CN104120165B

CN104120165B - The homocysteine detection kit that a kind of stability is strong

Info

Publication number: CN104120165B
Application number: CN201410202785.0A
Authority: CN
Inventors: 谭柏清; 董雯; 甘宜梧; 王绮
Original assignee: Biobase Biodustry Shandong Co Ltd
Current assignee: Biobase Biodustry Shandong Co Ltd
Priority date: 2014-05-14
Filing date: 2014-05-14
Publication date: 2016-03-23
Anticipated expiration: 2034-05-14
Also published as: CN104120165A

Abstract

The present invention relates to the homocysteine detection kit that a kind of stability is strong, comprise reagent 1 and reagent 2, the present invention is by the further improvement to reagent 1 and reagent 2, effectively can improve the stability of homocysteine detection kit, and the accuracy of reagent and sensitivity for analysis are not affected.Therefore, the homocysteine detection kit that stability provided by the invention is strong is conducive to further promoting the use of in the market.

Description

The homocysteine detection kit that a kind of stability is strong

Technical field

The present invention relates to clinical vitro detection reagent technique field, particularly a kind of homocysteine detection kit.

Background technology

Homocysteine (also known as homocysteine, homocysteine, Hcy) is the intermediate product in Methionine metabolism process.Homocysteine katabolism in vivo, has the greatest impact by two aspect factors.One is the shortage of some biological enzyme relevant with congenital heredity, and another is exactly the shortage of the nutritive elements such as folic acid in body, vitamin B6, vitamin B12.These two kinds shortages all can affect homocysteine and be converted into other materials, cause hyperhomocysteinemiainjury.The mensuration of Hcy, in the judgement to diseases such as cardiovascular disorder, uremia, atherosclerosis, diabetes, senile dementias, has important clinical meaning.

Homocysteine in blood plasma about 70% and albumin bound are mating type.Remaining sequestered is then main to be existed with two sulphur homocysteines with the homocysteine of disulfide-bonded-cysteine cpd form, only has and is present in blood plasma with reduced form homocysteine on a small quantity.We usually refer to total homotype semicystinol concentration investigating.

Affecting the topmost factor of homocysteine level no more than heredity lacks in food nutrition.The gene that inherited genetic factors mainly comprises N5 methyl tetrahydrofolate methylferase and cystathionine-beta-synthetase is undergone mutation, and causes the activity of enzyme to reduce, thus causes hyperhomocysteinemiainjury.Prove now the defect of N5 methyl tetrahydrofolate methylferase, in vivo when folic acid deficiency, the obvious rising of homotype semicystinol concentration investigating can have been caused.And according to the research of Mudd etc., the activity of the cystathionine-beta-synthetase caused by hereditary defect reduces, the concentration of homocysteine in blood plasma can be caused to exceed about ten times of normal value.

Show the research of nearly 1200 old men, in body, the level of homocysteine and diet have close relationship, and in blood plasma, the concentration of vitamin B6, B12 and folic acid is lower, and the concentration of its homocysteine is higher.Because the content of methionine(Met) in animal proteinum is about higher than vegetable-protein about three times, more homocysteine can be produced, therefore can cause damage to the arterial tissue of human body.

Other factors comprise sex, the age, renal function infringement and take the medicine affecting folic acid and vitamin B group, all can affect the level of homocysteine.Especially the infringement of kidney merit, the discharge of those sulfur-containing amino acid can be had a strong impact on, because the cystathionine-beta-synthetase of uriniferous tubules endotheliocyte is to the transformation of homocysteine, be considered to the pathways metabolism that non-protein mating type homocysteine in blood is very important.

It has been generally acknowledged that the horizontal normal value of the adult total Hcy of fasting plasma is 4 ~ 12 μm of ol/L, ideal value is <10 μm of ol/L, is then considered to homocysteine septicemia higher than 15 μm of ol/L; The horizontal normal value of the total Hcy of the elderly (>=60 years old) fasting plasma is 15 ~ 20 μm of ol/L, is considered to homocysteine septicemia higher than 20 μm of ol/L.

At present, the Hcy measuring method that laboratory is conventional has chromatography, euzymelinked immunosorbent assay (ELISA) and fluorescence polarization immunoassay.Chromatography has high specificity, the advantage such as highly sensitive, reproducible, generally acknowledge now that it is the prefered method measuring Plasma Hcy concentration, but the equipment needed because of chromatography (HPLC and GC-MS) is complicated and expensive, be difficult to adapt to the application of routine clinical chemical laboratory, and enzymoimmunoassay can realize automatization, and a large amount of sample can be measured, be therefore subject to clinical extensive welcome.

The principle of euzymelinked immunosorbent assay (ELISA) detection homocysteine is the Adenosylhomocysteinase EC3.3.1.1 based on small molecules capture technique (SMT).After Hcy is converted into sequestered, by with covalency substrate reactions, circulation amplify, produces adenosine simultaneously.Adenosine is hydrolyzed ammonification and xanthoglobulin immediately, and ammonia, under the effect of glutamate dehydrogenase, makes NADH be converted into NAD, and the Hcy concentration in sample is directly proportional to NADH conversion rate.

As shown in the figure:

。

ImBaunofenzymatic technique is a kind of without the need to pre-treatment sample, and technology and equipment is less demanding, and precision and the higher analytical procedure of specificity, because the method does not need expensive equipment, can automatization be realized, and a large amount of sample can be measured, therefore be subject to clinical extensive popularization.But owing to there is multiple enzyme in common homocysteine detection reagent, the stability of this reagent can be made to be affected, be unfavorable for the long-term preservation of reagent, thus wasting adverse consequences.

Summary of the invention

Be directed to conventional homocysteine detection kit (enzyme process) Problems existing, the invention provides the strong homocysteine detection kit of a kind of stability (enzyme process), this test kit is compared with conventional homocysteine detection kit (enzyme process), stability is better than the detection kit of routine, but accuracy and sensitivity for analysis unaffected, be conducive to improving the stability of reagent.

The present invention is achieved by the following measures:

The homocysteine detection kit (enzyme process) that a kind of stability is strong, comprises component 1(R1) and component 2(R2), during application, volume ratio is R1:R2=240:65.Each component raw material content is as follows:

Component 1(R1):

S-adenosylmethionine (SAM) 0.1-1mM

NADH0.3-3mM

Three (2 propyloic) phosphine hydrogenchloride (TCEP) 0.5-5mM

α-ketoglutaric acid 5-50mM

Sodium azide (NaN3) 0.1-10g/L

Vitamin C oxidase 2-10KU/L;

Component 2(R2):

Hcy methyltransgerase (HMTase) 1-10KU/L

Glutamate dehydrogenase (GLDH) 1-10KU/L

AdoHcy (SAH) lytic enzyme (SAHase) 2.5-25KU/L

Adenosine deaminase (ADA) 5-50KU/L

Bovine serum albumin (BSA) 20-40g/L

AMONYL 380LC 2-5g/L.

Beneficial effect of the present invention:

The homocysteine detection kit (enzyme process) that stability provided by the invention is strong, solve reagent this difficult problem unstable in dilute solution, thus effectively enhance the stability of test kit, but can not have an impact to the accuracy of reagent and sensitivity for analysis, be conducive to this reagent and further promote in the market.

Accompanying drawing explanation

Fig. 1 is that embodiment 1 detects data dependence figure with embodiment 2.

Fig. 2 is that embodiment 1 detects data dependence figure with embodiment 3.

Fig. 3 is that embodiment 1 detects data dependence figure with embodiment 4.

Embodiment

For a better understanding of the present invention, further illustrate below in conjunction with specific embodiment.

embodiment 1

Component 1

S-adenosylmethionine (SAM) 0.1mM

NADH0.3mM

Three (2 propyloic) phosphine hydrogenchloride (TCEP) 0.5mM

α-ketoglutaric acid 5mM

Component 2(R2):

Hcy methyltransgerase (HMTase) 1KU/L

Glutamate dehydrogenase (GLDH) 1KU/L

AdoHcy (SAH) lytic enzyme (SAHase) 2.5KU/L

Adenosine deaminase (ADA) 5KU/L

The homocysteine detection kit (enzyme process) that the present embodiment describes, in use, its measuring method adopts the automatic biochemistry analyzer with double reagent function, and as Toshiba 120 automatic analyser, operation is as following table:

Calculate: homotype semicystinol concentration investigating=(A mensuration/min ÷ A standard/min) × C standard.

embodiment 2

Component 1

S-adenosylmethionine (SAM) 1mM

NADH3mM

Three (2 propyloic) phosphine hydrogenchloride (TCEP) 5mM

α-ketoglutaric acid 50mM

Sodium azide (NaN3) 10g/L

Vitamin C oxidase 10KU/L

Component 2(R2):

Hcy methyltransgerase (HMTase) 10KU/L

Glutamate dehydrogenase (GLDH) 10KU/L

AdoHcy (SAH) lytic enzyme (SAHase) 25KU/L

Adenosine deaminase (ADA) 50KU/L

Bovine serum albumin (BSA) 40g/L

AMONYL 380LC 5g/L.

Concrete measuring method is with embodiment 1.

embodiment

Component 1(R1):

S-adenosylmethionine (SAM) 0.5mM

NADH2mM

Three (2 propyloic) phosphine hydrogenchloride (TCEP) 3mM

α-ketoglutaric acid 25mM

Sodium azide (NaN3) 5g/L

Vitamin C oxidase 5KU/L;

Component 2(R2):

Hcy methyltransgerase (HMTase) 5KU/L

Glutamate dehydrogenase (GLDH) 5KU/L

AdoHcy (SAH) lytic enzyme (SAHase) 15KU/L

Adenosine deaminase (ADA) 25KU/L

Bovine serum albumin (BSA) 30g/L

AMONYL 380LC 3g/L.

Concrete measuring method is with embodiment 1.

embodiment 4

Component 1

S-adenosylmethionine (SAM) 0.1mM

NADH0.3mM

Three (2 propyloic) phosphine hydrogenchloride (TCEP) 0.5mM

α-ketoglutaric acid 5mM

Sodium azide (NaN3) 7g/L

Vitamin C oxidase 8KU/L;

Component 2(R2):

Hcy methyltransgerase (HMTase) 1KU/L

Glutamate dehydrogenase (GLDH) 1KU/L

AdoHcy (SAH) lytic enzyme (SAHase) 2.5KU/L

Adenosine deaminase (ADA) 5KU/L

Bovine serum albumin (BSA) 25g/L

AMONYL 380LC 4g/L.

Concrete measuring method is with embodiment 1.

accuracy validation is tested:

Test kit and embodiment 1 test kit of Application Example 2,3,4 contrast, and detect 40 samples, the result of detection is as Fig. 1-Fig. 3.

Known by detecting data, the detected result dependency of embodiment 2,3,4 detection kit and embodiment 1 detection kit is respectively 0.9986,0.9986,0.9987, all be greater than 0.990 of requirement, dependency is relatively good, show that test kit of the present invention has high consistency with conventional homocysteine detection kit (enzyme process), prove that its accuracy is unaffected.

linear dependence confirmatory experiment

Homocysteine high level sample is found to be 46.25 μm of ol/L, serial dilution is carried out with physiological saline, the sample of preparation 6 different concns, be followed successively by the sample of 37.00 μm of ol/L, 27.75 μm of ol/L, 18.50 μm of ol/L, 9.25 μm of ol/L, 0 μm of ol/L concentration, each concentration level various kinds originally measures three times respectively, gets its mean value respectively.The reagent of embodiment 1,2,3,4 is utilized to detect respectively.It is as shown in the table for detected result:

Theoretical concentration (μm ol/L)	Embodiment 1 detected result (μm ol/L)	Embodiment 2 detected result (μm ol/L)	Embodiment 3 detected result (μm ol/L)	Embodiment 4 detected result (μm ol/L)
					46.25	46.03	45.48	47.07	46.86
37.00	37.90	37.63	36.24	36.78
					27.75	28.02	27.88	26.91	27.21
18.50	17.98	18.03	18.46	18.85
					9.25	9.19	9.37	9.22	9.31
0.00	0.17	0.17	0.12	0.09
					Correlation coefficient r	0.9994	0.9993	0.9990	0.9995

Detected result shows, and embodiment 1-embodiment 4 detected result dependency is all greater than 0.990, reaches product standard requirement, illustrates that in reagent, to add bovine serum albumin can not reduce the linear dependence that reagent detects.

stability confirmatory experiment

2 DEG C ~ 8 DEG C, store reagents in the light protected environment of non-corrosiveness gas, detect the stability of four kinds of embodiment reagent.Four kinds of reagent are monthly chosen same sample and are measured its absorbancy three times, average, and contrast, thus determine the steady time of reagent with fresh embodiment 1 reagent detected result.

Detect data as following table:

Time	Embodiment 1 reagent detected result	Embodiment 2 reagent detected result	Embodiment 3 reagent detected result	Embodiment 4 reagent detected result	Fresh embodiment 1 reagent detected result
						12 months	0.2457	0.2506	0.2487	0.2498	0.2479
13 months	0.3528	0.3587	0.3616	0.3632	0.3622
						14 months	0.4346	0.4188	0.4214	0.4204	0.4174
15 months	0.3328	0.3398	0.3382	0.3358	0.3407
						16 months	0.1967	0.2943	0.2967	0.3001	0.2957
17 months	0.0684	0.2734	0.2686	0.2691	0.2729
						18 months	0.0032	0.4826	0.4757	0.4811	0.4843
19 months	0.0002	0.3725	0.3733	0.3688	0.3698
						20 months	0.0001	0.4685	0.4596	0.4623	0.4703
21 months	0.0000	0.5374	0.5421	0.5417	0.5461
						22 months	0.0000	0.3284	0.3314	0.3321	0.3358
23 months	0.0000	0.3263	0.3198	0.3167	0.3265
						24 months	0.0000	0.2178	0.2213	0.2121	0.2235
25 months	0.0000	0.3357	0.3443	0.3500	0.4232
						26 months	0.0000	0.1365	0.1932	0.2397	0.5328

Experimental result shows, embodiment 1 reagent 2 DEG C ~ 8 DEG C, in the light protected environment of non-corrosiveness gas storage within 15 months, stablize, and embodiment 2,3,4 reagent 2 DEG C ~ 8 DEG C, in the light protected environment of non-corrosiveness gas storage within 24 months, stablize, illustrate and add the stability that bovine serum albumin effectively can improve homocysteine detection kit in reagent.

The homocysteine detection kit (enzyme process) that stability provided by the invention is strong, by the improvement to reagent 1 and reagent 2, effectively can improve the stability of homocysteine detection kit (enzyme process), and the accuracy of reagent and sensitivity for analysis are not affected.Therefore, the homocysteine detection kit (enzyme process) that stability provided by the invention is strong is conducive to further promoting the use of in the market.

Claims

1. the homocysteine detection kit that stability is strong, is characterized in that, comprises component 1 and component 2, and volume ratio is R1:R2=240:65,

Each component raw material content is as follows:

Component 1:

S-adenosylmethionine 0.1-1mM

NADH0.3-3mM

Three (2 propyloic) phosphine hydrogenchloride 0.5-5mM

α-ketoglutaric acid 5-50mM

Sodium azide 0.1-10g/L

Vitamin C oxidase 2-10KU/L;

Component 2:

Hcy methyltransgerase 1-10KU/L

Glutamate dehydrogenase 1-10KU/L

AdoHcy (SAH) lytic enzyme 2.5-25KU/L

Adenosine deaminase 5-50KU/L

Bovine serum albumin 20-40g/L

AMONYL 380LC 2-5g/L.