CN115896085A - Homocysteine hydrolase additive with high stability and strong interference resistance - Google Patents
Homocysteine hydrolase additive with high stability and strong interference resistance Download PDFInfo
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- CN115896085A CN115896085A CN202310145387.9A CN202310145387A CN115896085A CN 115896085 A CN115896085 A CN 115896085A CN 202310145387 A CN202310145387 A CN 202310145387A CN 115896085 A CN115896085 A CN 115896085A
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- homocysteine
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- homocysteine hydrolase
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Abstract
The invention provides a homocysteine hydrolase additive with high stability and strong interference resistance, which comprises the following components: the homocysteine hydrolase additive provided by the invention can improve the stability of homocysteine detection and has stronger anti-interference capability after being added into a homocysteine detection kit, and is mainly characterized in that novel amphoteric surfactants polyethylene glycol and polyoxyethylene benzyl styrene ether are combined with betaine and added into an S-adenosylhomocysteine hydrolase system, so that the stability of the reagent is improved and the integral anti-interference capability is improved.
Description
Technical Field
The invention relates to the field of homocysteine detection, in particular to a homocysteine hydrolase additive with high stability and strong interference resistance.
Background
Homocysteine is closely related to cardiovascular diseases and is an important risk factor for cardiovascular diseases, and high homocysteine can stimulate the vascular wall to cause arterial vessel injury, cause inflammation and plaque formation on the vascular wall, finally cause the blockage of cardiac blood flow, and can also cause birth defects such as neural tube deformity and congenital deformity.
The diagnosis method comprises the following steps: the earliest method for detecting homocysteine is amino acid analysis, and the method for detecting homocysteine in serum by Ueland and the like is improved, and the following methods are commonly used at present.
Isotope method: the method established by Refsum et al in 1985. The method comprises condensing 14C-labeled adenosine with HCY, separating by chromatography, and measuring HCY concentration by liquid scintillation counting radiation intensity. The method has high sensitivity and strong specificity, but has fussy operation and radioactive pollution, and cannot be popularized and used.
Chromatography: in 1987, stabler first reported gas chromatography-mass spectrometry for homocysteine. The method can simultaneously detect cysteine, methionine, cystathionine, methylglycine and other substances. Although the sensitivity and the specificity are good, the instrument is expensive and cannot be popularized. High Performance Liquid Chromatography (HPLC) is a mature and widely used method at present, and has the defects of various variations of sample treatment, chromatographic conditions, sample detection and quantification, so that the standardization is difficult. Fiskertrand et al first performed full-automatic high performance liquid chromatography in 1993 on plasma and urine for HCY and thiol determination. HPLC can be divided into various methods according to the mode of derivatization (pre-or post-column derivatization), detection methods (fluorescence, electrochemistry). Accurate determination of homocysteine using HPLC requires good equipment, high technical experience and appropriate time for applying HPLC methods, and additional selection and preparation of internal quality controls are also important.
Immunological methods: the method applies specific anti-S-adenosylhomocysteine monoclonal technology, adopts fluorescence polarization method or immunity method to determine HCY, adopts full-automatic fluorescence polarization immunity technology by American Yapek company, uses AXSYM instrument to detect HCY, and has the reaction principle that: HCY is present in normal human plasma in approximately 1% reduced form, 70% bound to albumin, and 30% formed small molecule disulfides. Under the action of a pretreatment solution containing dithiothreitol, HCY, mixed disulfide, protein-bound type and the like of the plasma sample are reduced into a free HCY form (tHCY): tHCY is converted to SAH in the presence of S-adenosyl-L-homocysteine (SAH) hydrolase and excess adenosine; pre-diluted SAH mixture, anti-SAH monoclonal antibody and labeled fluorescent S-adenosine-L-cysteine tracer are incubated together, and the total HCY level of the sample can be measured by automatically detecting the change of polarized light by an instrument.
In the prior art, the Hcy cycloenzyme method detection kit is mostly used in cystathionine cycloenzyme method, but the cystathionine cycloenzyme method still has the defects of poor stability and the like.
Disclosure of Invention
The invention aims to provide a homocysteine hydrolase additive with high stability and strong interference resistance, which comprises a buffer solution and a stabilizing solution, wherein the stabilizing solution comprises S-adenosyl homocysteine hydrolase, betaine, a preservative and a surfactant.
Preferably, the buffer is a biological buffer comprising bovine serum albumin and AEP-HBC.
Preferably in any of the above embodiments, the preservative is sodium azide.
In any of the above embodiments, preferably, the surfactant is polyethylene glycol and polyoxyethylene benzyl styrene ether.
In any of the above embodiments, preferably, the stabilizing solution comprises the following components: s-adenosyl homocysteine hydrolase, betaine, sodium azide, polyethylene glycol and polyoxyethylene benzyl styrene ether.
Preferably in any of the above embodiments, the additive comprises: 1.5-1.8KU/L of S-adenosyl homocysteine hydrolase, 2.5-3.5g/L of betaine, 0.4-0.6g/L of sodium azide, 2.8-3.2g/L of polyethylene glycol, 2.8-3.2g/L of polyoxyethylene benzyl styrene ether, 5wt% of bovine serum albumin and 5g/L of AEP-HBC.
The invention also provides a homocysteine detection kit, which comprises the additive.
Preferably, the kit comprises two components, R1 and R2;
the R1 component comprises: 30-40KU/L of lactate dehydrogenase, 0.73-0.79mmol/L of L-serine, 0.45-0.50mmol/L of reduced coenzyme I, and 0.48-0.52mmol/L of tris (2-hydroxyethyl) phosphine chloride;
the R2 component comprises: 1.8-2.0KU/L cystathionine-beta-synthetase, 0.8-1.0KU/L cystathionine-beta-lyase, 1.5-1.8KU/L S-adenosyl homocysteine hydrolase, 2.5-3.5g/L betaine, 0.4-0.6g/L sodium azide, 2.8-3.2g/L polyethylene glycol, 2.8-3.2g/L polyoxyethylene benzyl styrene ether, 5wt% bovine serum albumin and 5g/L AEP-HBC.
The invention has the beneficial effects that: the homocysteine hydrolase additive provided by the invention can improve the stability for homocysteine detection and has stronger anti-interference capability after being added into a homocysteine detection kit, and the main reason is that the novel amphoteric surfactant polyethylene glycol, polyoxyethylene benzyl styrene ether and betaine are mutually combined and added into an S-adenosine homocysteine hydrolase system, so that the stability of the reagent is improved, and the integral anti-interference capability is improved.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Example one
The homocysteine hydrolase additive with high stability and strong interference resistance comprises a buffer solution and a stabilizing solution, wherein the stabilizing solution comprises S-adenosyl homocysteine hydrolase, betaine, a preservative and a surfactant.
Preferably, the buffer is a biological buffer comprising bovine serum albumin and AEP-HBC.
In any of the above embodiments, preferably, the preservative is sodium azide.
In any of the above embodiments, preferably, the surfactant is polyethylene glycol and polyoxyethylene benzyl styrene ether.
In any of the above embodiments, preferably, the stabilizing solution comprises the following components: s-adenosyl homocysteine hydrolase, betaine, sodium azide, polyethylene glycol and polyoxyethylene benzyl styrene ether.
Preferably in any of the above embodiments, the additive comprises: 1.5-1.8KU/L of S-adenosyl homocysteine hydrolase, 2.5g/L of betaine, 0.4g/L of sodium azide, 2.8g/L of polyethylene glycol, 2.8g/L of polyoxyethylene benzyl styrene ether, 5wt% of bovine serum albumin and 5g/L of AEP-HBC.
The invention also provides a homocysteine detection kit, which comprises two components of R1 and R2;
the R1 component comprises: lactate dehydrogenase 30KU/L, L-serine 0.73mmol/L, reduced coenzyme I0.45 mmol/L, and tris (2-hydroxyethyl) phosphine chloride 0.48mmol/L;
the R2 component comprises: 1.8KU/L cystathionine-beta-synthetase, 0.8KU/L cystathionine-beta-lyase, 1.5-1.8KU/L S-adenosylhomocysteine hydrolase, 2.5g/L betaine, 0.4g/L sodium azide, 2.8g/L polyethylene glycol, 2.8g/L polyoxyethylene benzyl styrene ether, 5wt% bovine serum albumin and 5g/L AEP-HBC.
Example two
The homocysteine hydrolase additive with high stability and strong interference resistance comprises a buffer solution and a stabilizing solution, wherein the stabilizing solution comprises S-adenosyl homocysteine hydrolase, betaine, a preservative and a surfactant.
Preferably, the buffer is a biological buffer comprising bovine serum albumin and AEP-HBC.
Preferably in any of the above embodiments, the preservative is sodium azide.
In any of the above embodiments, preferably, the surfactant is polyethylene glycol and polyoxyethylene benzyl styrene ether.
In any of the above embodiments, it is preferred that the stabilizing solution comprises the following components: s-adenosyl homocysteine hydrolase, betaine, sodium azide, polyethylene glycol and polyoxyethylene benzyl styrene ether.
Preferably in any of the above embodiments, the additive comprises: 1.5-1.8KU/L of S-adenosyl homocysteine hydrolase, 2.8g/L of betaine, 0.4g/L of sodium azide, 2.9g/L of polyethylene glycol, 2.9g/L of polyoxyethylene benzyl styrene ether, 5wt% of bovine serum albumin and 5g/L of AEP-HBC.
The invention also provides a homocysteine detection kit, which comprises two components of R1 and R2;
the R1 component comprises: lactate dehydrogenase 32KU/L, L-serine 0.74mmol/L, reduced coenzyme I0.46 mmol/L, and tris (2-hydroxyethyl) phosphine chloride 0.49mmol/L;
the R2 component comprises: 1.8KU/L cystathionine-beta-synthetase, 0.8KU/L cystathionine-beta-lyase, 1.5-1.8KU/L S-adenosylhomocysteine hydrolase, 2.8g/L betaine, 0.4g/L sodium azide, 2.9g/L polyethylene glycol, 2.9g/L polyoxyethylene benzyl styrene ether, 5wt% bovine serum albumin and 5g/L AEP-HBC.
EXAMPLE III
The homocysteine hydrolase additive with high stability and strong interference resistance comprises a buffer solution and a stabilizing solution, wherein the stabilizing solution comprises S-adenosyl homocysteine hydrolase, betaine, a preservative and a surfactant.
Preferably, the buffer is a biological buffer comprising bovine serum albumin and AEP-HBC.
Preferably in any of the above embodiments, the preservative is sodium azide.
In any of the above embodiments, preferably, the surfactant is polyethylene glycol and polyoxyethylene benzyl styrene ether.
In any of the above embodiments, it is preferred that the stabilizing solution comprises the following components: s-adenosyl homocysteine hydrolase, betaine, sodium azide, polyethylene glycol and polyoxyethylene benzyl styrene ether.
Preferably in any of the above embodiments, the additive comprises: 1.7KU/L of S-adenosyl homocysteine hydrolase, 3g/L of betaine, 0.5g/L of sodium azide, 3g/L of polyethylene glycol, 3g/L of polyoxyethylene benzyl styrene ether, 5wt% of bovine serum albumin and 5g/L of AEP-HBC.
The invention also provides a homocysteine detection kit, which comprises two components of R1 and R2;
the R1 component comprises: 35KU/L of lactate dehydrogenase, 0.76mmol/L of L-serine, 0.48mmol/L of reduced coenzyme I and 0.5mmol/L of tris (2-hydroxyethyl) phosphine chloride;
the R2 component comprises: 1.8KU/L cystathionine-beta-synthetase, 0.9KU/L cystathionine-beta-lyase, 1.7KU/L S-adenosylhomocysteine hydrolase, 3g/L betaine, 0.5g/L sodium azide, 3g/L polyethylene glycol, 3g/L polyoxyethylene benzyl styrene ether, 5wt% bovine serum albumin and 5g/L AEP-HBC.
Example four
The homocysteine hydrolase additive with high stability and strong interference resistance comprises a buffer solution and a stabilizing solution, wherein the stabilizing solution comprises S-adenosyl homocysteine hydrolase, betaine, a preservative and a surfactant.
Preferably, the buffer is a biological buffer comprising bovine serum albumin and AEP-HBC.
Preferably in any of the above embodiments, the preservative is sodium azide.
In any of the above embodiments, preferably, the surfactant is polyethylene glycol and polyoxyethylene benzyl styrene ether.
In any of the above embodiments, it is preferred that the stabilizing solution comprises the following components: s-adenosyl homocysteine hydrolase, betaine, sodium azide, polyethylene glycol and polyoxyethylene benzyl styrene ether.
Preferably in any of the above embodiments, the additive comprises: 1.8KU/L of S-adenosyl homocysteine hydrolase, 3.4g/L of betaine, 0.6g/L of sodium azide, 3.1g/L of polyethylene glycol, 3.1g/L of polyoxyethylene benzyl styrene ether, 5wt% of bovine serum albumin and 5g/L of AEP-HBC.
The invention also provides a homocysteine detection kit, which comprises two components of R1 and R2;
the R1 component comprises: lactate dehydrogenase 38KU/L, L-serine 0.77mmol/L, reduced coenzyme I0.49 mmol/L, and tris (2-hydroxyethyl) phosphine chloride 0.51mmol/L;
the R2 component comprises: 1.8-2.0KU/L cystathionine-beta-synthetase, 0.8-1.0KU/L cystathionine-beta-lyase, 1.8KU/L S-adenosylhomocysteine hydrolase, 3.4g/L betaine, 0.6g/L sodium azide, 3.1g/L polyethylene glycol, 3.1g/L polyoxyethylene benzyl styrene ether, 5wt% bovine serum albumin and 5g/L AEP-HBC.
EXAMPLE five
The homocysteine hydrolase additive with high stability and strong interference resistance comprises a buffer solution and a stabilizing solution, wherein the stabilizing solution comprises S-adenosyl homocysteine hydrolase, betaine, a preservative and a surfactant.
Preferably, the buffer is a biological buffer comprising bovine serum albumin and AEP-HBC.
In any of the above embodiments, preferably, the preservative is sodium azide.
In any of the above embodiments, preferably, the surfactant is polyethylene glycol and polyoxyethylene benzyl styrene ether.
In any of the above embodiments, it is preferred that the stabilizing solution comprises the following components: s-adenosyl homocysteine hydrolase, betaine, sodium azide, polyethylene glycol and polyoxyethylene benzyl styrene ether.
Preferably in any of the above embodiments, the additive comprises: 1.8KU/L of S-adenosyl homocysteine hydrolase, 3.5g/L of betaine, 0.6g/L of sodium azide, 3.2g/L of polyethylene glycol, 3.2g/L of polyoxyethylene benzyl styrene ether, 5wt% of bovine serum albumin and 5g/L of AEP-HBC.
The invention also provides a homocysteine detection kit, which comprises two components of R1 and R2;
the R1 component comprises: 40KU/L of lactate dehydrogenase, 0.79mmol/L of L-serine, 0.50mmol/L of reduced coenzyme I and 0.52mmol/L of tris (2-hydroxyethyl) phosphine chloride;
the R2 component comprises: cystathionine-beta-synthetase 2.0KU/L, cystathionine-beta-lyase 1.0KU/L, S-adenosylhomocysteine hydrolase 1.8KU/L, betaine 3.5g/L, sodium azide 0.6g/L, polyethylene glycol 3.2g/L, polyoxyethylene benzyl styrene ether 3.2g/L, bovine serum albumin 5wt% and AEP-HBC5g/L.
Stability test
The reagents in the example 3 are evenly divided into 10 groups, wherein the reagent amount of each group is 37mL for R1 and 10mL for R2; and 10 groups of similar measurement kits approved by national food and drug administration in common markets are taken as comparison, the kit is placed in a refrigerator at 2-8 ℃, a group of reagents are taken out on the same day every month to detect HCY (concentration 40 mu mol/L) L, and the detection results are shown in table 1, so that the stability of the reagent can be remarkably improved by the novel amphoteric surfactant cocoamidopropyl betaine.
TABLE 1
| Group of | Control group | EXAMPLE III |
| 1 month | 38.82 | 39.94 |
| 2 months old | 38.65 | 39.76 |
| 3 months old | 37.77 | 39.44 |
| 4 months old | 37.02 | 39.22 |
| For 5 months | 36.81 | 39.19 |
Interference experiment
The reagent prepared in example 3 has no significant interference with the test results when S-adenosylmethionine (SAM) ≦ 35 μmol/L, glutathione ≦ 0.7mmol/L, cystathionine concentration ≦ 32 μmol/L, adenosine ≦ 170 μmol/L, blood ammonia ≦ 65 μmol/L, triglycerides ≦ 2800mg/dL, and ascorbic acid ≦ 25 mmol/L.
Although embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are exemplary and not to be construed as limiting the present invention, and that those skilled in the art may make variations, modifications, substitutions and alterations within the scope of the present invention without departing from the spirit and scope of the present invention. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (8)
1. The homocysteine hydrolase additive with high stability and strong interference resistance is characterized by comprising a buffer solution and a stabilizing solution, wherein the stabilizing solution comprises S-adenosyl homocysteine hydrolase, betaine, a preservative and a surfactant.
2. The homocysteine hydrolase additive according to claim 1 where the buffer is biological buffer including bovine serum albumin and AEP-HBC.
3. The homocysteine hydrolase additive according to claim 2, wherein the preservative is sodium azide.
4. The additive according to claim 3, wherein the surfactant is polyethylene glycol or polyoxyethylene benzyl styrene ether.
5. The homocysteine hydrolase additive according to claim 4 with high stability and anti-interference ability, wherein the stabilizing solution comprises the following components: s-adenosyl homocysteine hydrolase, betaine, sodium azide, polyethylene glycol and polyoxyethylene benzyl styrene ether.
6. The homocysteine hydrolase additive according to claim 5 with high stability and high interference resistance, wherein the additive comprises: 1.5-1.8KU/L of S-adenosyl homocysteine hydrolase, 2.5-3.5g/L of betaine, 0.4-0.6g/L of sodium azide, 2.8-3.2g/L of polyethylene glycol, 2.8-3.2g/L of polyoxyethylene benzyl styrene ether, 5wt% of bovine serum albumin and 5g/L of AEP-HBC.
7. A homocysteine detection kit, characterized in that it comprises the additive of claim 1 or 6.
8. The homocysteine detection kit according to claim 7, which comprises two components of R1 and R2;
the R1 component comprises: 30-40KU/L of lactate dehydrogenase, 0.73-0.79mmol/L of L-serine, 0.45-0.50mmol/L of reduced coenzyme I, and 0.48-0.52mmol/L of tris (2-hydroxyethyl) phosphine chloride;
the R2 component comprises: 1.8-2.0KU/L cystathionine-beta-synthetase, 0.8-1.0KU/L cystathionine-beta-lyase, 1.5-1.8KU/L S-adenosyl homocysteine hydrolase, 2.5-3.5g/L betaine, 0.4-0.6g/L sodium azide, 2.8-3.2g/L polyethylene glycol, 2.8-3.2g/L polyoxyethylene benzyl styrene ether, 5wt% bovine serum albumin and 5g/L AEP-HBC.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202310145387.9A CN115896085A (en) | 2023-02-21 | 2023-02-21 | Homocysteine hydrolase additive with high stability and strong interference resistance |
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| CN202310145387.9A CN115896085A (en) | 2023-02-21 | 2023-02-21 | Homocysteine hydrolase additive with high stability and strong interference resistance |
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