CN105445469A - Detection kit of ischemia modified albumins - Google Patents
Detection kit of ischemia modified albumins Download PDFInfo
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- CN105445469A CN105445469A CN201410436469.XA CN201410436469A CN105445469A CN 105445469 A CN105445469 A CN 105445469A CN 201410436469 A CN201410436469 A CN 201410436469A CN 105445469 A CN105445469 A CN 105445469A
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Abstract
The invention relates to a detection kit of ischemia modified albumins. The kit concretely comprises cobalt ions and dithiothreitol. A stabilizer is added to the kit, so the storage stability of the kit is increased; and a solubilizer is also added, so pollution of reagents to a colorimetric device is removed.
Description
Technical field
The present invention relates to clinical examination field, particularly, relate to a kind of ischemia modified albumin IMA detection kit of improvement.
Background technology
Ischemia modified albumin IMA (Ischemia-modifiedalbumin; IMA) refer to when tissue ischemia and Reperfu-sion occur; the albumin that N end amino acid is modified (binding site is occupied by copper ion or is acetylation or lacks), is characterized in declining with the binding ability of transition metal (nickel, copper, cobalt).
Namely IMA raises rapidly in 5-10 minute after myocardial ischemia, gets back to baseline in 6-12h, is an early stage index after myocardial ischemia occurs before meronecrosis.The index of blocking with other reflecting myocardium is as compared with CK-MB, Myo, cTn, and its kinetic curve shows, its time zone produced is more forward, judges that myocardial ischemia sensitivity is higher.
Clinically, IMA is mainly used in the early diagnosis of acute myocardial ischemia, and acute coronary syndrome (ACS) early diagnosis and risk stratification.Sinha research shows relative cardiogram (ECG) and TnT, and IMA is the highest as myocardial ischemia index sensitivity.Be combined with ECG or TnT and diagnose ACS, can detection sensitivity be significantly improved.Also there are some researches show that IMA is not only the good index judging myocardial ischemia, and its level raised is also relevant to the order of severity of myocardial ischemia simultaneously.Also have report to point out, the specificity of IMA is lower, and the diseases such as acute infection, end stagerenaldisease, prostatic disorders and cirrhosis can cause IMA to raise.
At present, IMA, as the index evaluating myocardial ischemia, is approved by U.S. FDA, is also the index of a unique judgement myocardial ischemia simultaneously.Therefore, this project has broad application prospects.
The main detection method of current IMA is albumin cobalt Binding experiment (albumin-cobaltbindingassay, ACB).Its main measuring principle is:
The first step:
Excessive Co
2++ albumin → Co-albumin+residue Co
2+
Excessive Co
2++ IMA → do not combine;
Second step:
Residue Co
2++ developer → color products.
Ischemia modified albumin IMA is to Co
2+binding ability lower than normal albumin, Human Serum Albumin and excessive Co
2+in conjunction with after, remaining free Co
2+react with organic chromogenic thing (dithiothreitol (DTT) and analog or azo developer etc.) and generate coloured (red or blue) product, under certain wavelength, carry out colorimetric, its absorbance and Co
2+concentration, IMA content are directly proportional, and compare with standard items, can calculate the concentration of IMA in sample.
In ACB experiment, after cobalt ions is combined with developer, the compound of formation is water-soluble very poor.Particularly when the IMA concentration contained in sample is higher or detect dummy, the complex concentration that reaction generates is high, can directly separate out in course of reaction and be adsorbed in the colorimetric device of instrument.This pollutes instrument, and then affects the analytical performance of whole instrument.
Use the assay methods such as ELISA, HPLC/MS in addition in addition, but due to inconvenient operation, use seldom.
Summary of the invention
According to certain embodiments of the present invention, provide a kind of method improved albumin cobalt Binding experiment Instrumental and pollute, it comprises step: 1) provide sample to be tested, 2) sample to be tested is contacted with the first reagent, 3) afterwards sample to be tested is contacted with the second reagent.Be to be understood that, any albumin cobalt Binding experiment as known in the art (ACB experiment) was all applicable to the method that the present invention improves pollution, such as but not limited to " CardiovascularBiomarkers:PathophysiologyandDiseaseManage ment " Springer publishing house 2010; " BiomarkersinHeartDisease " JamesdeLemos.JohnWiley & Sons, those described in 2009.
In the embodiment that some are concrete, the first pack contains: damping fluid and Co2+; Second pack contains: damping fluid and developer.Compare with albumin cobalt Binding experiment of the prior art, one of method difference of the present invention is also to comprise solubilizer in reaction system.In the embodiment that some are concrete, in the first reagent and/or in the second reagent, even and/or in the 3rd reagent or in each reagent, contain solubilizer; Solubilizer is selected from following one or more: albumin, methyl alcohol, ethanol, propylene glycol, glycerine, isopropyl alcohol, isoamylol and dimethyl sulfoxide (DMSO).In one preferred embodiment, solubilizer is albumin (such as BSA) or glycerine.In the embodiment that some are concrete, when solubilizer is albumin, albuminous content is 0.01-20%, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 8, 9, 10, 15, 16, 18, 20% or above-mentioned any two end values between scope, preferred 0.05-2%, more preferably 0.1-1%, most preferably 0.2%, some preferred embodiment in, the content of albumin in the 1st reagent is 0.2%.When solubilizer is the solubilizer beyond albumin, its content is 0.1-20%, such as 0.1,0.15,0.2,0.25,0.3,0.35,0.4,0.45,0.5,0.55,0.6,0.65,0.7,0.75,0.8,0.85,0.9,0.95,1.0,1.5,1.6,1.7,1.8,1.9,2.0,2.5,3.0,3.5,4,4.5,5,5.5,6,6.5,7,8,9,10,15,16,18,20% or above-mentioned any two end values between scope, preferred 0.2-10%, more preferably 0.5-5%; In one preferred embodiment, the content of glycerine in the 2nd reagent is 2%.
Although be not bound by any theory, can understand like this, method of the present invention, by introducing solubilizer, increases compound dissolubility in a solvent, and then removes reaction product to the pollution of instrument colorimetric device.
Any sample to be tested being applicable to albumin cobalt Binding experiment in prior art all can be used for the method for the application, is selected from whole blood, serum, blood plasma and urine.
In the embodiment that some are concrete, the reagent (such as the second reagent) at stabilizing agent place also comprises stabilizing agent.Stabilizing agent is preferably three (2-carboxyethyl) phosphine-hydrochloride.The content of stabilizing agent is 0.05-100mM, such as 0.05,0.1,0.2,0.3,0.5,1.0,2.0,3.0,5.0,6.0,8.0,10,12,15,20,25,30,40,50,60,70,80,90, scope between 100mM or above-mentioned any two end values, preferred 0.2-20mM, most preferably 1mM.Although be not bound by any theory, can understand like this, because developer (as dithiothreitol (DTT)) is reducing substances, when using as liquid reagent box, place easily by the dioxygen oxidation in air for a long time, and then causing losing coloration ability.Therefore, method of the present invention, by adding stability component, increases the stability of developer at fluid matrix, increases the storage stability of reagent.
In the embodiment that some are concrete, damping fluid be selected from following one or more: Good's damping fluid, phosphate buffer, glycine buffer and acetate buffer, buffer concentration is 10-500mM, 10-50mM, 50-100mM, 100-200mM, 200-300mM, 300-500mM.
In the embodiment that some are concrete, Co
2+be selected from: cobalt chloride, cobaltous sulphate, cobalt nitrate, cobalt borate; Described Co
2+content be 0.01-10mM, preferred 0.05-2mM, more preferably 0.1 – 0.5mM, most preferably 0.3mM.
In the embodiment that some are concrete, developer is dithiothreitol (DTT), and the content of described developer is 0.1-100mM, preferred 1-10mM, more preferably 2-5mM, most preferably 3mM.
Embodiment there is provided a kind of ischemia modified albumin IMA detection kit according to of the present invention other, it comprises:
Wherein solubilizer is selected from following one or more: albumin, methyl alcohol, ethanol, propylene glycol, glycerine, isopropyl alcohol, isoamylol and dimethyl sulfoxide (DMSO).
In the embodiment that some are concrete, kit also comprises stabilizing agent.Stabilizing agent is preferably three (2-carboxyethyl) phosphine-hydrochloride.The content of stabilizing agent is 0.05-100mM, preferred 0.2-20mM, more preferably 0.5-5mM, most preferably 1mM.
Compare with IMA detection kit of the prior art, one of kit difference of the present invention is also to comprise solubilizer in kit.In the embodiment that some are concrete, in the first reagent and/or in the second reagent, even and/or in the 3rd reagent or in each reagent, contain solubilizer.Solubilizer is selected from following one or more: albumin, methyl alcohol, ethanol, propylene glycol, glycerine, isopropyl alcohol, isoamylol and dimethyl sulfoxide (DMSO).In one preferred embodiment, solubilizer is albumin or glycerine.In the embodiment that some are concrete, when solubilizer is albumin, albuminous content is 0.01-20%, preferred 0.05-2%, more preferably 0.1-1%, most preferably 0.2%; In one preferred embodiment, the content of albumin in the 1st reagent is 0.2%.When solubilizer is the solubilizer beyond albumin, its content is 0.1-20%, preferred 0.2-10%, more preferably 0.5-5%, most preferably 2%; In one preferred embodiment, the content of glycerine in the 2nd reagent is 2%.
In some embodiments, the damping fluid in kit be selected from following one or more: Good's damping fluid, phosphate buffer, glycine buffer and acetate buffer.Damping fluid in first reagent and the second reagent can be the same or different, preferably identical.In some embodiments, Co
2+be selected from cobalt chloride, cobaltous sulphate, cobalt nitrate, cobalt borate; Described Co
2+content be 0.05-2mM, more preferably 0.1 – 0.5mM, most preferably 0.3mM.In some embodiments, developer is dithiothreitol (DTT), and the content of described developer is 1-10mM, more preferably 2-5mM, most preferably 3mM.
Technician is appreciated that described kit can be prepared into any suitable form, and the form of three reagent or double reagent, is prepared into the form of double reagent usually.Kit can be powdered reagent, is dissolved in water before namely using; Also can be liquid reagent, directly use.Technician is further appreciated that in order to avoid developing the color in advance, Co
2+different reagent is arranged in from developer.Such as in some embodiments, Co
2+be arranged in the first reagent, and developer is arranged in the second reagent.In some embodiments, developer and stabilizing agent are arranged in same reagent.
In some embodiments, kit can also comprise the auxiliary element such as surfactant and/or antiseptic, and concentration is respectively between 0.01 to 2%, and this can be determined voluntarily by technician.
According to a concrete embodiment, ischemia modified albumin IMA detection kit of the present invention, comprises or by forming as follows:
Reagent 1:
Good ' s damping fluid 50mM,
Cobalt chloride 0.3mM,
TritonX-4050.1％；
Reagent 2:
The embodiment concrete according to another, ischemia modified albumin IMA detection kit of the present invention, comprises or by forming as follows:
Reagent 1:
Reagent 2 composition and concentration:
Good ' s damping fluid 100mM,
Dithiothreitol (DTT) 5mM,
Three (2-carboxyethyl) phosphine-hydrochloride 5mM.
Embodiment there is provided a kind of preparation method of ischemia modified albumin IMA detection kit according to of the present invention other, prepare kit of the present invention according to conventional method well known in the art, it is characterized in that comprising the step adding solubilizer in reagent.Solubilizer is selected from following one or more: albumin, methyl alcohol, ethanol, propylene glycol, glycerine, isopropyl alcohol, isoamylol and dimethyl sulfoxide (DMSO).In one preferred embodiment, solubilizer is albumin or glycerine.In the embodiment that some are concrete, when solubilizer is albumin, albuminous content is 0.01-20%, preferred 0.05-2%, more preferably 0.1-1%, most preferably 0.2%; In one preferred embodiment, the content of albumin in the 1st reagent is 0.2%.When solubilizer is the solubilizer beyond albumin, its content is 0.1-20%, preferred 0.2-10%, more preferably 0.5-5%, most preferably 2%; In one preferred embodiment, the content of glycerine in the 2nd reagent is 2%.
When detecting IMA, the residue cobalt ions that produces can use method well known in the art, the method such as can be given out light by galvanochemistry, chemistry, also can by the instrument detection reaction comprising ion analyser.
The kit of detection ischemia modified albumin IMA provided by the invention, can use, not need other instrument on automatic clinical chemistry analyzer and semi-automatic biochemical analyzer, measurement result is accurate, and speed is fast, reproducible, good stability, antijamming capability is strong, is convenient to promote the use of.
Accompanying drawing explanation
Fig. 1 shows concentration of specimens range of linearity when kit 2 of the present invention measures sample.
Embodiment
To further illustrate the present invention by following non-limiting example below, as well known to those skilled in the art, without departing from the spirit of the invention, can make many amendments to the present invention, such amendment also falls into scope of the present invention.If no special instructions, the experiment material used all can easily obtain from commercial company following experimental technique.
Embodiment 1: the preparation method of kit of the present invention
According to following composition, kit is mixed with the form of liquid double reagent.
First of kit 1. kit of the present invention consists of double reagent kit, and its composition is as follows:
Reagent 1 composition and concentration:
Good ' s damping fluid: 50mM, pH8.0
Cobalt chloride: 0.3mM,
TritonX-405:0.1％；
Reagent 2 composition and concentration:
The 2nd of kit 2. kit of the present invention consists of double reagent kit, and its composition is as follows:
Reagent 1 composition and concentration:
Reagent 2 composition and concentration:
Good ' s damping fluid: 100mM,
Dithiothreitol (DTT): 5mM,
Three (2-carboxyethyl) phosphine-hydrochloride: 5mM.
The using method of embodiment 2. kit
This detection method is rate method.
1) serum sample is provided,
2) after being mixed with 20 μ L serum samples by 150 μ L reagent 1,5min is hatched for 37 DEG C, absorbance A 1 under detection 505nm,
3) add 50 μ L reagent 2, then hatch 5min, absorbance A 2 under 505nm during detection 5min.Thus calculate the concentration of IMA in sample.Other suitable detection mode well known in the art and operation steps also can use.
Test case 1. study on the stability
This kit 1 is put into 2-8 DEG C 10 months, investigate reagent stability.The change of Main Basis blank absorbency is investigated.Experimental result is in table 1.Can find out, having good stability of this kit.
Table 1: the stability result of kit 1 of the present invention
Test case 2: repeatability, the range of linearity and antifouling property
(1) use the same routine serum sample of kit 2 of the present invention horizontal survey simultaneously 20 times, detection kit measures repeatability during sample, the results are shown in Table 2.
Table 2: kit 2 of the present invention measures repeated result during serum sample
(2) use serum high level and low value sample, detect with kit 2 of the present invention, verify concentration of specimens range of linearity during kits sample of the present invention, the results are shown in Figure 1.Visible, kits repeatability of the present invention is high, and measure linear scope is wide.
(3) kit 1 or 2 is measured respectively on BeckmanAU series model the blank absorbency of R1, ensure that each batch is detected the same cuvette of use, investigate the absorbance change of cuvette before and after detecting.
Table 3: kit 2 of the present invention is on the impact (solubilizer is at R1) of reagent contamination performance
Table 4: embodiment of the present invention kit 1 is on the impact (solubilizer is at R2) of reagent contamination performance
Can obviously find out, kit of the present invention, after doing once mensuration, contrast colors cup did not affect, and contrast agent box then contrast colors cup produces obvious adsorption fouling, and then affects the analytical performance of whole detecting instrument.
Claims (13)
1. an ischemia modified albumin IMA detection kit, it comprises:
Wherein solubilizer is selected from following one or more: albumin, methyl alcohol, ethanol, propylene glycol, glycerine, isopropyl alcohol, isoamylol and dimethyl sulfoxide (DMSO); The preferred albumin of solubilizer or glycerine.
2. ischemia modified albumin IMA detection kit according to claim 1, also comprises: stabilizing agent, and described stabilizing agent is three (2-carboxyethyl) phosphine-hydrochloride;
The content of stabilizing agent is 0.05-100mM, preferably 0.05,0.1 the scope, 0.2,0.3,0.5,1.0,2.0,3.0,5.0,6.0,8.0,10,12,15,20,25,30,40,50,60,70,80,90, between 100mM or above-mentioned any two end values, more preferably 0.2-20mM, more preferably 0.5-5mM, most preferably 1mM.
3. ischemia modified albumin IMA detection kit according to claim 1, wherein:
Albuminous content counts 0.01-20% by mass/volume, preferably 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 8, 9, 10% or above-mentioned any two end values between scope, more preferably 0.05-2%, more preferably 0.1-1%, most preferably 0.2%,
Solubilizer level beyond albumin by volume/volume counts 0.1-20%, preferably 0.1,0.15,0.2,0.25,0.3,0.35,0.4,0.45,0.5,0.55,0.6,0.65,0.7,0.75,0.8,0.85,0.9,0.95,1.0,1.5,1.6,1.7,1.8,1.9,2.0,2.5,3.0,3.5,4,4.5,5,5.5,6,6.5,7,8,9,10,15% or above-mentioned any two end values between scope, more preferably 0.2-10%, more preferably 0.5-5%, most preferably 2%.
4. ischemia modified albumin IMA detection kit according to claim 1, wherein:
Damping fluid be selected from following one or more: Good's damping fluid, phosphate buffer, glycine buffer and acetate buffer;
Co
2+be selected from: cobalt chloride, cobaltous sulphate, cobalt nitrate, cobalt borate; Described Co
2+content be 0.05-2mM, more preferably 0.1 – 0.5mM, most preferably 0.3mM;
Developer is dithiothreitol (DTT), and the content of described developer is 1-10mM, more preferably 2-5mM, most preferably 3mM.
5. ischemia modified albumin IMA detection kit according to claim 1, described kit is the form of three reagent or double reagent; Wherein Co
2+different reagent is positioned at from developer.
6. ischemia modified albumin IMA detection kit according to claim 1, also comprises surfactant and/or antiseptic, by mass/volume concentration respectively between 0.01 to 2%.
7. an ischemia modified albumin IMA detection kit, it comprises or by forming as follows:
Reagent 1:
Good ' s damping fluid 50mM,
Cobalt chloride 0.3mM,
TritonX-405 is by mass/volume 0.1%;
Reagent 2:
8. an ischemia modified albumin IMA detection kit, it comprises or by forming as follows:
Reagent 1:
Reagent 2 composition and concentration:
Good ' s damping fluid 100mM,
Dithiothreitol (DTT) 5mM,
Three (2-carboxyethyl) phosphine-hydrochloride 5mM.
9. prepare a method for the ischemia modified albumin IMA detection kit as described in any one of claim 1-8, it is characterized in that method comprises the step adding solubilizer in reagent;
Described solubilizer is selected from following one or more: albumin, methyl alcohol, ethanol, propylene glycol, glycerine, isopropyl alcohol, isoamylol and dimethyl sulfoxide (DMSO);
Albumin is 0.01-20% by the content of mass/volume, preferred 0.05-2%, more preferably 0.1-1%, most preferably 0.2%;
Solubilizer beyond albumin is 0.1-20% by mass/volume content, preferred 0.2-10%, more preferably 0.5-5%, most preferably 2%.
10. improve the method that albumin cobalt Binding experiment Instrumental pollutes, it comprises step:
1) provide sample to be tested,
2) make sample to be tested contact with the first reagent,
3) after, sample to be tested is contacted with the second reagent;
Wherein
First pack is containing damping fluid and Co
2+;
Second pack is containing damping fluid and developer;
Wherein also contain solubilizer in the first reagent and/or the second reagent;
Described solubilizer is selected from following one or more: albumin, methyl alcohol, ethanol, propylene glycol, glycerine, isopropyl alcohol, isoamylol and dimethyl sulfoxide (DMSO);
Sample to be tested is selected from: whole blood, serum, blood plasma and urine.
11. methods improved albumin cobalt Binding experiment Instrumental and pollute according to claim 10, described second reagent also comprises stabilizing agent,
Stabilizing agent is preferably three (2-carboxyethyl) phosphine-hydrochloride; The content of stabilizing agent is 0.05-100mM, preferred 0.2-20mM, more preferably 0.5-5mM, most preferably 1mM.
12. methods improved albumin cobalt Binding experiment Instrumental and pollute according to claim 10, wherein:
Albumin is 0.01-20% by the content of mass/volume, preferred 0.05-2%, more preferably 0.1-1%, most preferably 0.2%;
Solubilizer beyond albumin is 0.1-20% by mass/volume content, preferred 0.2-10%, more preferably 0.5-5%, most preferably 2%.
The method that 13. improvement albumin cobalt Binding experiment Instrumentals according to any one of claim 10-12 pollute, wherein:
Damping fluid be selected from following one or more: Good's damping fluid, phosphate buffer, glycine buffer and acetate buffer, buffer concentration is 10-500mM;
Co
2+be selected from: cobalt chloride, cobaltous sulphate, cobalt nitrate, cobalt borate; Described Co
2+content be 0.01-10mM, preferred 0.05-2mM, more preferably 0.1 – 0.5mM, most preferably 0.3mM;
Developer is dithiothreitol (DTT), and the content of described developer is 0.1-100mM, preferred 1-10mM, more preferably 2-5mM, most preferably 3mM.
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| CN108152225A (en) * | 2017-11-29 | 2018-06-12 | 武汉生之源生物科技股份有限公司 | The assay method and detection kit of a kind of ischemia modified albumin IMA |
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