT state, detect the abnormal of sugar tolerance.Existing report in 1858 shows, has the inositol excretion (diabetic subject exceeds 10 times of normal peoples) of high density in diabetic subject's urine, and prompting can be used as one of index of impaired glucose tolerance.And, the people's such as Hejin report shows, before and after the 75g glucosieloading test, there is good dependency in the blood glucose value after the increasing amount (Δ UMI) of urine mysoinositol excretion and glucose load, be can reflect on an empty stomach cannot clear and definite glucose load after one of the good index of hyperglycemia state.In addition, the dependency of inositol and diabetic nerve injury and diabetic nephropathy also more and more receives publicity.

At present, the detection method of inositol adopts the methods such as high-efficient liquid phase technique (HPLC), vapor-phase chromatography (GC) or mass spectroscopy (MS) more, except the instrument of needs costliness, and trivial operations, test speed is slow, and need to have technician to carry out operation.

[summary of the invention]

The present invention is in order to overcome the deficiencies in the prior art, and a kind of test kit of mensuration urine inositol of simple to operate, quick, sensitive, accurate quantitative analysis is provided.

To achieve these goals, the present invention has designed a kind of test kit of measuring urine inositol, comprises reagent R1 and reagent R2, wherein:

Reagent R1, comprises damping fluid; Thio-NAD ⁺1.0g/L; Stablizer 1.0g/L; Sanitas 0.5g/L;

Reagent R2, comprises damping fluid; NADH 5-6g/L; Myo-Inositol dehydrogenase 50-100KU/L; Stablizer 0.1-0.2g/L; Sanitas 0.5g/L.

The pH value of described reagent R1 and reagent R2 is respectively 5-12.

In described reagent R1, damping fluid is Tris damping fluid 0.1mol/L or MES damping fluid 20g/L.

In described reagent R1, stablizer is a kind of in bovine serum albumin, N.F,USP MANNITOL or amino acid.

In described reagent R1, sanitas is a kind of in sodiumazide, Thiomersalate or gentamicin.

In described reagent R2, also comprise sequestrant 0.8g/L.

In described reagent R2, damping fluid is Tris damping fluid 0.1mol/L or GOOD buffer reagent 10g/L.

In described reagent R2, stablizer is a kind of in bovine serum albumin, N.F,USP MANNITOL or amino acid.

In described reagent R2, sanitas is a kind of in sodiumazide, Thiomersalate or gentamicin.

Described sequestrant is ethylenediamine tetraacetic acid (EDTA).

The present invention compared with the existing technology, it is simple, convenient to prepare, without specific apparatus, on the common full-automatic or semi-automatic biochemical analyzer of clinical labororatory, get final product complete operation, whole process only needs 10 minutes, significantly saved manpower and material resources, made clinically great amount of samples to be measured and become possibility simultaneously.The present invention, for measuring the enzyme circulating reaction reagent of urine mysoinositol, is had to higher stability, can be widely used in clinical diagnosis, and reduce the waste of reagent.

[accompanying drawing explanation]

Fig. 1 is the curvilinear trend figure that test kit mysoinositol solution of the present invention and inositol are measured reagent absorbancy after mixing.

Fig. 2 is kit measurement glycated serum protein y=0.989x+0.726 of the present invention, R ²the linearity range of=0.992 o'clock.

Fig. 3 is kit measurement glycated serum protein y=1.038x-1.634 of the present invention, R ²the linearity range of=0.991 o'clock.

[embodiment]

Below in conjunction with accompanying drawing, the invention will be further described.

Embodiment 1:

The reagent that employing completes after preparation detects, accuracy, precision and linear setting requirement have been reached, consider that stability that reagent stores and myo-Inositol dehydrogenase wherein, NADH are unstable under solution state, this product is made to double reagent product, reagent (R1) is 3:1 with the solution state volume ratio of reagent (R2), and concrete composition distribution and compound method are as follows:

Reagent R1:

Reagent R2 (during lyophilized powder state)

Reagent R2a (solution)

GOOD buffer reagent 10g/L

Bovine serum albumin 1.0g/L

NaN ₃ 0.2g/L

Reagent R2b (lyophilized powder)

NADH 5g/L

Myo-Inositol dehydrogenase 50KU/L

Reagent R2 (during solution state)

After the composition of reagent (R1) and reagent (R2) is distributed, first according to the mode of liquid double reagent, prepare, can obtain product, if reagent (R2) is lyophilized powder, before using, need reagent (R2a) to add in reagent (R2b), form each moiety for setting reagent (R2) solution of concentration.The freeze-drying process of reagent (R2) is as follows:

First solution is cooled to-40 ℃ from 25 ℃, this is cooling maintains 4 hours, guarantee that the solution temperature in bottle is even, then under certain vacuum tightness, (under the pressure of 20pa) is slowly warmed up to 25 ℃, this process need 3-4 hour, then under the vacuum tightness of the temperature of 25 ℃ and 20pa pressure, be dried.Above process is controlled by vacuum-freeze-dry machine completely, need to be approximately 37 hours, until the temperature of lyophilized powder and the temperature of Freeze Drying Equipment kiln when consistent, represent that lyophilized powder has reached fully dry state, drying process finishes, finally in vial, be filled with nitrogen, cover bottle stopper, complete the freeze-drying of whole reagent R2.

Experiment one:

Equipment: Hitachi's 7170 type automatic biochemistry analyzers

According to urine inositol, measure the reaction principle of reagent, consider reagent sensitivity and inhale the relation that affects and automatic clinical chemistry analyzer tankage and the restriction at present between sample ratio, adopt sample: reagent (R1): reagent (R2)=12 μ L: 180 μ L: the ratio of 60 μ L is tested.

First adopt ultraviolet-visible pectrophotometer under 405nm wavelength and 37 ℃ hatch under, after certain density inositol is mixed according to reaction ratio with the single reagent of preparation, carry out absorbancy and change scanning, can be observed inositol solution and after mixing, have obvious absorbancy ascendant trend with inositol mensuration reagent, it is fixing that its slope keeps within a certain period of time, and result as shown in Figure 1.

Therefore consider the surge time of the mixed potential of hydrogen of double reagent and temperature, on Hitachi's automatic biochemistry analyzer, chosen the content that the absorbancy of the 6th minute to the 10th minute changes to detect inositol.

Below definite reaction system parameter:

Specimen types: human urine; Consumption: 12 μ L;

Reagent dosage: reagent (R1) 180 μ L, reagent (R2) 60 μ L;

Reaction conditions: urine 12 μ L, after mixing with reagent (R1), at 37 ℃, hatch 5 minutes, then after adding reagent (R2), mix and hatch 1 minute, next adopt the wavelength of 405nm to measure, METHOD FOR CONTINUOUS DETERMINATION 4 minutes, measured 1 time every 1 minute, obtained absorbancy pace of change (Δ A/min).

Calibration steps: the solution that employing inositol is mixed with 100 μ mol/L is as calibration object.

While adopting mentioned reagent to measure fresh patient to urinate the content of inositol, on Hitachi's 7170 automatic biochemistry analyzers, detect, with HPLC method in contrast, result as shown in Figure 2.

In Fig. 2, curve represents the regression curve of measurement result, and ordinate zou is measurement result of the present invention, and X-coordinate is the measurement result of HPLC method, and measure unit is μ mol/L.

Statistics y=0.989x+0.726;

Measure number of cases: 40 examples;

Coefficient R=0.9959, R ²=0.992;

Design parameter sees the following form:

Sample numbering	Present method	HPLC
			1	69.52	68.15
2	63.09	61.18
			3	57.30	56.86
4	42.98	43.35
			5	48.02	49.28
6	49.90	51.90
			7	28.89	28.21
8	30.94	30.01
			9	45.05	44.47
10	70.44	71.06

11	37.65	36.99
			12	40.12	38.47
13	68.03	65.50
			14	55.48	53.30
15	47.00	48.14
			16	49.44	49.95
17	47.23	46.29
			18	31.89	31.03
19	40.45	41.68
			20	57.40	57.12
21	53.96	54.02
			22	58.47	58.80
23	41.51	40.12
			24	65.99	66.76
25	71.65	72.15
			26	39.60	39.98
27	45.65	45.37
			28	48.61	47.19
29	26.78	26.26
			30	57.31	56.76
31	23.52	22.92
			32	35.09	34.80
33	57.47	55.56
			34	30.02	31.01
35	48.06	46.57
			36	42.19	44.42
37	67.10	69.78
			38	32.31	32.15
39	62.64	63.33
			40	43.17	42.84

As can be seen here, adopt enzyme circulation method of the present invention to measure inositol, there is good dependency with HPLC method.

With inositol preparation maximum, be the linear criterion product of 400 μ mol/L, as follows through mentioned reagent measurement result:

Linear criterion solution (μ mol/L)	Measurement result (μ mol/L)
		0.0	0.18
80.0	81.22
		160.0	159.27
240.0	245.88
		320.0	321.70

400.0

393.26

In Fig. 3, ordinate zou is the concentration of linear criterion solution of the present invention, the concentration value of X-coordinate for adopting present method to record, and measure unit is μ mol/L, wherein R ²=0.991, as can be seen here, adopt reagent of the present invention, the determination of the upper limit value that can reach that enzyme circulation method is measured inositol is 400 μ mol/L.

Embodiment 2:

Reagent R1:

Reagent R2:

Use the measuring method identical with exemplifying embodiment 1, result is as following table: (measure unit: μ mol/L)

Sample numbering	Present method	HPLC
			1	42.60	41.21
2	60.16	58.31
			3	47.60	46.85
4	32.25	33.44
			5	51.22	50.19
6	52.97	51.21
			7	45.29	47.35
8	67.11	65.17

9	27.68	27.16
			10	34.33	35.72
11	64.72	62.09
			12	30.15	32.88
13	71.26	69.92
			14	28.19	29.03
15	49.51	48.99
			16	58.29	59.10
17	52.32	52.85
			18	68.23	66.84
19	37.54	36.91
			20	51.70	52.57
21	58.25	58.59
			22	65.78	66.92
23	39.79	39.41
			24	48.17	47.10
25	57.21	56.55
			26	26.31	26.79
27	45.13	45.67
			28	72.89	71.67
29	41.96	40.78
			30	53.66	55.02
31	35.49	34.77
			32	30.01	31.60
33	42.71	44.29
			34	32.81	32.33
35	43.17	42.17
			36	62.09	60.12
37	60.22	61.55
			38	48.90	46.27
39	55.26	53.89
			40	23.57	24.90

Statistics: y=1.038x-1.634

Measure number of cases n=40 example

Coefficient R=0.9956, R ²=0.9912.

Claims

1. measure a test kit for urine inositol, comprise reagent R1 and reagent R2, wherein:

Reagent R2, comprises damping fluid; NADH5-6g/L; Myo-Inositol dehydrogenase 50-100KU/L; Stablizer 0.1-0.2g/L; Sanitas 0.5g/L,

And in described reagent R1, stablizer is a kind of in bovine serum albumin, N.F,USP MANNITOL or amino acid,

2. the test kit of mensuration urine inositol according to claim 1, is characterized in that: the pH value of described reagent R1 and reagent R2 is respectively 5-12.

3. the test kit of mensuration urine inositol according to claim 1, is characterized in that: in described reagent R1, damping fluid is Tris damping fluid 0.1mol/L or Tris damping fluid 20g/L.

4. the test kit of mensuration urine inositol according to claim 1, is characterized in that: in described reagent R1, stablizer is bovine serum albumin.

5. the test kit of mensuration urine inositol according to claim 1, is characterized in that: in described reagent R1, sanitas is a kind of in sodiumazide, Thiomersalate or gentamicin.

6. the test kit of mensuration urine inositol according to claim 1, is characterized in that: in described reagent R2, also comprise sequestrant 0.8g/L.

7. the test kit of mensuration urine inositol according to claim 1, is characterized in that: in described reagent R2, damping fluid is Tris damping fluid 0.1mol/L or GOOD buffer reagent 10g/L.

8. the test kit of mensuration urine inositol according to claim 1, is characterized in that: in described reagent R2, stablizer is bovine serum albumin.

9. the test kit of mensuration urine inositol according to claim 1, is characterized in that: in described reagent R2, sanitas is a kind of in sodiumazide, Thiomersalate or gentamicin.

10. the test kit of mensuration urine inositol according to claim 6, is characterized in that: described sequestrant is ethylenediamine tetraacetic acid (EDTA).