CN105181970B - Stable kit for detecting homocysteine - Google Patents
Stable kit for detecting homocysteine Download PDFInfo
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- CN105181970B CN105181970B CN201510540641.0A CN201510540641A CN105181970B CN 105181970 B CN105181970 B CN 105181970B CN 201510540641 A CN201510540641 A CN 201510540641A CN 105181970 B CN105181970 B CN 105181970B
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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Abstract
The invention relates to a stable kit for detecting homocysteine, and belongs to the technical field of medical science examination and determination. The stable kit for detecting homocysteine comprises a reagent 1 and a reagent 2; the reagent 1 comprises the following components: 1-200mmol/L of an HCY reducing agent, 1.0-100mmol/L of serine, 0.5-0.8g/L of NADH, and 50-200KU/L of LDH; the reagent 2 comprises the following components: 0.1-10g/L of a stabilizing agent, 1-100KU/L of cystathionine beta-synthase, and 1-100KU/L of cystathionine beta-catabolic enzyme. The stable kit for detecting homocysteine has the advantages of convenient determination, fast detection speed, high sensitivity, high accuracy, good stability, wide linear scope and long storage time. The two reagents does not need preparation, are liquid-state reagents, and can be directly used. The kit can be stored below 4 DEG C for at least one year. Furthermore, the raw material cost is low. Clinic examination need is completely satisfied.
Description
Technical field
The present invention relates to a kind of stable homocysteine detection kit, belongs to medical test determination techniques field.
Background technology
Homocysteine is sulfur-containing amino acid, is generated by methionine demethylation in the cell.Recent studies have indicated that, together
Type cysteine changes thrombin function by producing superoxides and peroxide vascular endothelial cell injury, increases blood
Bolt formability, promotes atherosclerosiss and thrombosiss, increases cardiovascular disease incidence rate and mortality rate, therefore determines
Clinically meaning is particularly significant for the concentration of homocysteine in blood.
Determining the method for homocysteine in blood plasma has many kinds, including high performance liquid chromatography (HPLC), cyclophorase
Method, fluorescence polarization immunoassay (FPIA), enzyme-linked immunosorbent assay (EIA), amino-acid analyzer algoscopy, chromatography of ions
Method, capillary GC-MS, electrochemical process etc..
Wherein, high performance liquid chromatography (HPLC)：The homocysteine of form of ownership in blood sample is first made using reducing agent
It is transformed into reduction form, the fluorescent material complex of half moon bright propylhomoserin of derivative generation homotype one is then carried out with fluorescent agent, after derivative
Sample carry out chromatography.Because absorption affinity difference of the chromatographic stationary phases to different material, therefore mobile phase are eluted
Order it is also different, the different material in sample is separated according to this principle, with homotype half under fluorescence detector detection eluting
The fluorescence intensity of the fluorescent material complex of cystine one, it is compared and is calculated with standard substance/interior target ratio, it is possible to
Determine the level of blood total homocysteine.The method is classical reference method, but complex operation, take relatively long, and
Cost is larger, is gradually replaced by additive method.
Enzymatic cycling：At present mainly with HcyMetase and the cyclic amplification of S-Adenosylhomocysteine synthase
Effect, along with ADA Adenosine deaminase, purine nucleoside phosphorylase, xanthine oxidase, peroxidase etc., or adenosine deamination
The chromogenic reactions such as enzyme, glutamte dehydrogenase determine content of homocysteine.The advantage of the method is can directly to go up biochemistry
Instrument is used, quickly, accurately convenient.But because the toolenzyme for using is more, continuous catalytic reaction step is complicated, reagent quality and
Reaction linearly all needs constantly correction, and working service is required.Due to the stability of each protease it is different with Active pharmaceutical, therefore survey
Determine it is more difficult in reagent manufacture find preferable steady-state conditionss, therefore stability is subject to certain restrictions.And determine it is main affect because
Element is blood ammonia, and in addition dhdps enzyme is expensive, is not easy to obtain on the market, causes cost huge.
Fluorescence polarization immunoassay (FPIA)：Measuring principle is first with dithiothreitol, DTT by the Hcy of combined state in specimen
After being reduced into free form, while adding adenosine, Hcy is converted in the presence of s- adenosine-L- homocysteine hydrolases
For S- adenosines-L- homocysteines (SAH), be subsequently adding anti-SAH monoclonal antibodies and fluorescently-labeled SAH antigens, SAH with
The anti-SAH antibody of fluorescent labeled antigen competition binding, forms the immune complex of macromole, finally determines its fluorescence polarization degree.This
Method requires that blood specimen delivers in time laboratory, it is to avoid interference of the release of Hcy to measurement result in hemocyte.FPIA methods are determined
Hcy has preferable precision, has good dependency between serum and plasma results, but FPIA methods are normal compared with suitable laboratory
Rule application, is not suitable for industrialized production.
Likewise, the chromatography of ions is also mainly used in experimentation with electrocapillary phoresis method, seldom clinically use, and hair
Thin electrophoresis method is similar to efficient liquid-phase chromatography method, also exists and operates the time-consuming longer and test data variation of more complicated, test
Than it is larger the shortcomings of.
Enzyme-linked immunosorbent assay (EIA)：The method is clinically to determine homocysteine common method, and advantage is behaviour
Make comparisons simple and fast, and reproducible, good stability, but the method major part needs manual operations, take it is longer, and
Expensive reagents.
The content of the invention
The purpose of the present invention is to be directed to the above-mentioned problems in the prior art, it is proposed that a kind of sensitivity is high, thermally-stabilised
Good, storage time length the stable homocysteine detection kit of property.
The purpose of the present invention can be realized by following technical proposal：A kind of stable homocysteine detectable
Box, including reagent 1 and reagent 2,
The component of the reagent 1 is：HCY reducing agents：1-200mmol/L, serine：1.0-100mmol/L, NADH：
0.5-0.8g/L, LDH：50-200KU/L；
The component of the reagent 2 is：Stabilizer：0.1-10g/L, cystathionine β-synthase：1-100KU/L, cystathionine beta-
Catabolic enzyme：1-100KU/L.
In the homocysteine detection kit of aforementioned stable, the HCY reducing agents described in reagent 1 are TCEP, mercapto
The mixture of guanidine-acetic acid, EDTA.It is most of aoxidizing when homocysteine is gathered in the cell, and into after blood circulation
Type is present, and and protein binding, reduced form homocysteine only accounts for 1% in blood.Determine total homocysteine in blood
Content, typically need to be reduced to reduced form homocysteine using reducing agent by oxidized form homocysteine.HCY reducing agents
Reagent is a lot, but has been because reproducibility, so being easily oxidized, all unstable, in addition some reagents are in prior art
Dry powder or for 3 reagents, in use extremely inconvenience.Its reducing agent is relatively steady relative to other for TCPE in reducing agent of the present invention
It is fixed, and the PH for using is widest in area.
Preferably, described TCEP, TGA, EDTA 5-7 in mass ratio:1.5-2.5:1 mixing.HCY is reduced
The reason for concentration of agent is limited to 1-200mmol/L is that reductant concentration is too small can not completely reduce the HCY in serum, lead
Cause Lower result.If HCY concentration is big, subsequent reactions can be affected, so as to affect whole result.The concentration of HCY reducing agents is
The concentration of TCEP, TGA, EDTA press the consumption adjustment of TCEP.TCPE mixes according to the above ratio with TGA and EDTA to be made
With, it is more stable, so that reagent is more stable.
In the homocysteine detection kit of aforementioned stable, the stabilizer described in reagent 2 is soluble starch
With the mixture of glutathion.Homocysteine detection kit is typically using glycerol, ethylene glycol, BSA etc. in prior art
Conventional reagent does stability, causes stablizing effect general, and the soluble starch and glutathion adopted in the present invention all contains
There is the enzyme of more sulfydryl, when individually doing stability and exist, heat stability is poor, but found by constantly test, can
When soluble starch and glutathion are used collectively as stabilizer, homocysteine detectable can be increased substantially
Stablizing effect, can reach and at least preserve more than 1 year at 4 DEG C.
Preferably, the soluble starch and glutathion in mass ratio 10：0.8-2 mixes.The concentration of stabilizer is
The concentration of soluble starch, glutathion presses the consumption adjustment of soluble starch.If the excessive concentration of stabilizer, cost compared with
Greatly, if but stabilizer concentration it is too low, do not reach due effect.
In the homocysteine detection kit of aforementioned stable, reagent 1 and reagent 2 also include buffer 10-
200mmol/L, wherein buffer are TRIS, Good's buffer, MOPS, HEPES, triethanolamine, kaliumphosphate buffer, phosphoric acid
One or more in sodium buffer.
In the homocysteine detection kit of aforementioned stable, reagent 1 and reagent 2 also include protective agent, protection
Agent accounts for the 0.1-20% of the mass percent of reagent 1 or reagent 2, and wherein protective agent is BSA, sucrose, trehalose, glycerol, second two
One or more in alcohol, Sorbitol, PEG6000.
In the homocysteine detection kit of aforementioned stable, reagent 1 and reagent 2 also include surfactant,
Surfactant accounts for the 0.01-1% of the mass percent of reagent 1 or reagent 2, and wherein surfactant is TritonX series, tells
One or more in warm series, brij-35, two Laurel sulfuric ester of glycerols.
In the homocysteine detection kit of aforementioned stable, reagent 1 and reagent 2 also include preservative 0.1-
5g/L, wherein, preservative is one or more in sodium azide, proclin300, MIT, gentamycin sulfate, thimerosal.
The stable homocysteine detection kit principle of the present invention is：Oxidized form HCY is converted to free HCY, trip
From HCY is under CBS catalysis and serine reaction generates L-cystathionine；L-cystathionine generates HCY, acetone acid again under CBL catalysis
And NH3；The acetone acid that the circular response is generated can be detected with lactate dehydrogenase L DH and NADH, and NADH is transformed into NAD+Speed
Rate is directly proportional to HCY contents in sample.
The reaction equation of reaction principle is as follows：
NADH reductions speed is determined at 340nm can measure the content of homocysteine.
The stable homocysteine detection kit of the present invention can make liquid reagent box.
Compared with prior art, the present invention has following advantage：
The stable homocysteine detection kit of the present invention determines convenient, and double reagent, without the need for preparing, is liquid reagent,
Can directly use, and detection speed is fast, sensitivity is high, and accuracy is high, good stability, and the range of linearity is wide, and storage time is long, can
At least preserved at 4 DEG C more than 1 year with reaching, in addition cost of material is relatively low, fully meet the needs of Clinical Laboratory.
Description of the drawings
Fig. 1 is that the stable homocysteine detection kit of the present invention is same with the serum of the import reagent box of comparative example 1
The correlation curve of type cysteine content.
Fig. 2 is the heat stability of the stable homocysteine detection kit of the present invention and the test kit of comparative example 2,6,7
Curve.
Fig. 3 is the reducing agent heat of the stable homocysteine detection kit of the present invention and the test kit of comparative example 3,4,5
Stability curve.
Specific embodiment
The specific embodiment that the following is the present invention is described with reference to the drawings, and technical scheme is further retouched
State, but the present invention is not limited to these embodiments.
The test condition of content of homocysteine is in kit measurement serum of the present invention：Method：Performance rate method；It is master/slave
Wavelength：340nm/405nm；The reagent 2 of sample/reagent 1/： 16.5/250/25；Temperature：37℃；The time of serum+reagent 1：1-
5min；Add the response time after reagent 2：3-5min；Correction type：Linearly；Calibration steps：Two-point calibration；The Direction of Reaction：To
Under.
Determination step：
The μ l of specimen 16.5
The μ l of reagent 1 250
Mix, be placed in 37 DEG C of incubation 1-5min
The μ l of reagent 2 25
Mix, after 37 DEG C of incubation 1.5min, continuous detecting 1-3min calculates Δ A/min.
Result of calculation
Sample requirement：
1st, sample is Diagnostic Value of Fasting Serum or blood plasma.
2nd, specimen should under cryogenic transport preservation, and cold preservation and Quick spin are needed after collection, can at 2-8 DEG C of specimen sealing
Preserve 48h.If detection is delayed needs cold preservation serum.
Embodiment 1
The stable homocysteine detection kit of the present embodiment includes reagent 1 and reagent 2,
The component of reagent 1 is：TRIS：100mmol/L, TCEP：50mmol/L, TGA：10mmol/L, EDTA：
10mmol/L, serine：100mmol/L, NADH：0.5g/L, BSA：5g/L, Hydrazoic acid,sodium salt：1g/L, Triton X-100：0.1%,
LDH：50KU/L；
The component of reagent 2 is：TRIS：100mmol/L, BSA：5g/L, soluble starch：1g/L, glutathion：1g/L,
Hydrazoic acid,sodium salt：1g/L, Triton X-100：0.1%, cystathionine β-synthase：100KU/L, cystathionine beta-catabolic enzyme：100KU/L.
Embodiment 2
The stable homocysteine detection kit of the present embodiment includes reagent 1 and reagent 2, and the component of reagent 1 is：
TRIS：100mmol/L, TCEP：80mmol/L, TGA：20mmol/L, EDTA：10mmol/L, serine：80mmol/
L, NADH：0.5g/L, sucrose：10g/L, gentamycin sulfate：1g/L, Triton X-100：0.1%, LDH：80KU/L；
The component of reagent 2 is：TRIS：100mmol/L, sucrose：10g/L, soluble starch：2g/L, glutathion：1g/
L, gentamycin sulfate：1g/L, tween 80：0.1%, cystathionine β-synthase：80KU/L, cystathionine beta-catabolic enzyme：80KU/
L。
Embodiment 3
The stable homocysteine detection kit of the present embodiment includes reagent 1 and reagent 2, and the component of reagent 1 is：Phosphorus
Acid buffer：100mmol/L, TCEP：80mmol/L, TGA：50mmol/L, EDTA：10mmol/L, serine：
100mmol/L, NADH：0.5g/L, BSA：5g/L, Hydrazoic acid,sodium salt：1g/L, Triton X-100：0.1%, LDH：100KU/L；
The component of reagent 2 is：Phosphate buffer：100mmol/L, BSA：5g/L, soluble starch：2g/L, glutathion：
1g/L, Hydrazoic acid,sodium salt：1g/L, Triton X-100：0.1%, cystathionine β-synthase：50KU/L, cystathionine beta-catabolic enzyme：
100KU/L。
Comparative example 1
Commercially available import homocysteine detection kit.
Comparative example 2
Commercially available common domestic homocysteine detection kit.
Comparative example 3
There was only one kind with the reducing agent differed only in reagent 1 of homocysteine detection kit in embodiment 1
TCEP：50mmol/L, other are same as Example 1, are not repeated herein.
Comparative example 4
There was only TCEP with the reducing agent in reagent 1 that differs only in of homocysteine detection kit in embodiment 1：
50mmol/L and EDTA：10mmol/L, other are same as Example 1, are not repeated herein.
Comparative example 5
There was only TCEP with the reducing agent in reagent 1 that differs only in of homocysteine detection kit in embodiment 1：
50mmol/L and TGA：10mmol/L, other are same as Example 1, are not repeated herein.
Comparative example 6
Only have with the stabilizer differed only in reagent 2 of homocysteine detection kit in embodiment 1 solvable
Property starch：1g/L, other are same as Example 1, are not repeated herein.
Comparative example 7
There was only paddy Guang with the stabilizer differed only in reagent 2 of homocysteine detection kit in embodiment 1
Sweet peptide：1g/L, other are same as Example 1, are not repeated herein.
1st, will be the other embodiment of the present invention (this is sentenced as a example by embodiment 1, or) in the embodiment of the present invention and right
Ratio 1 is made comparisons, and content of homocysteine in 30 parts of serum samples of test, testing result is as shown in table 1；Then with comparative example
1 import reagent box test result is vertical coordinate, with the test result of embodiment 1 as abscissa, does regression analyses, and both dependencys are such as
Shown in Fig. 1.
Table 1：The dependency of embodiment 1 and comparative example 1
Knowable to table 1 and Fig. 1：The test kit serologic test deviation maximum of embodiment 1 and comparative example 1 is 9.5%, is returned
Equation is Y=0.9954X-0.1642, R2=0.9985, coefficient R is 0.9993, shows the test kit of the embodiment of the present invention 1
Dependency with the commercially available import homocysteine detection kit of comparative example 1 is strong, illustrates the test kit of the present invention and can substitute
Common test kit on market.
2nd, will be the other embodiment of the present invention (this is sentenced as a example by embodiment 1, or) in the embodiment of the present invention and right
Reagent in ratio 2,6,7 is put into together in 37 DEG C of water baths, observes daily change of sensitivity, and daily change of sensitivity is such as
Shown in table 2, heat stability curve is as shown in Figure 2.
Table 2：The change of sensitivity of reagent in embodiment 1 and comparative example 2,6,7
Knowable to table 2 and Fig. 2, the present invention stabilizer that simultaneously addition soluble starch and glutathion are compounded can be notable
Improve the stability of homocysteine detection kit.
3rd, will be the other embodiment of the present invention (this is sentenced as a example by embodiment 1, or) in the embodiment of the present invention and right
Reagent in ratio 3,4,5 is put into together in 37 DEG C of water baths, the content of TCEP in monitoring reagent, the change such as table of TCEP contents
Shown in 3, the stability of reducing agent is as shown in Figure 3 in reaction reagent.
Table 3：The change of the content of the TCEP of reagent in embodiment 1 and comparative example 3,4,5
Knowable to table 3 and Fig. 3, the present invention adds the HCY reducing agents that TCEP, TGA, EDTA are mixed to show
Write the stability for improving homocysteine detection kit.
Specific embodiment described herein is only explanation for example spiritual to the present invention.Technology neck belonging to of the invention
The technical staff in domain can be made various modifications to described specific embodiment or supplement or substituted using similar mode, but and
Do not deviate by the spirit of the present invention or surmount scope defined in appended claims.
It is ripe to this area although having made a detailed description and being cited some specific embodiments to the present invention
For practicing technical staff, as long as it is obvious that can make various changes without departing from the spirit and scope of the present invention or correct.
Claims (7)
1. a kind of stable homocysteine detection kit, it is characterised in that including reagent 1 and reagent 2,
The component of the reagent 1 includes：HCY reducing agents：1-200mmol/L, serine：1.0-100mmol/L, NADH：0.5-
0.8g/L, LDH：50-200KU/L；
The component of the reagent 2 includes：Stabilizer：0.1-10g/L, cystathionine β-synthase：1-100KU/L, cystathionine beta-point
Solution enzyme：1-100KU/L, stabilizer is the mixture of soluble starch and glutathion, the soluble starch and glutathion
In mass ratio 10：0.8-2 mixes.
2. stable homocysteine detection kit according to claim 1, it is characterised in that described in reagent 1
HCY reducing agents be TCEP, TGA, the mixture of EDTA.
3. stable homocysteine detection kit according to claim 2, it is characterised in that TCEP, sulfydryl second
Acid, EDTA 5-7 in mass ratio:1.5-2.5:1 mixing.
4. stable homocysteine detection kit according to claim 1, it is characterised in that reagent 1 and reagent 2
Also include buffer 10-200mmol/L, wherein buffer be TRIS, Good's buffer, MOPS, HEPES, triethanolamine,
One or more in kaliumphosphate buffer, sodium phosphate buffer.
5. stable homocysteine detection kit according to claim 4, it is characterised in that reagent 1 and reagent 2
Also include protective agent, protective agent accounts for the 0.1-20% of the mass percent of reagent 1 or reagent 2, and wherein protective agent is BSA, sugarcane
One or more in sugar, trehalose, glycerol, ethylene glycol, Sorbitol, PEG6000.
6. stable homocysteine detection kit according to claim 5, it is characterised in that reagent 1 and reagent 2
Also include surfactant, surfactant accounts for the 0.01-1% of the mass percent of reagent 1 or reagent 2, and wherein surface is lived
Property agent be TritonX series, TWEEN Series, brij-35, two Laurel sulfuric ester of glycerols in one or more.
7. stable homocysteine detection kit according to claim 6, it is characterised in that reagent 1 and reagent 2
Also include preservative 0.1-5g/L, wherein, preservative is sodium azide, proclin300, MIT, gentamycin sulfate, thimerosal
In one or more.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106434854A (en) * | 2016-09-29 | 2017-02-22 | 北京世纪沃德生物科技有限公司 | Kit for detecting homocysteine |
| CN106248666A (en) * | 2016-10-06 | 2016-12-21 | 济南天舜生物技术有限公司 | Homocysteine (HCY) detectable that a kind of stability is strong |
| CN106970230B (en) * | 2017-03-29 | 2019-06-11 | 广州市伊川生物科技有限公司 | A kind of homocysteine detection reagent box |
| CN107153044A (en) * | 2017-07-20 | 2017-09-12 | 青岛浩铂生物科技有限公司 | The homocysteine kit and its detection method of a kind of modified form |
| CN107271691A (en) * | 2017-08-10 | 2017-10-20 | 威特曼生物科技（南京）有限公司 | Homocysteine detection kit and its application method |
| CN108828222A (en) * | 2018-04-13 | 2018-11-16 | 济南贝韩生物技术有限公司 | A kind of homocysteine detection kit of strong antijamming capability |
| CN109001462A (en) * | 2018-07-04 | 2018-12-14 | 浙江伊利康生物技术有限公司 | A kind of homocysteine detection kit |
| CN108828215B (en) * | 2018-08-30 | 2019-05-14 | 中拓生物有限公司 | A kind of glutathione reductase assay kit and its preparation method and application |
| CN110261601B (en) * | 2019-07-16 | 2022-07-12 | 三诺生物传感股份有限公司 | Homocysteine detection kit |
| CN111766387B (en) * | 2020-07-10 | 2022-02-01 | 北京华益精点生物技术有限公司 | Preparation method of quality control card and quality control card |
| CN112051354B (en) * | 2020-08-05 | 2022-06-14 | 武汉生之源生物科技股份有限公司 | Lipase determination kit and preparation method thereof |
| CN112595851B (en) * | 2020-11-25 | 2024-10-11 | 北京安图生物工程有限公司 | Homocysteine assay kit with strong stability and preparation method thereof |
| CN114689415B (en) * | 2020-12-31 | 2024-12-03 | 桂林优利特医疗电子有限公司 | A homocysteine detection kit with good stability and preparation method |
| CN114088692A (en) * | 2021-11-13 | 2022-02-25 | 普十生物科技(北京)有限公司 | Reduction reagent for HCY detection, kit and use method thereof |
| CN114935619A (en) * | 2022-06-13 | 2022-08-23 | 苏州帕诺米克生物科技有限公司 | Method and kit for detecting water-soluble vitamins |
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| US6635438B2 (en) * | 1999-11-02 | 2003-10-21 | Catch, Inc. | Enzymatic cycling assays for homocysteine and cystathionine |
| CN104198726B (en) * | 2014-08-14 | 2016-07-06 | 上海睿康生物科技有限公司 | A kind of stable homocysteine detection kit |
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