CN106443014B - Detection kit of ischemia modified albumin IMA and preparation method thereof - Google Patents
Detection kit of ischemia modified albumin IMA and preparation method thereof Download PDFInfo
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- CN106443014B CN106443014B CN201610833076.1A CN201610833076A CN106443014B CN 106443014 B CN106443014 B CN 106443014B CN 201610833076 A CN201610833076 A CN 201610833076A CN 106443014 B CN106443014 B CN 106443014B
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- reagents
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- 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
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/32—Cardiovascular disorders
Abstract
The present invention relates to biotechnologies, disclose a kind of detection kit of ischemia modified albumin IMA and preparation method thereof.The kit includes R1 reagents and R2 reagents, wherein it is 20% 80% that stabilizer in the R2, which includes ethanol amine, nonionic surfactant and 2,2 ˊ lignocaines, two sulphur dihydrochloride, and the mass volume ratio of the ethanol amine,.In embodiments of the present invention, by adding in ethanol amine, nonionic surfactant and 2 in R2 reagents, 2 ˊ lignocaines, two sulphur dihydrochloride, dithiothreitol (DTT) (DTT) in kit R2 in this way is just not easy to be aoxidized, and half-life period is long so that the stability of the detection kit of ischemia modified albumin IMA is high, sample correlations are good, strong antijamming capability.
Description
Technical field
The present invention relates to biotechnology, the detection kit of more particularly to a kind of ischemia modified albumin IMA and its preparation
Method.
Background technology
Ischemia modified albumin IMA (ischemia modified albumin, IMA) is when tissue ischemia/Reperfu- sion occurs
When, since free radical etc. destroys sero-abluminous amino acid sequence, and cause albumin with transition metal (including Cu, Co
And Ni) binding ability change, it is this to occur then to claim ischemic with the albumin that transition metal binding ability changes due to ischemic
Modified albumin (IMA).
In recent years, myocardial injury markers have rapid development.ECG (electrocardiogram), troponin (cTn), flesh red eggs
The detection of (Myo), Creatine Kinase MB (CK-MB) quality are clinically widely used in vain, and gradually substitution is former
The detection of some enzymatic activitys.But for the diagnosis of acute coronary syndrome (Acute Coronary Syndromes, ACS)
Problem is not yet properly settled.
ACS has many characteristics, such as that morbidity is anxious, variation is fast, clinical manifestation and danger are inhomogenous, and early diagnosis is difficult.Traditional
Biomarker such as troponin (cTn), myoglobins (Myo), creatine kinase isozyme (CK-MB) only occur bad in cardiac muscle
It is just increased when dead, but at this moment brings irreversible pathological lesion to patient.Therefore, being badly in need of one kind can be with reflecting myocardium ischemic
Early stage sensitivity biochemical indicator for early diagnosing.
IMA is research hotspot in recent years, and the research for ACS early diagnosis opens new road.IMA and tradition cardiac muscle
Necrosis marker is different, and the sensitivity that IMA detects ACS patient's myocardial ischemia is 2 times of ECG, 4 times of cTn.Available for evaluating
Early stage invertibity myocardial ischemia.IMA can be used for the risk stratification and guiding treatment of ACS.2003, since IMA is in Acute myocardial
High negative predictive value in ischemia diagnosis, FDA recommend its exclusion index for being ACS.IMA combinations electrocardiogram and troponin inspection
It surveys as a result, contributing to the early diagnosis of ACS, early intervention treatment improves the prognosis of patient and reduces case fatality rate.
At present IMA predominantly detect method be albumin cobalt Binding experiment (albumin-cobalt binding assay,
ACB).Detection IMA kits are made of liquid double reagent, and the first reagent is buffer solution and cobalt ions reagent, and the second reagent is slow
Fliud flushing and dithiothreitol (DTT) (DTT) reagent.But since DTT is a kind of very strong reducing agent in itself, it is easy to oxidation by air,
And half-life period is shorter, therefore stability and corkage stability are poor, reagent interference free performance is poor, sample in the presence of being used for a long time for reagent
The shortcomings of this correlation is poor.
In conclusion it is high, good, strong antijamming capability the ischemia modified albumin IMA of sample correlations to provide a kind of stability
The problem of detection kit is our urgent need to resolve.
Invention content
A kind of detection kit for being designed to provide ischemia modified albumin IMA of embodiment of the present invention and its preparation side
Method so that the stability of the detection kit of ischemia modified albumin IMA is high, sample correlations are good, strong antijamming capability.
In order to solve the above technical problems, embodiments of the present invention provide a kind of detection reagent of ischemia modified albumin IMA
Box, including R1 reagents and R2 reagents, wherein, each component and concentration in R1 reagents are respectively：
The concentration of buffer solution：20-500mM；
The concentration of cobalt ions：0.1-10g/L；
The concentration of stabilizer：0.1-10mM；
The concentration of preservative：0.01-5mM；
Each component and concentration in R2 reagents are respectively：
The concentration of buffer solution：20-500mM；
The concentration of dithiothreitol (DTT)：0.01-10mM；
The concentration of stabilizer：0.1-10mM；
The concentration of preservative：0.01-5mM；
Stabilizer in R2 includes ethanol amine, nonionic surfactant and 2,2 ˊ-lignocaine, two sulphur dihydrochloride,
And the mass volume ratio of ethanol amine is 20%-80%.
Embodiments of the present invention additionally provide a kind of method for the detection kit for preparing ischemia modified albumin IMA, comprising
Following steps：
Prepare R1 reagents：Following components is taken to stir evenly；
The concentration of buffer solution：20-500mM；
The concentration of cobalt ions：0.1-10g/L；
The concentration of stabilizer：0.1-10mM；
The concentration of preservative：0.01-5mM；
Prepare R2 reagents：Following components is taken to stir evenly；
The concentration of buffer solution：20-500mM；
The concentration of dithiothreitol (DTT)：0.01-10mM；
The concentration of stabilizer：0.1-10mM；
The concentration of preservative：0.01-5mM；
Stabilizer in R2 includes ethanol amine, nonionic surfactant and 2,2 ˊ-lignocaine, two sulphur dihydrochloride,
And the mass volume ratio of ethanol amine is 20%-80%.
Embodiment of the present invention in terms of existing technologies, passes through addition ethanol amine, non-ionic surface in R2 reagents
Activating agent and 2,2 ˊ-lignocaine, two sulphur dihydrochloride, DTT is just not easy to be aoxidized in such R2 reagents, extends DTT and partly declines
Phase further increases the stability of reagent, wherein 2,2 ˊ-lignocaine, two sulphur dihydrochloride is a kind of heparin antagonists, so as to
Increase the antijamming capability of reagent, improve reagent sample correlations.And the non-ionic surface active added in simultaneously and 2,2 ˊ-diethyl
Two sulphur dihydrochloride of amino, ethanol amine play synergistic protective effect so that the stabilization of the detection kit of ischemia modified albumin IMA
Property it is high, sample correlations are good, strong antijamming capability.
In addition, the ethanol amine in R2 reagents includes one kind or more in monoethanolamine, diethanol amine and triethanolamine
Kind.
In addition, the stabilizer in R1 reagents includes dihydroxysuccinic acid, ethanol amine, nonionic surfactant and glycerine
In it is one or more.
In addition, the stabilizer in R2 further includes one or both of dihydroxysuccinic acid, glycerine.
In addition, the mass volume ratio of ethanol amine is 50%-80%.
In addition, the buffer solution in R1 reagents and R2 reagents is each independently comprising glycine buffer, phosphoric acid salt buffer
One or more of liquid, acetate buffer, zwitterionic buffer and citric acid-sodium citrate buffer solution.
In addition, the buffer solution in R1 reagents and R2 reagents includes zwitterionic buffer and acetate buffer each independently
Liquid.
In addition, buffer solution in R1 reagents and R2 reagents a concentration of 20-200mM each independently, R1 reagents and R2 reagents
In the pH value of buffer solution be each independently 6-10.
In addition, the preservative in R1 reagents and R2 reagents includes Sodium azide, wherein, a concentration of 0.02-2mM of Sodium azide.
Description of the drawings
The comparison correlation detection curve of kit in Fig. 1 first embodiment of the invention.
Specific embodiment
To make the object, technical solutions and advantages of the present invention clearer, below in conjunction with attached drawing to each reality of the present invention
The mode of applying is explained in detail.However, it will be understood by those skilled in the art that in each embodiment of the present invention,
In order to make the reader understand this application better, many technical details are proposed.But even if without these technical details and base
In the various changes and modifications of following embodiment, the application technical solution claimed can also be realized.
The first embodiment of the present invention is related to a kind of detection kits of ischemia modified albumin IMA, include R1 reagents and R2
Reagent, wherein,
The component of R1 reagents and a concentration of：
The component of R2 reagents and a concentration of：
It is worth noting that, the ethanol amine in R2 reagents is included in monoethanolamine, diethanol amine and triethanolamine
It is one or more.
A kind of formula in the market routinely applied of the formula for FDA accreditations in present embodiment.
The preparation method of R1 reagents and R2 reagents is conventional method, i.e. the component in R1 reagents and R2 reagents adds respectively
Enter after distilled water that respectively mixing stirs evenly.
Kit in present embodiment carries out the actual conditions of ischemia modified albumin measuring such as on Biochemical Analyzer
Under：(by taking Hitachi 7100 as an example)
(this kit is applicable not only to Hitachi 7100, applies also for semi-automatic, the full-automatic biochemical of other brands and model
Analyzer, design parameter can be adjusted according to instrument).
Measure wavelength：Dominant wavelength 505nm (near or)；Commplementary wave length 700nm (near or)
Assay method：Two-point method；Rise reaction
Measuring temperature：37℃
Method using ischemia modified albumin IMA in the kit measurement sample in present embodiment is as follows：
Sample (sample of detection includes distilled water, standard items and clinical sample) plus R1 reagent mixings, 37 DEG C of incubation 5min
Absorbance A 0 is read afterwards, adds in R2 reagent mixings immediately, after 37 DEG C are reacted 5min, reads absorbance A 1, Δ A=A1-A0.Wherein
20 μ l, R1 reagent dosage of sample dosage, 200 μ l, R2 reagent dosage, 50 μ l.
IMA contents are calculated as follows in kit measurement sample in present embodiment：
Second embodiment of the present invention is related to a kind of detection kit of ischemia modified albumin IMA, includes R1 reagents and R2
Reagent, wherein,
The component of R1 reagents and a concentration of：
The component of R2 reagents and a concentration of：
The same first embodiment of preparation method of kit in present embodiment, the same first embodiment of detection method.
Third embodiment of the present invention is related to a kind of detection kit of ischemia modified albumin IMA, includes R1 reagents and R2
Reagent, wherein,
The component of R1 reagents and a concentration of：
The component of R2 reagents and a concentration of：
The same first embodiment of preparation method of kit in present embodiment, the same first embodiment of detection method.
The 4th embodiment of the present invention is related to a kind of detection kit of ischemia modified albumin IMA, includes R1 reagents and R2
Reagent, wherein,
The component of R1 reagents and a concentration of：
The component of R2 reagents and a concentration of：
The same first embodiment of preparation method of kit in present embodiment, the same first embodiment of detection method.
To involved in embodiment of the present invention to 4 kinds of kits carry out corkage stability and long-time stability investigate：
Open stability：Kit corkage in first embodiment to the 4th embodiment is placed on 2 DEG C of -8 DEG C of guarantors
After depositing 30 days, the quality-control product that ischemia modified albumin IMA is measured by the Biochemical Analyzer detection method is taken out, each concentration repeats to survey
It is 3 times fixed, calculate the relative deviation of testing result and sign value.The results are shown in Table 1：
Table 1：
As can be seen from Table 1 the corkage stability of first embodiment and second embodiment significantly better than third embodiment and
4th embodiment.This shows to add in stabilizer in R2 agent prescriptions With 2,2 ˊ-lignocaine
The stability of reagent, which is significantly better than, after two sulphur dihydrochlorides is not added with stabilizer.Simultaneously by the ratio of ethanol amine in the 4th embodiment
50% is reduced to hereinafter, the stability of reagent is also decreased obviously.
Long-time stability：12 are preserved by the kit in first embodiment to the 4th embodiment is placed in 2 DEG C -8 DEG C
After a month, the quality-control product that ischemia modified albumin IMA is measured by Biochemical Analyzer detection side method is taken out, each concentration repeats to survey
It is 3 times fixed, calculate the relative deviation of testing result and sign value.As a result as shown in Table 2-4：
Table 2：
Table 3：
Table 4：
It can be seen that from table 2-4, stabilizer added in first embodiment of the invention and second embodimentWith 2,2 ˊ-lignocaine, two sulphur dihydrochloride simultaneously ethanol amine mass volume ratio example 50% with
The stability of reagent is preferable when upper, and reagent is still stablized after storage 12 months.
Anti-interference evaluation is carried out to 4 kinds of detection boxes involved in embodiment of the present invention：
It is dry to investigate anti-interference mainly investigation haemolysis (hemoglobin), (chyle) high in fat, high bilirubin and the VC of reagent etc.
Disturb influence of the factor to testing result.It prepares a certain amount of matrix plasma and is divided into multigroup interference for adding various concentration inward
Object investigates pattern detection value and the deviation of former base matter plasma sample detected value after addition chaff interferent.Testing result such as table 5-6 institutes
Show：
Table 5：
Table 6：
First embodiment and second embodiment are to chyle in sample as can be seen from Table 5<15mmol/L、Vc<
600mg/L, hemoglobin<5g/L, bilirubin<300mg/L, the influence to measurement result can be neglected.Third embodiment and
Influence of four embodiments to every chaff interferent is slightly sensitive, and reagent is larger by the interference of bilirubin in the third embodiment.Cause
This can be seen that the formula interference free performance of first embodiment and second embodiment is preferable.
Table 6 measures different anti-coagulants pattern detection results for embodiment and shows first embodiment to anticoagulant heparin agent
Sample measures result is consistent with serum result, and correlation is influenced for 0.99 without conspicuousness.Third embodiment is to anticoagulant heparin agent
The significant interference of sample, anticoagulant heparin sample and serum sample maximum deviation equally find out that first is real 40.9%
It applies and stabilizer is added in modeIt is done with the antiheparin of 2,2 ˊ-lignocaine, two sulphur dihydrochloride reagent
The better performances disturbed.
Correlation experiment is compared kit prepared by first embodiment with commercial reagent box：
Using the kit prepared in first embodiment, detected with common certain generally acknowledged ischemia modified albumin IMA in the market
Kit (colorimetric method) carries out control test, while has detected 50 clinical serum samples, and clinical sample comes from the Sixth Man people
Hospital.Respectively to sample replication 2 times, average value is calculated respectively, and linear regression analyses are carried out to 50 pattern detection results,
Calculate the related coefficient of two kinds of kit testing results.Testing result is as shown in Figure 1, wherein, that Y-axis represents is the first embodiment party
The testing result (U/ml) of kit in formula, what X-axis represented is the testing result (U/ml) for comparing reagent, i.e. commercial reagent box
Testing result.
The results show that correlation coefficient r=0.99 (R2=0.977) of two kinds of kit testing results, equation of linear regression
According to U.S. clinical Laboratory Standard association (CLSI) documentation requirements (r>0.975), the deficiency decorated white egg of this practicality present invention
White assay kit and commercial reagent box have good clinical sample correlation, it can be seen that ischemia modified albumin IMA of the present invention
Assay kit can clinically replace other reagent agent boxes to use, easy to use, reduce testing cost, be more suitable for clinical big
Scale uses.
The 5th embodiment of the present invention is related to a kind of preparation method of the detection kit of ischemia modified albumin IMA, the party
Method comprises the steps of：
Prepare R1 reagents：Following components is taken to stir evenly；
The concentration of buffer solution：20-500mM；
The concentration of cobalt ions：0.1-10g/L；
The concentration of stabilizer：0.1-10mM；
The concentration of preservative：0.01-5mM；
Prepare R2 reagents：Following components is taken to stir evenly；
The concentration of buffer solution：20-500mM；
The concentration of dithiothreitol (DTT)：0.01-10mM；
The concentration of stabilizer：0.1-10mM；
The concentration of preservative：0.01-5mM；
Stabilizer in the R2 includes ethanol amine, nonionic surfactant and 2,2 ˊ-two sulphur disalt of lignocaine
Hydrochlorate, and the mass volume ratio of the ethanol amine is 20%-80%.
It will be understood by those skilled in the art that the respective embodiments described above are to realize specific embodiments of the present invention,
And in practical applications, can to it, various changes can be made in the form and details, without departing from the spirit and scope of the present invention.
Claims (9)
1. a kind of detection kit of ischemia modified albumin IMA, which is characterized in that including R1 reagents and R2 reagents, wherein,
Each component and concentration in the R1 reagents are respectively：
Each component and concentration in the R2 reagents are respectively：
Stabilizer in the R2 includes ethanol amine, nonionic surfactantCO-630 and 2,2 ˊ-diethyl
Two sulphur dihydrochloride of amino, and the mass volume ratio of the ethanol amine is 50%-80%.
2. detection kit according to claim 1, which is characterized in that the ethanol amine in the R2 reagents includes an ethyl alcohol
It is one or more in amine, diethanol amine and triethanolamine.
3. detection kit according to claim 1, which is characterized in that the stabilizer in the R1 reagents includes dihydroxy
It is one or more in succinic acid, ethanol amine, nonionic surfactant and glycerine.
4. detection kit according to claim 1, which is characterized in that the stabilizer in the R2 further includes dihydroxy amber
One or both of amber acid, glycerine.
5. detection kit according to claim 1, which is characterized in that the buffer solution in the R1 reagents and R2 reagents is each
From independently include glycine buffer, phosphoric acid phthalate buffer, acetate buffer, zwitterionic buffer and citric acid-
One or more of sodium citrate buffer solution.
6. detection kit according to claim 5, which is characterized in that the buffer solution in the R1 reagents and R2 reagents is each
From independently comprising zwitterionic buffer and acetate buffer.
7. detection kit according to claim 1, which is characterized in that the R1 reagents and the buffer solution in R2 reagents
Concentration is each independently 20-200mM.
8. detection kit according to claim 1, which is characterized in that the preservative packet in the R1 reagents and R2 reagents
Containing Sodium azide, wherein, a concentration of 0.02-2mM of the Sodium azide.
9. a kind of preparation method of the detection kit of ischemia modified albumin IMA, which is characterized in that comprise the steps of：
Prepare R1 reagents：Following components is taken to stir evenly；
The concentration of buffer solution：20-500mM；
The concentration of cobalt ions：0.1-10g/L；
The concentration of stabilizer：0.1-10mM；
The concentration of preservative：0.01-5mM；
Prepare R2 reagents：Following components is taken to stir evenly；
The concentration of buffer solution：20-500mM；Ph value of buffer solution：5.5；
The concentration of dithiothreitol (DTT)：0.01-10mM；
The concentration of stabilizer：0.1-10mM；
The concentration of preservative：0.01-5mM；
Stabilizer in the R2 includes ethanol amine, nonionic surfactantCO-630 and 2,2 ˊ-diethyl
Two sulphur dihydrochloride of amino, and the mass volume ratio of the ethanol amine is 50%-80%.
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| CN108152225B (en) * | 2017-11-29 | 2020-11-06 | 武汉生之源生物科技股份有限公司 | Determination method and detection kit for ischemia modified albumin |
| CN109142749A (en) * | 2018-08-11 | 2019-01-04 | 金华市强盛生物科技有限公司 | A kind of cardic fatty acid binding protein detection kit |
| CN113238060B (en) * | 2021-05-08 | 2022-10-11 | 迈克生物股份有限公司 | Kit for predicting or diagnosing myocarditis |
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| CN103760357B (en) * | 2013-11-04 | 2016-02-03 | 山东博科生物产业有限公司 | A kind of ischemia modified albumin IMA detection kit |
| CN104198729B (en) * | 2014-08-27 | 2015-06-03 | 宁波瑞源生物科技有限公司 | Steady ischemia modified albumin kit |
| CN105021609A (en) * | 2015-07-06 | 2015-11-04 | 广州金域医学检验中心有限公司 | Ischemia-modified albumin detection reagent and detection method thereof |
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