CN106198995A - A kind of homotype semicystionl diagnostic kit and the assay method of homotype semicystinol concentration investigating - Google Patents
A kind of homotype semicystionl diagnostic kit and the assay method of homotype semicystinol concentration investigating Download PDFInfo
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- CN106198995A CN106198995A CN201610473516.7A CN201610473516A CN106198995A CN 106198995 A CN106198995 A CN 106198995A CN 201610473516 A CN201610473516 A CN 201610473516A CN 106198995 A CN106198995 A CN 106198995A
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- reagent
- homotype
- homocysteine
- diagnostic kit
- stabilizer
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- 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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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
Abstract
The present invention relates to the assay method of a kind of homotype semicystinol concentration investigating, oxidized form homocysteine generates α batanone acid, ammonia and hydrogen sulfide, and ammonia, under the effect of glutamte dehydrogenase, makes NADH be converted into NAD+, come quantitatively by measuring NADH absorbance rate at 340nm；A kind of homotype semicystionl diagnostic kit, including reagent I and reagent II, reagent I contains reduced coenzyme Ⅰ, glutamte dehydrogenase, three (2 carboxyethyl) phosphonium salt hydrochlorate, alpha Ketoglutarate, surfactant, buffer, stabilizer, and reagent II contains HCYase, pyridoxal 5-phosphate, buffer, stabilizer.The invention has the beneficial effects as follows: establish the test kit of homocysteine in liquid double reagent detection human serum, it is not necessary to additionally configure fluorescence detector, it is simple to the detection of great amount of samples；Detection method will not disturbing by the acetone acid in sample, methionine and cysteine, price is less expensive.
Description
Technical field
The present invention relates to the assay method of a kind of homotype semicystionl diagnostic kit and homotype semicystinol concentration investigating.
Background technology
Homocysteine α, γ-lyases (homocysteine α, γ-lyases, HCYase, EC4.4.1.11) is
A kind of pyridoxal 5-phosphate (PLP) relies on enzyme, also known as L-Methionine γ-lyases, belongs to γ protein family.L-Methionine can be catalyzed
And the alpha, gamma of homocysteine-elimination reaction and γ-substitution reaction, catalysis Cys and the α of derivant, β eliminate
Reaction and beta substitution reaction.The enzyme being catalyzed this type of reaction finds in escherichia coli, named L-Methionine γ-lyase
(Methioninelyase), nowadays find, such as stench vacation list by its named methionine Gamma-catenase and in multiple biology
Pole bacterium, trichomonal vaginitis, gas list embrace Pseudomonas, arabidopsis etc..Although source difference, the molecular weight of HCYase is mostly at 170kDa
Left and right, is made up of at the homotype subunit of 40-48KD four molecular size ranges；There is pyridoxal 5-phosphate dependency, the ultraviolet of albumen
Absorb and a characteristic peak being combined with pyridoxal 5-phosphate also can occur in addition to having absworption peak at 280nm at 420nm.
The metabolism of HCYase sulfur-containing amino acid in vivo plays a significant role, and is developed to as new antitumoral
Medicine and the diagnostic reagent of plasma homocysteine, its mechanism of action and the potential using value in treatment cause people
Very big concern.Additionally it plays a significant role in the metabolism of sulfur-containing amino acid in protozoon body, can be anti-with it for target position exploitation
Parasite medicine.
Methionine is a kind of essential amino acids, is the main methyl group provider of body.It is that tumor cell is special that methionine relies on
Have, show that tumor cell cannot grow in the environment removing methionine.Methionine relies in people's tumor patient extensive
Existing, the Methionine metabolism of nearly all mankind's tumor cell all changes: the tumor that normal cell, non-methionine rely on is thin
Born of the same parents, methionine rely on tumor cell in methionine concentration reduce successively, and methionine concentration keep constant be tumor cell
Growth is necessary.HCYase can be catalyzed the alpha, gamma-cracking reaction of L-Methionine, generates α-batanone acid, methyl-sulfuric acid and ammonia, energy
Effectively reduce methionine concentration.Compared with classic chemotherapy method, HCYase has tumour-specific as new type antineoplastic medicine
Height, the features such as toxic and side effects is little, demonstrate huge clinical practice potentiality.
From McCully since 1969 propose the viewpoint that homocysteine is relevant with atherosclerosis, domestic
Outer scholar expands substantial amounts of research around this problem.Recent study confirm, hyperhomocysteinemiainjury with as people
The multiple cardiovascular disease such as the atherosclerosis of class the first killer, hypertension, myocardial infarction have close contact, are
One independent hazard factor of cardiovascular disease.In blood plasma, Hcy concentration often reduces by 3 μm ol/L, and ischemic heart desease incidence rate subtracts
Few 11%, stroke incidence reduces 19%.Homocysteine clinically can be as cardiovascular disease, especially coronary artery
The significant risk index of Atherosclerotic Agents and myocardial infarction, the elevated-levels of its concentration is directly proportional to the danger of disease.Therefore,
Study a kind of simple, quick, sensitive detection method significant.
2000, Tan reported a kind of method of investigating humotype semi-cystinol by enzyme biochemical.HCYase is catalyzed homotype
Cysteine is decomposed into α-batanone acid, hydrogen sulfide and ammonia, catabolite hydrogen sulfide and fluorescein N, N-dibutyl phenylenediamine
(DBPDA) at FeCl3Sour environment under formed pigment phenothiazine chloride-based compound, the latter quantitative determines at 660nm
And the content of HCY is directly proportional in its absorptance and sample.But this method needs to detect on particle fluorescence detector, is not suitable for
The application of clinical diagnosis detection and popularization.And this method is due at FeCl3Acid condition under develop the color, it is contemplated that HCYase and glimmering
Light element developer DBPDA can not stably be saved in sour environment, and the test kit therefore using the method is three reagent mostly, behaviour
Make loaded down with trivial details.
Summary of the invention
The technical problem to be solved in the present invention is: based on the problems referred to above, and the present invention provides a kind of homocysteine diagnosis
Test kit and the assay method of homotype semicystinol concentration investigating.
The present invention solves the technical scheme that its technical problem used: the mensuration of a kind of homotype semicystinol concentration investigating
Method, under (2-carboxyethyl) phosphonium salt hydrochlorate effect, oxidized form homocysteine is converted into sequestered homocysteine,
Under the effect of homocysteine alpha, gamma-lyases, generate α-batanone acid, ammonia and hydrogen sulfide；Ammonia is at the work of glutamte dehydrogenase
Under with, NADH is made to be converted into NAD+, the concentration of quantitative homocysteine is carried out by measuring NADH absorptance at 340nm.
A kind of homotype semicystionl diagnostic kit, including reagent I and reagent II two parts, wherein reagent I contains reduction
Live in type cozymase (NADH), glutamte dehydrogenase (GLDH), three (2-carboxyethyl) phosphonium salt hydrochlorate (TCEP), α-ketoglutaric acid, surface
Property agent, buffer, stabilizer；Reagent II contains HCYase, pyridoxal 5-phosphate (PLP), buffer, stabilizer.
Further, in every liter of reagent I solution, the content of each component is:
In every liter of reagent II solution, the content of each component is:
Further, in every liter of reagent I solution, the content of each component is:
In every liter of reagent II solution, the content of each component is:
Further, surfactant be triton x-100, B66, polysorbas20, dodecylbenzene sodium sulfonate, glycine betaine,
One or more in cetyl trimethylammonium bromide.
Further, stabilizer is sodium azide, nitrine lithium, PC300, sodium benzoate, potassium sorbate, P-hydroxybenzoic acid third
One or more in ester, Polyethylene Glycol, polyvinyl alcohol, disodiumedetate.
The invention has the beneficial effects as follows: (1) establishes the reagent of homocysteine in liquid double reagent detection human serum
Box, can operate, it is not necessary to additionally configure fluorescence detector, it is simple to great amount of samples on full-automatic or semi-automatic biochemical analyzer
Detection；
(2) in HCYase method detection human serum, the method for homocysteine will not be by the acetone acid in sample, first sulfur
Propylhomoserin and the interference of cysteine, the price of this method is more less expensive than the price of the method for other detection homocysteines, is beneficial to
Homocysteine detection purpose is promoted.
Accompanying drawing explanation
The present invention is further described below in conjunction with the accompanying drawings.
Fig. 1 is the range of linearity figure of test kit prepared by the present invention；
Fig. 2 is the dependency graph of the test kit prepared of the present invention and homocysteine Enzymatic cycling detection clinical sample.
Detailed description of the invention
Presently in connection with specific embodiment, the invention will be further described, and following example are intended to illustrate rather than
Limitation of the invention further.
1. the test kit of HCY content in preparation detection human serum
R1 reagent: in 1L 100mM Tris-HCl buffer, adds 10KU/L GLDH, 5mM TCEP, 0.5mM
NADH, 15mM α-ketoglutaric acid, 0.01% triton x-100,0.05% sodium azide, stir and be R1 reagent.
R2 reagent: in 1L 100mM Tris-HCl buffer, add 20KU/L HCYase, 0.05mM PLP,
0.05% sodium azide, stirs and is R2 reagent.
Homocysteine calibration object: in 3mM HCl solution, adds 28 μMs of HCY
Detection method:
As a example by Hitachi 7080 biochemistry analyzer: measuring wavelength is 340nm, and assay method is KINETIC METHOD.Sample size
20uL, R1 reagent 220uL, R2 reagent 30ul, calibrating mode is linear determination, and the Direction of Reaction is upwards.Calibration object mixes with R1
After, read absorbance A1 reaction the 16th, add R2, read absorbance A2 at 31, calculate absorbance difference δ A=
A2-A1.Take sample to be tested, be measured in the same method the absorbance difference of sample, substituted into formula HCY content (μm mol/L)=(mensuration
Pipe absorbance difference/calibration pipe absorbance difference) content of HCY in × calibration object, the HCY in sample to be tested can be calculated
Content.
2. range of linearity detection: take clinical high level pooled serum (60 μm ol/L) and carry out doubling dilution with normal saline,
To the dilute serum of 4 concentration, the dilute serum duplicate detection of each concentration 3 times, take its average, measured value is entered with theoretical value
Row linear regression analysis.As Fig. 1 statistical analysis shows that the regression equation of the method is: y=0.988x-0.377R2=0.999P <
0.05, the method is in 3-60 μm ol/L internal linear preferably (being shown in Table 1).
Table 1: the test kit range of linearity of the present invention
Test kit the most of the present invention is relevant to homocysteine detection reagent box (enzyme Enzymatic cycling) detection clinical sample
Property detection
50 example clinical samples are selected, respectively with this law and homocysteine detection reagent box (enzyme in 3～60 μm ol/L
Enzymatic cycling) (Shaoxing metformin company) detection, the regression equation obtaining this law and enzyme parameters is: y=0.913x+
1.110R2=0.996P < 0.05 (see Fig. 2).
Test kit anti-interference the most of the present invention is tested
Take fresh mix serum, be divided into 7 equal portions, then take 6 equal portions and add different interfering materials respectively, interfering material
Concentration is as shown in table 2.The homocysteine detection examination that matched group is sold with ShaoXing,ZheJiang metformin bio tech ltd
Agent box (enzyme parameters), the test kit that experimental group is prepared with embodiment in the present invention 1 is as detectable, respectively in detection serum
HCY content.Matched group the results are shown in Table 2 with the disturbance material in experimental group detection serum.Relative deviation (%)=(interference sample
The mensuration average of this mensuration average-matched group is noiseless sample) mensuration average × 100% of the noiseless sample of/matched group
Table 2 test kit of the present invention compares with matched group test kit interference free performance
From above-mentioned table 2 result, the materials such as acetone acid, blood ammonia, methionine, cysteine are not had by test kit of the present invention
There is significantly interference, and matched group testing result is disturbed by acetone acid, methionine and cysteine.This illustrates the present invention
Test kit is better than market test kit on sale at anti-interference aspect, improves the accuracy of testing result, has the market competitiveness.
The present invention utilizes the homocysteine detectable of homocysteine alpha, gamma-lyases preparation, detects homotype
The range of linearity of cysteine is in 3-60 μm ol/L.In this range of linearity, (enzyme circulates for reagent of the present invention and homocysteine
Method) reagent dependency is good.And the inventive method effectively reduces the dry of the materials such as acetone acid, methionine, cysteine
Disturb impact, thus improve the accuracy of detection homocysteine.The test kit of the present invention can be full-automatic or semi-automatic
Biochemical instruments completes detection, and easy and simple to handle, price is cheap relative to other homotype cysteine detecting methods, beneficially the market of reagent
Promote.
With the above-mentioned desirable embodiment according to the present invention for enlightenment, by above-mentioned description, relevant staff is complete
Entirely can carry out various change and amendment in the range of without departing from this invention technological thought.The technology of this invention
The content that property scope is not limited in description, it is necessary to determine its technical scope according to right.
Claims (6)
1. an assay method for homotype semicystinol concentration investigating, is characterized in that: under three (2-carboxyethyl) phosphonium salt hydrochlorate effect, oxygen
Change type homocysteine is converted into sequestered homocysteine, under the effect of homocysteine alpha, gamma-lyases, raw
Become α-batanone acid, ammonia and hydrogen sulfide；Ammonia, under the effect of glutamte dehydrogenase, makes NADH be converted into NAD+, by measuring NADH
Absorptance at 340nm carrys out the concentration of quantitative homocysteine.
2. a homotype semicystionl diagnostic kit, is characterized in that: including reagent I and reagent II two parts, wherein reagent I contains
Have reduced coenzyme Ⅰ, glutamte dehydrogenase, three (2-carboxyethyl) phosphonium salt hydrochlorate, α-ketoglutaric acid, surfactant, buffer,
Stabilizer；Reagent II contains HCYase, pyridoxal 5-phosphate, buffer, stabilizer.
A kind of homotype semicystionl diagnostic kit the most according to claim 2, is characterized in that:
In every liter of reagent I solution, the content of each component is:
In every liter of reagent II solution, the content of each component is:
A kind of homotype semicystionl diagnostic kit the most according to claim 3, is characterized in that:
In every liter of reagent I solution, the content of each component is:
In every liter of reagent II solution, the content of each component is:
A kind of homotype semicystionl diagnostic kit the most according to claim 2, is characterized in that: described surfactant
For the one in triton x-100, B66, polysorbas20, dodecylbenzene sodium sulfonate, glycine betaine, cetyl trimethylammonium bromide
Or it is several.
A kind of homotype semicystionl diagnostic kit the most according to claim 2, is characterized in that: described stabilizer is folded
Nitrogen sodium, nitrine lithium, PC300, sodium benzoate, potassium sorbate, propyl p-hydroxybenzoate, Polyethylene Glycol, polyvinyl alcohol, ethylenediamine
One or more in tetraacethyl disodium.
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| CN201610473516.7A CN106198995A (en) | 2016-06-24 | 2016-06-24 | A kind of homotype semicystionl diagnostic kit and the assay method of homotype semicystinol concentration investigating |
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Cited By (7)
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| 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 |
| CN110261601A (en) * | 2019-07-16 | 2019-09-20 | 三诺生物传感股份有限公司 | A kind of homocysteine detection kit |
| CN110308282A (en) * | 2019-06-21 | 2019-10-08 | 中生北控生物科技股份有限公司 | A kind of stable homocysteine Enzymatic cycling detection kit |
| CN110346575A (en) * | 2018-04-04 | 2019-10-18 | 南京东纳生物科技有限公司 | A kind of homocysteine fluorescence immune chromatography detection kit and its detection method |
| CN110646417A (en) * | 2019-10-24 | 2020-01-03 | 福建医科大学 | Rapid pyridoxal phosphate determination method taking nanogold as chromogenic probe |
| CN111366715A (en) * | 2018-12-25 | 2020-07-03 | 深圳迈瑞生物医疗电子股份有限公司 | Anti-heparin interference agent and anti-heparin interference kit |
| CN114280190A (en) * | 2021-12-27 | 2022-04-05 | 无锡市江原实业技贸有限公司 | Kit for detecting related substances of bicysteine |
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| CN114280190A (en) * | 2021-12-27 | 2022-04-05 | 无锡市江原实业技贸有限公司 | Kit for detecting related substances of bicysteine |
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Application publication date: 20161207 |