CN104330575B - A kind of Troponin I detection reagent and preparation method thereof - Google Patents
A kind of Troponin I detection reagent and preparation method thereof Download PDFInfo
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Abstract
The present invention relates to a kind of Troponin I detection reagent and preparation method thereof, it is formed with reagent one and reagent two, wherein, the volume ratio of the reagent one and reagent two is 5: 1；The reagent one includes：20~50mmol/L buffer solution, 2%w/v coagulant, 2%w/v BSA, 0.1%~0.5%w/v preservative；The reagent two includes：0.1%~0.5%w/v is marked with colloid gold particle, 2%~5%w/v stabilizer, 20~50mmol/L buffer solution, the 0.1%~0.5%w/v preservative of anti-human cTnI antibody；The mark has a diameter of 60nm~90nm of the colloid gold particle of cTnI antibody.Many advantages, such as present invention has workable, high analyte sensitivity, High Linear and quickly and easily facilitated.
Description
【Technical field】
The present invention relates to a kind of Troponin I detection reagent and preparation method thereof, and in particular to one kind is used to detect serum
Or cTnI homogeneous phase sol particle type cTnI immunoassay reagents and preparation method thereof in blood plasma, belong to external in medicine equipment
Diagnostic field.
【Background technology】
Troponin I molecular weight is 23000, is that actin suppresses subunit, it has 3 kinds of hypotypes：It is fast skeletal muscle hypotype, slow
Skeletal muscle hypotype and myocardium hypotype.This 3 kinds of TnI hypotypes come from 3 kinds of different genes respectively.Myocardium hypotype (cTnI) is relative two kinds
Skeletal muscle hypotype there are about cTnI after 40% non-homology AMI releasing pattern (free form still with other troponin knots
Synthesising complex, oxidised form still reduces form) also imperfectly understand so far.
To the diagnosis of myocardial damage in the clinical biochemistry indications of many diagnosing acute myocardial infarctions (AMI), CK-MB (blood
Clear away heart-fire creatase) once it had been considered as once " goldstandard " for diagnosing AMI, have been widely used for many years.With to cardiac troponin
(cTn) further investigate, either to the specificity or diagnostic sensitivity of cardiac muscle, CK-MB status all receives serious challenge.
CTn is considered as determination mark best at present, and just progressively substitution CK-MB turns into AMI diagnosis " goldstandard ".
Myocardial cell injury will necessarily occur for the patient with various coronary artery diseases, and the clinical manifestation of some patients can
Can be not in full conformity with WHO on AMI diagnostic criteria (unstable angina is exactly one of them), but with some myocardial damages
Mark (such as cTnT) is raised, so as to cause intracellular constituent to leak into Peripheral Circulation.This causes cardiac muscle cell
The detection of damage markers is possibly realized.CTnT and cTnI (3~6h) blood level after AMI are raised quickly, and CK-MB (3~
8h) quite or a little earlier, they are determined specificity and sensitivity are apparently higher than CK-MB.CTn has the considerably long diagnostic window phase
(cTnI7~9 day, cTnT is longer).Diagnosis of the cTn to acute chest pain patient (no matter whetheing there is Skeletal muscle injury) is superior to CK-MB.
Research shows：CTnI and cTnT can identify what CK-MB can not be detected without significant difference in terms of the diagnosis to AMI
Myocardial damage.For cTnT, cTnI shows relatively low initial sensitivity and higher specificity.The relative value just risen
For, cTnT is higher than cTnI；The frequency that cTnT rises in patients with unstable angina is higher than cTnI.The death in 30 days after AMI
In terms of the forecast of rate, cTnT is better than cTnI.
Either myocardial infarction of the unstable angina still without Q ripples, the cTnT most prognostic values of initial 24 hours.It is right
The follow-up discovery of unstable coronary artery disease patient, cTnT and all normal person of exercise test two, only the 1% of dead or AMI；
If abnormal, dead or AMI is up to 50%.The follow-up investigation of acute coronary illness (including myocardial infarction) patient is found,
CTnT is less than the death rate only 4% of 0.1 μ g/L patient, and Comparatively speaking, the death rate of the patient more than 0.1 μ g/L is then big 3 times,
The percentage suffered a shock is big 3 times, and the percentage for occurring congested heart failure also increases by 1 times.To cTnI observational study
Similar result is obtained.In unstable coronary artery disease patient, the patient death rate that cTnI is more than 0.1 μ g/L is small compared with cTnI
It is big more than 3 times in the death rate of 0.1 μ g/L patient.Therefore, any acute coronary illness patient measures cTn simultaneously and increased,
It should be regarded as high risk.
【The content of the invention】
To solve the above problems, high it is an object of the invention to provide a kind of sensitivity for analysis, the detection range of linearity is big, makes
With the quick Troponin I detection reagent based on the even phase immunization of collaurum of convenient and detection.
The second object of the present invention is to provide a kind of preparation method of Troponin I detection reagent.
To realize above-mentioned first purpose, the technical scheme that the present invention takes is：A kind of Troponin I detection reagent, its by
Reagent one and reagent two, which coordinate, to be formed, wherein, the volume ratio of the reagent one and reagent two is 5: 1；The reagent I includes：
20~50mmol/L buffer solution, 2%w/v coagulant, 2%w/v BSA, 0.1%~0.5%w/v preservative；It is described
Reagent two includes：0.1%~0.5%w/v is marked with the colloid gold particle of anti-human cTnI antibody, 2%~5%w/v stabilization
Agent, 20~50mmol/L buffer solution, 0.1%~0.5%w/v preservative；It is described to mark the colloid for having cTnI antibody
A diameter of 60nm~90nm of gold grain.
The present invention Troponin I detection reagent be further：Buffer solution in the reagent one and reagent two is specially
Tris-HCl buffer solutions, HEPES buffer solution, borate buffer solution or glycine buffer containing 0.5%~5.0%w/vBSA
In one or more.
The present invention Troponin I detection reagent be further：The reagent one, the pH value of reagent two are 7.0~9.0.
The present invention Troponin I detection reagent be further：The BSA concentration is 1%w/v, reagent one, reagent two
PH value is 8.0 ± 0.2.
The present invention Troponin I detection reagent be further：Coagulant in the reagent one for PEG6000 or
It is at least any of in Brij-35.
The present invention Troponin I detection reagent be further：The a diameter of 75nm of colloid gold particle in the reagent two
~85nm, granule content is 0.08~0.8mg/mL.
The present invention Troponin I detection reagent be further：The colloid gold particle content is 0.4mg/mL.
The present invention Troponin I detection reagent be also：The mark has cTnI antibody to be with the immune work of difference
The mixture of the multiple clone strain monoclonal antibodies in property site.
To realize above-mentioned second purpose, the technical scheme that the present invention takes is：A kind of preparation of Troponin I detection reagent
Method, it comprises the following steps：
1) coagulant, BSA and preservative, are added to buffer solution, reagent one is made；
2) gold chloride and trisodium citrate, are boiled into obtained colloidal gold solution according to the heating of mass ratio 1: 1；By cTnI antibody
It is added in colloidal gold solution, and adds stabilizer, buffer solution, preservative, reagent two is made；
3), reagent one and reagent two are used cooperatively according to 5: 1 volume ratio, Troponin I detection reagent is produced.
The preparation method of Troponin I detection reagent of the present invention is further：The step 2) in, prepare gold chloride with
During citric acid three sodium solution, 0.2 μm of membrane filtration is used.
Compared with prior art, the present invention has the advantages that：
1. the present invention utilizes the even phase particle characteristics of collaurum, by specific cTnI antibody labelings in colloid gold particle surface,
When there is cTnI in detecting system or detection environment, the antibody on colloid gold particle surface i.e. by corresponding antigen capture,
And antigen-antibody complex is formed, the polymerization or accumulation of local colloid gold particle are in turn resulted in, makes the even phase reagent printing opacity of collaurum
Spectrum is moved from red to blue color spectrum, absorbance at reduction and 660nm of this mobile key reaction for absorbance at 540nm
Rise, so as to reach the purpose of cTnI antigens in a quantitative detection corpse or other object for laboratory examination and chemical testing, exempt from while avoiding the enhancing of similar detection project latex
Epidemic disease turbidimetry reagent produces the latex microsphere cross-linking agent absorption difficult cleaning of shortcoming of cuvette after the reaction.
2. the present invention can be used for cTnI contents in detection serum or blood plasma, it is adaptable to the light splitting used during clinical detection
The instruments such as photometer, semi-automatic biochemical analyzer and automatic clinical chemistry analyzer.
3. present invention detection cTnI sensitivity can reach 0.1ng/mL, the range of linearity is detected up to 0.1~30ng/mL,
With high analyte sensitivity, the features such as high specific.
【Brief description of the drawings】
Fig. 1 is the colloid gold label thing calibration graph for variable grain size.
Fig. 2 is that reagent of the present invention is linearly compared figure with the analysis of commercially available related reagent.
【Embodiment】
Firstly, it is necessary to which the w/v being related in explanation, the present invention is designated as " w/v ", unit is " g/mL ".
The present invention is a kind of Troponin I detection reagent, and it is formed with reagent one and reagent two, wherein, the examination
The volume ratio of agent one and reagent two is 5: 1.
The reagent one includes：20~50mmol/L buffer solution, 2%w/v coagulant, 2%w/v BSA,
0.1%~0.5%w/v preservative.Wherein, the buffer solution of the reagent one is specially to contain 0.5%~5.0%w/vBSA's
One or more in Tris-HCl buffer solutions, HEPES buffer solution, borate buffer solution or glycine buffer.The reagent
One pH value is 7.0~9.0, and pH value preferably is 8.0 ± 0.2.The BSA concentration is 2%w/v.The coagulant is
It is at least any of in PEG6000 or Brij-35.
The reagent two includes：0.1%~0.5%w/v be marked with the colloid gold particle of anti-human cTnI antibody, 2%~
5%w/v stabilizer, 20~50mmol/L buffer solution, 0.1%~0.5%w/v preservative.Wherein, the mark has
A diameter of 60nm~90nm of the colloid gold particle of people's cTnI antibody.The buffer solution of the reagent two be specially containing 0.5%~
One kind in 5.0%w/vBSA Tris-HCl buffer solutions, HEPES buffer solution, borate buffer solution or glycine buffer or
It is several.The pH value of the reagent two is 7.0~9.0, and pH value preferably is 8.0 ± 0.2.Preferential colloid gold particle is a diameter of
75nm~85nm, particle is unsuitable too small, otherwise reaction speed can be caused slack-off；Particle is also unsuitable excessive, and label can be caused to gather
Collection sedimentation further influences measurement result.The colloid gold particle granule content is 0.08~0.8mg/mL, and preferential particle contains
Measure as 0.4mg/mL.The mark has cTnI antibody to resist or with the multiple clone strain monoclonal antibodies of different immunocompetence sites for more
Mixture.
The preparation method of the Troponin I detection reagent is as follows：
1) coagulant, BSA and preservative, are added to buffer solution, reagent one is made；
2) gold chloride and trisodium citrate, are boiled into obtained colloidal gold solution according to the heating of mass ratio 1: 1；By cTnI antibody
It is added in colloidal gold solution, and adds stabilizer, buffer solution, preservative, reagent two is made；Wherein, configuration gold chloride and lemon
During lemon three sodium solution of acid, 0.2 μm of membrane filtration is used；
3), reagent one and reagent two are used cooperatively according to 5: 1 volume ratio, Troponin I detection reagent is produced.
It is below the specific embodiment of Troponin I detection reagent of the invention.
Embodiment one
The preparation of colloid gold particle (following all operations need to be completed in the dustless space of high-cleanness, high)
1) preparation：All glass containers used first are washed with abluent and rinsed well with flowing water, then use silicon
Change reagent soaked overnight, it is standby pair so the inner surface of gold vessel ware processed does silicidation, then with distilled water flushing totally；
2) 1% (w/v) gold chloride and 1% (w/v) sodium citrate solution are prepared, 0.2 μm of membrane filtration is used；
3) 1 piece of magnetic stir bar is put into the 1000ml round-bottomed flasks cleaned up, 1000ml ultra-pure waters are added；
4) certain volume concentration is added for 1% (w/v) gold chloride, 600rpm or so high-speed stirreds, and it is heated to solution boiling
Rise, then rapid is that 1% sodium citrate solution is added rapidly in flask by certain volume concentration, keeps heating and is stirred vigorously
10min, solution colour first turns black, and then gradually becomes aubergine；
5) cool down：Close heater switch and continue to stir 10min, then stop stirring and be cooled to room temperature, it is extensive with ultra-pure water
Original volume is arrived again；
6) colloid gold particle of different-grain diameter can be controlled according to the ratio for adding gold chloride and sodium citrate, finally with
Electronics particle size analyzer detects 10mL colloidal gold solutions, shows that result is used as the particle size results of gold grain using particle size analyzer.
Embodiment two
The preparation of reagent one
The formula of reagent one is as follows：Tris-Hcl buffer solutions, 0.9%NaCl (w/v), 2%BSA (w/v), 0.1%NaN3
(w/v), 0.05%Tween20 (w/v), can be adjusted according to the concentration of Tris quality control buffer solution according to HCl usage amount
The pH value of whole buffer solution.
Embodiment three
The preparation of label
1) the 1 milliliter of collaurum prepared liquid is taken to be added in 1.5 milliliters of centrifuge tube；
2) colloidal gold solution in centrifuge tube is adjusted between pH8.0~10.0 with 10% solution of potassium carbonate；
3) by a certain amount of antibody into above-mentioned centrifuge tube, shake 30 minutes；
4) 100uL 10% NaCl solution is added into above-mentioned solution, is mixed, observes whether each pipe has face after standing 2h
Color change or Precipitation, when without color change and without Precipitation show add antibody molecule be less than albumen greatest requirements
Amount；
5) 500ml colloidal gold solutions are added in triangular flask, adjusts the pH value of solution with 10% potassium carbonate while stirring
Adjustment process is detected to 8.0~10.0, and with accurate pH test paper；
6) the antibody consumption of determination is added in above-mentioned solution by described method, continues to stir 30 minutes, ultrafiltration is received
Collect label.
Example IV
The preparation of reagent two
The large biological molecule dissolved in the buffer solution of reagent two and chemical substance are Tris-Hcl buffer solutions, 0.9% (w/v)
NaCl, 2% (w/v) BSA, 0.1% (w/v) NaN3,0.05% (w/v) Tween20,5% (w/v) sucrose or trehalose, 2%
(w/v) glycine, 2% (w/v) PEG6000, this part buffer is consistent with reagent one, repeated no more again.
The reagent cushioning liquid prepared is collected to obtained label with ultrafiltration to mix, final control label gold
Granule density is 0.4mg/mL.
Embodiment five
The preparation of calibration object
Commercially available people source cTnI albumen sterling is dissolved or diluted with 2% (w/v) BSA solution, 1200ng/mL calibrations are made
Product, the concentration gradient required for is diluted with 0.9% (w/v) NaCl when using.
Embodiment six
The selection of variable grain degree colloid gold particle
Mark is completed according to the colloid gold particle for preparing different-grain diameter of colloid gold particle in above example reagent is made
Two, select same buffer concentration and pH to be tested.
Experimental method：
1st, calibration object, reagent one and reagent two are mixed successively according to volume ratio for 3: 250: 50, fully reacted at 37 DEG C.
2nd, absorbance at 540 ± 10nm is recorded on 7180 automatic clinical chemistry analyzers.
3rd, compare variable grain size colloid gold label thing calibration curve and finally determine optimal granular size.
According to calibration curve judge selection 75nm~85nm scopes gold grain it is more suitable, bulky grain sensitivity for analysis compared with
The good HOOK effects that are still also easy to produce are unfavorable for detection, and little particle is substantially not so good as 75nm~85nm in the analysis ability of Spring layer
Particle.
Accompanying drawing one is that reagent of the present invention is relatively schemed with commercially available relevant item reagent analysis linear ratio.
Test method：By taking a clinical high level sample as an example, by its doubling dilution such as Gradient, respectively with contrast agents and this
Invention reagent is detected, and is fitted it linearly, as a result such as accompanying drawing two：
The linear data result of table 1
The linear y=0.9637x-0.623 r=0.9900 of reagent of the present invention
The linear y=0.7416x-5.8988 r=0.8362 of contrast method reagent
The linear result of reagent of the present invention is can be seen that from related data statistics be substantially better than contrast agents linearly test knot
Really.
Embodiment seven
(4) interference test
A certain amount of bilirubin, bovine hemoglobin, fat emulsion and rheumatoid factor are each added in normal human serum,
Sample initial value is first determined, interfering material is added in the sample according to following table interfering material concentration afterwards, only adds a kind of every time
Chaff interference, makes it up to the concentration shown in following table, is measured while being calculated according to measurement result and Volume Changes inverse extension rate
Result afterwards, the results are shown in Table 2.
The anti-interference testing result of table 2
Shown according to data above, reagent of the present invention meets clinical detection requirement, explanation in this chaff interference existence range
Reagent of the present invention has good interference free performance.
Embodiment above is only the preferred embodiment of this creation, all in this wound not to limit this creation
Any modification, equivalent substitution and improvements for being done etc. within the spirit and principle of work, should be included in this creation protection domain it
It is interior.
Claims (1)
1. a kind of preparation method of Troponin I detection reagent, it is characterised in that：The Troponin I detection reagent is by reagent
One and reagent two coordinate and form, wherein, the volume ratio of the reagent one and reagent two is 5: 1；The reagent I includes：20~
50mmol/L buffer solution, 2%w/v coagulant, 2%w/v BSA, 0.1%~0.5%w/v preservative；The reagent
Two include：0.1%~0.5%w/v is marked with the colloid gold particle of anti-human cTnI antibody, 2%~5%w/v stabilizer, 20
~50mmol/L buffer solution, 0.1%~0.5%w/v preservative；It is described to mark the colloid gold particle for having cTnI antibody
A diameter of 75nm~85nm, granule content is 0.4mg/mL；The mark has cTnI antibody to be with the immune work of difference
The mixture of the multiple clone strain monoclonal antibodies in property site；
The preparation method comprises the following steps：
1) preparation：All glass containers used first are washed with abluent and rinsed well with flowing water, are then tried with silication
Agent soaked overnight, pair so the inner surface of gold vessel ware processed does silicidation, then it is clean with distilled water flushing, it is standby；
2) 1% (w/v) gold chloride and 1% (w/v) sodium citrate solution are prepared, 0.2 μm of membrane filtration is used；
3) 1 piece of magnetic stir bar is put into the 1000ml round-bottomed flasks cleaned up, 1000ml ultra-pure waters are added；
4) certain volume concentration is added for 1% (w/v) gold chloride, 600rpm high-speed stirreds, and it is heated to solution boiling, Ran Houxun
Certain volume concentration is that 1% sodium citrate solution is added rapidly in flask by speed, is kept heating and is stirred vigorously 10min, molten
Liquid color first turns black, and then gradually becomes aubergine；
5) cool down：Close heater switch and continue to stir 10min, then stop stirring and be cooled to room temperature, returned to ultra-pure water
Original volume；
6) colloid gold particle of different-grain diameter can be controlled according to the ratio for adding gold chloride and sodium citrate, finally with electronics
Particle size analyzer detects 10mL colloidal gold solutions, shows that result is used as the particle size results of gold grain using particle size analyzer.
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| CN104698167A (en) * | 2015-03-20 | 2015-06-10 | 黑龙江大学 | Enterobacter sakazakii detection reagent and preparation method thereof |
| CN106885911A (en) * | 2015-12-16 | 2017-06-23 | 山东博科生物产业有限公司 | A kind of stabilization, the cardiac muscle troponin I reagent of strong antijamming capability and detection method |
| CN105628930A (en) * | 2015-12-22 | 2016-06-01 | 山东博科生物产业有限公司 | Troponin I detection reagent with high sensitivity through latex enhanced turbidimetric Immunoassay |
| CN105785050A (en) * | 2016-04-18 | 2016-07-20 | 石河子大学 | Comprehensive buffer solution for detecting brucellosis by means of colloidal immuno-gold immuno-filtration assay |
| CN106093418A (en) * | 2016-05-26 | 2016-11-09 | 安徽伊普诺康生物技术股份有限公司 | A kind of test kit measuring Troponin I and preparation method thereof |
| CN107966567B (en) * | 2017-11-21 | 2018-12-18 | 浙江夸克生物科技有限公司 | A kind of haptoglobin assay kit |
| CN109212199A (en) * | 2018-08-11 | 2019-01-15 | 金华市强盛生物科技有限公司 | A kind of Troponin I detection kit |
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| US6284194B1 (en) * | 1998-03-11 | 2001-09-04 | Albert E. Chu | Analytical assay device and methods using surfactant treated membranes to increase assay sensitivity |
| US8003407B2 (en) * | 2004-07-29 | 2011-08-23 | Relia Diagnostic Systems, Llc | Lateral flow system and assay |
| JP2007322310A (en) * | 2006-06-02 | 2007-12-13 | Nippon Kayaku Co Ltd | Method for detecting or measuring substance to be analyzed in specimen |
| CN101699287B (en) * | 2009-09-08 | 2013-04-24 | 北京利德曼生化股份有限公司 | Homogeneous phase sol particle type cystatin C measuring kit and preparation method thereof |
| EA013943B1 (en) * | 2010-01-11 | 2010-08-30 | Общество С Ограниченной Ответственностью Научно-Производственное Объединение "Биотест" (Ооо Нпо "Биотест") | The assay kit “cardio test” for immunochromatographic detection of heart fatty acid binding protein and troponin i in whole blood samples for express diagnosis of myocardial infarction |
| CN102053153A (en) * | 2010-11-25 | 2011-05-11 | 西安微通生物技术有限公司 | Dot immuno gold directed infiltration detection kit and application thereof |
| CN102207507A (en) * | 2011-03-24 | 2011-10-05 | 武汉璟泓万方堂医药科技有限公司 | Semi-quantitative detecting test paper of cardiac troponin and preparation method thereof |
| CN102539787A (en) * | 2012-01-10 | 2012-07-04 | 上海一滴准生物科技有限公司 | Rapid troponin I serum detection reagent kit (colloidal golden method) |
| CN102749454B (en) * | 2012-06-11 | 2015-01-07 | 宁波鼎鑫生物科技有限公司 | Full-scale C-reactive protein (CRP) colloidal gold immunoturbidimetric assay kit |
| CN103837677A (en) * | 2013-12-25 | 2014-06-04 | 北京乐普医疗科技有限责任公司 | Test strip for detecting heart markers, preparation method and detection method |
| CN104391124B (en) * | 2014-11-17 | 2016-03-30 | 中国人民解放军第三0五医院 | A kind of myoglobins detects the preparation method of reagent |
| CN104330576B (en) * | 2014-11-17 | 2017-10-03 | 中国人民解放军第三0五医院 | A kind of cardic fatty acid binding protein detection reagent and preparation method thereof |
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