CN103389385A - Latex-coated troponin detection kit - Google Patents
Latex-coated troponin detection kit Download PDFInfo
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- CN103389385A CN103389385A CN2013103447737A CN201310344773A CN103389385A CN 103389385 A CN103389385 A CN 103389385A CN 2013103447737 A CN2013103447737 A CN 2013103447737A CN 201310344773 A CN201310344773 A CN 201310344773A CN 103389385 A CN103389385 A CN 103389385A
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Abstract
The invention provides a latex-coated troponin detection kit and relates to the field of in vitro diagnostic medical immunology. The latex-coated troponin detection kit is composed of a reagent R1, a reagent R2 and a calibrator, wherein the reagent R1 is a Tris buffer system composed of 30-80 m mol/l of Tris buffer solution, 70-140 m mol/l of polyethylene glycol 6000-10000 and 15-30 m mol/l of ethylenediamine tetraacetic acid disodium; the reagent R2 is composed of sensitized particles of cTnI antibody-coated polystyrene latex particles and 40-100 mmol/l of Tris buffer solution; the calibrator is 0-130 U/ml bovine serum matrix, the latex-coated troponin detection kit also includes 0.2-2.2% of antiseptic and 1-10% of sterilizer, and the percentage is percentage by volume of the bovine serum matrix. The latex-coated troponin detection kit is good in repeatability, stable in reagents, simple and convenient to operate, high in sensitivity, good in specificity, rapid to measure and accurate and reliable in measurement result.
Description
Technical field
The present invention relates to medical immunology in-vitro diagnosis field, be specifically related to the coated troponin detection kit of a kind of latex.
Background technology
Acute myocardial infarction (AMI) is one of higher disease of M ﹠ M.In the zymetology index of many diagnosis AMI, the creatine kinase isozyme (CK-MB) that contains M and B subunit once once had been considered to " goldstandard " diagnosed and widespread use for many years., along with the research that deepens continuously to myocardial injury markers, find gradually some sensitivitys, specificity mark preferably.Wherein, cardiac troponin (cTn) just progressively replaces CK-MB with its excellent sensitivity and specificity, becomes new definite mark of judgement myocardial damage necrosis.
CTn detects clinical diagnosis, the harmfulness be mainly used in treating myocardial ischemia damage such as acute coronary syndrome (comprising recessive angina pectoris and unstable angina and AMI) etc. and estimates and the prognosis judgement, can be used in addition MI after the thromboembolism treatment effect judge; Left heart failure, congested cardiac insufficiency that the estimation for the treatment of myocardial ischemia damage area, clinical diagnosis myocarditis, myocardium wound (openheart surgery), peri-operation period cardiac complication, Severe sepsis or septicopyemia cause, and the clinical observation on the therapeutic effect of some medicine.CTn comprises cardiac muscle troponin I (cTnI) and two kinds of hypotypes of serum cardiac troponin T (cTnT).A large amount of clinical research data confirmations, cTnI or the cTnT clinical value when detecting myocardial damage is identical.
It is occur in recent years a kind of comparatively stable, body fluid albumen homogeneous phase immunoturbidimetry detection method accurately that latex particle strengthens turbidimetry.The PETIA method is divided into two kinds substantially.A kind of is the scattering turbidimetry detection method; Another kind is the turbid detection method of transmittance.The ultimate principle of these two kinds of methods is closely similar, it is all the surface-crosslinked monoclonal antibody at the polymer latex microballoon,, can flock together rapidly at short notice after antigen is combined when the crosslinked microballoon that antibody is arranged, change astigmatic performance or the light transmission of reactant liquor.And reactant liquor astigmatism performance or the change of light transmission (being absorbance) and the concentration of tested antigen have stronger correlativity, can reflect within the specific limits the concentration of tested antigen.The PETIA detection method is to carry out the mensuration of antigen, antibody response and result in homogeneous reaction system.After antigen, antibody response, directly the absorbance of assaying reaction liquid, save the ELISA method and repeatedly hatched and washed the loaded down with trivial details operation stepss such as plate, and a few minutes just can obtain result, and are time saving and energy saving.In addition, the interference of many manual operation factors and the extraneous factors such as reagent, environment has also correspondingly been avoided in the simplification of nano immune turbidimetry operation steps, and stability and repeatability are all better, can reflect more truly the content of measured matter.Although the sensitivity of immunoturbidimetry is more weaker than ELISA method, the lower limit of foot many marker proteins in human normal plasma being detected, can meet the clinical detection requirement fully.
The common method of measuring at present cTnI has: euzymelinked immunosorbent assay (ELISA) absorption method, chemoluminescence method, enzyme connection fluorometry, colloidal gold immunity chromatography and immunoturbidimetry.At present method relatively more commonly used is colloidal gold immunity chromatography, but the method susceptibility is bad, and repeatability is bad, especially can only be as qualitative examination, for the observation of curative effect of clinical patient, certain limitation is arranged.
Summary of the invention
The purpose of this invention is to provide the coated troponin detection kit of a kind of latex, its good reproducibility, stable reagent, easy and simple to handle, highly sensitive, specificity is good, measure fast, measurement result accurately and reliably.
In order to solve the existing problem of background technology, the present invention is by the following technical solutions: it is comprised of reagent R1, reagent R2 and calibration object three parts, reagent R1 is the Tris buffer system, and it consists of Tris damping fluid 30-80mmol/l, Macrogol 6000-10000 70-140mmol/l and disodium ethylene diamine tetraacetate 15-30mmol/l; Reagent R2 is coated polystyrene latex particle sensitization particle, Tris damping fluid 40-100mmol/l of cTnI antibody; Calibration object is 0-130U/ml cow's serum matrix, also comprises antiseptic 0.2-2.2%, stabilizing agent 1-10%, and above-mentioned number percent is the number percent of cow's serum matrix volumetric usage.
Polystyrene latex particle in described reagent R2 is that the latex particle diameter is between 150-190nm.
In the coated process of described reagent R2 antibody, cTnI antibody and polystyrene latex coating quality are than being 1/10-5/10.
In the coated process of described reagent R2 antibody, the confining liquid component comprises the glycocoll of 0.5%-1.5% skimmed milk power, 20-60mm.
In the coated process of described reagent R2 antibody, latex dilution component comprises Tris damping fluid 30-80mmol/l, 0.5%-1.5% skimmed milk power, 0.9%NaN3.
Described calibration object is five calibration objects that cardiac troponin cTnI concentration is respectively 1.56,3.13,6.25,12.5,25.0ng/ml.
Described cow's serum matrix, by without HBV, HIV, HAV, HCV, the TP infection sources, is added glycerine, sucrose, BSA, sweet mellow wine, sorbierite simultaneously as protective agent, and Sodium azide is as antiseptic.
The present invention has following beneficial effect: its good reproducibility, stable reagent, easy and simple to handle, highly sensitive, specificity is good, measure fast, measurement result accurately and reliably.
Description of drawings:
Fig. 1 is kit calibration curve figure in the present invention;
Fig. 2 is for adopting reagent of the present invention and external certain renowned company's reagent serum correlativity comparison chart;
Fig. 3 is table 1: implement 1 kit response parameter table middle of the present invention in the present invention;
Fig. 4 is kit lowest detectable limit experimental data table of the present invention in table 2: embodiment 3;
Fig. 5 is the lowest detectable limit experimental data table of external certain renowned company's kit in table 3: embodiment 3;
Fig. 6 is the sensitivity experiment tables of data of kit of the present invention in table 4: embodiment 4;
Fig. 7 is the sensitivity experiment tables of data of external certain renowned company's kit in table 5: embodiment 4.
Embodiment:
This embodiment is taked following technical scheme: it is comprised of reagent R1, reagent R2 and calibration object three parts, reagent R1 is the Tris buffer system, and it consists of Tris damping fluid 30-80mmol/l, Macrogol 6000-10000 70-140mmol/l and disodium ethylene diamine tetraacetate 15-30mmol/l; Reagent R2 is coated polystyrene latex particle sensitization particle, Tris damping fluid 40-100mmol/l of cTnI antibody; Calibration object is 0-130U/ml cow's serum matrix, also comprises antiseptic 0.2-2.2%, stabilizing agent 1-10%, and above-mentioned number percent is the number percent of cow's serum matrix volumetric usage.
Polystyrene latex particle in described reagent R2, be characterized as the latex particle diameter between 150-190nm.
In the coated process of described reagent R2 antibody, cTnI antibody and polystyrene latex coating quality are than being 1/10-5/10.
In the coated process of described reagent R2 antibody, the confining liquid component comprises the glycocoll of 0.5%-1.5% skimmed milk power, 20-60mm.
In the coated process of described reagent R2 antibody, latex dilution component comprises Tris damping fluid 30-80mmol/l, 0.5%-1.5% skimmed milk power, 0.9%NaN3.
Described calibration object is five calibration objects that cardiac troponin cTnI concentration is respectively 1.56,3.13,6.25,12.5,25.0ng/ml.
Described cow's serum matrix, by without HBV, HIV, HAV, HCV, the TP infection sources, is added glycerine, sucrose, BSA, sweet mellow wine, sorbierite simultaneously as protective agent, and Sodium azide is as antiseptic.
This embodiment has following beneficial effect: its good reproducibility, stable reagent, easy and simple to handle, highly sensitive, specificity is good, measure fast, measurement result accurately and reliably.
Embodiment 1:
The mensuration kit is composed as follows: the required primary raw material of detection kit of the present invention and standard is as follows:
1 cTnI antibody, the outsourcing Abcam cTnI of company antigen, the preparation method makes according to conventional antibody, this antibody only in people cTnI reaction with other antigens without immunological cross-reaction, more than tiring and reaching 1:16.
2 polystyrene latexs, buy the company from German FIT; The latex particle particle diameter between 150-190nm, is 500nm testing the detection predominant wavelength of corresponding reagent.
3 recombinant human cTnI(are available from Abcam company), need standard items and quality-control product for the preparation of this reagent place.
Reagent is formulated as follows:
Reagent R1:
Tris damping fluid 70mmol/l
Macrogol 6000 120mmol/l
Disodium ethylene diamine tetraacetate 10mmol/l
NaN3 0.9%
Various compositions add under can room temperature successively, perhaps add simultaneously, or respectively separately packing and with detect before in instant preparation.
Reagent R2:
The polystyrene latex grain of 190nm and cTnI antibody mix (damping fluid adopts the Tris damping fluid) with the mass ratio of 100:30, both are mixed rear 37 ℃ of absorption 8 hours, the antibody that does not connect is removed in dialysis afterwards, add confining liquid (glycocoll of 0.1% skimmed milk power, 0.2mm), sealed 2 hours.The centrifugal supernatant that goes, (0.05% skimmed milk power, 0.9%NaN3) to 0.18% for Tris damping fluid 55mmol/l, disodium ethylene diamine tetraacetate 10.5mmol/l with the latex dilution.
The preparation of cow's serum matrix calibration object:
With treated cow's serum, add BAS 0.1%, NaN3 0.9% obtains the calibration object dilution.Be dissolved in the solution of the similar human serum matrix of preparation with restructuring cTnI, prepare the standard items (1.56ng/ml, 3.13ng/ml, 6.25 ng/ml, 12.50 ng/ml, 25.0 ng/ml) of variable concentrations.
The cTnI detection kit that the present embodiment is described, be applicable to various types of full automatic biochemical apparatus, and take Hitachi's 7170 full automatic biochemical apparatus as example, it operates as table 1.Analytical approach: Two point end assay, i.e. reagent R1; The consumption of R2 is respectively 150ul and 50ul, sample size 20ul; 150ul reagent R1 adds the 20ul sample to add 50ulR2 after 37 ℃ of 3min, postpones 100 seconds beginning read points, approximately 120 seconds reading duration; Detect wavelength predominant wavelength 500nm respectively.
Embodiment 2: the correlation test that detects reagent
1 correlation test
Use the cTnI latex intensified type reagent of this law invention reagent (specifically filling a prescription with embodiment 1) and contrast agents A company, adopt automatic 7170 automatic clinical chemistry analyzers to measure simultaneously by each autoregressive parameter 50 parts of human serums (comprising normal and monstrosity), measured value is carried out correlation analysis.According to above-mentioned " the cTnI assay method " in parameter measure measurement result and see Fig. 2, X, Y-axis is measured value (the content ng/ml of cTnI),
Result by Fig. 2 finds out, the relevant of two kinds of reagent is R2=0.9985, and regression equation is y=0.9678x+0.0104.It is good that result shows that this reagent and import reagent are measured patients serum's correlativity, has good specificity and accuracy.
In addition, above experiment is that 7170 full automatic biochemical apparatus that adopt Hitachi, Ltd to make carry out, but reagent of the present invention is not limited to above-mentioned instrument, also is applicable to other full-automatic or semi-automatic biochemical analyzers.
Embodiment 3: the lowest detectable limit test
This experiment purpose is to detect the minimum check-up inducing degree of reagent when the test clinical sample.
Adopt experimental example 1 reagent, contrast agents, standard items, blank solution (being generally normal saline solution and Purified Water), normal human serum sample, low value sample (sample of numerical value in reagent range of linearity lower limit ± 1/3).
Machine: Hitachi's 7170 automatic biochemistry analyzers.
Operation steps: use normal saline solution or deionized water dissolving low value sample, then 50% be diluted to 5 points, and zero point each test sample 5 times together, calculating mean value, try to achieve SD numerical value.
Result is resolved:, according to detecting data, calculates SD numerical value and CV numerical value, calculates respectively 1SD, and 2SD, from minimum, the numerical value of its mean value-2SD is exactly the minimum check-up inducing degree of reagent more than zero point mean value+2SD.
Table 2,3 results show, when reagent of the present invention is measured dilution 1/16,1/8,1/4,1/2 serum, measure the numerical value of mean value-2SD all greater than mean value+2SD at zero point, show that reagent lowest detectable limit of the present invention can reach 0.3ng/ml at least.And contrast A company reagent is measured dilution 1/16,1/8,1/4,1/2 serum, and relatively serum mean value-2SD with zero point, mean value+2SD was big or small.Find out in table that our company's reagent lowest detection is limited to 0.25 ng/ml left and right, A company reagent lowest detection is limited to the 0.5ng/ml left and right.
Embodiment 4: sensitivity experiment
This experiment purpose is to detect the absorbance changing value of reagent when test physiological saline and certain density management serum.
Adopt experimental example 1 reagent, contrast agents, standard items, blank solution, 0.9% normal saline solution, the absorbance changing value during the management serum of concentration.
Machine: Hitachi's 7170 automatic biochemistry analyzers.
Operation steps: use normal saline solution, low value sample, each test sample 5 times, calculate absorbance.
Table 4,5 shows, when reagent of the present invention is measured physiological saline, absorbance is changed to 0.0008(1/10000A), when theoretical concentration is the 1.80ng/ml serum sample, absorbance is changed to 112.6(1/10000A), and A company reagent while measuring physiological saline absorbance be changed to 0.0004(10000A), when theoretical concentration is the 1.80ng/ml serum sample, absorbance is changed to 90.2(1/10000A), show that reagent sensitivity of the present invention will be significantly higher than A company reagent.
Claims (7)
1. the coated troponin detection kit of a latex, it is characterized in that it is comprised of reagent R1, reagent R2 and calibration object three parts, reagent R1 is the Tris buffer system, and it consists of Tris damping fluid 30-80mmol/l, Macrogol 6000-10000 70-140mmol/l and disodium ethylene diamine tetraacetate 15-30mmol/l; Reagent R2 is coated polystyrene latex particle sensitization particle, Tris damping fluid 40-100mmol/l of cTnI antibody; Calibration object is 0-130U/ml cow's serum matrix, also comprises antiseptic 0.2-2.2%, stabilizing agent 1-10%, and above-mentioned number percent is the number percent of cow's serum matrix volumetric usage.
2. the troponin detection kit that latex is coated, is characterized in that the polystyrene latex particle in described reagent R2, is characterized as the latex particle diameter between 150-190nm.
3. the coated troponin detection kit of a latex, is characterized in that in the coated process of described reagent R2 antibody that cTnI antibody and polystyrene latex coating quality are than being 1/10-5/10.
4. the troponin detection kit that latex is coated, is characterized in that in the coated process of described reagent R2 antibody, the confining liquid component comprises the glycocoll of 0.5%-1.5% skimmed milk power, 20-60mm.
5. the troponin detection kit that latex is coated, is characterized in that in the coated process of described reagent R2 antibody, latex dilution component comprises Tris damping fluid 30-80mmol/l, 0.5%-1.5% skimmed milk power, 0.9%NaN3.
6. the troponin detection kit that latex is coated, is characterized in that described calibration object is five calibration objects that cardiac troponin cTnI concentration is respectively 1.56,3.13,6.25,12.5,25.0ng/ml.
7. the coated troponin detection kit of a latex; it is characterized in that described cow's serum matrix is by without HBV, HIV, HAV, HCV, the TP infection sources; add simultaneously glycerine, sucrose, BSA, sweet mellow wine, sorbierite as protective agent, Sodium azide is as antiseptic.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103940986A (en) * | 2014-03-24 | 2014-07-23 | 安徽省煦棠医疗科技有限公司 | Preparation of troponin I specific locus antibody and detection kit thereof |
| CN104142406A (en) * | 2014-08-22 | 2014-11-12 | 山东博科生物产业有限公司 | Stable troponin I detection kit |
| CN104181308A (en) * | 2014-08-22 | 2014-12-03 | 山东博科生物产业有限公司 | Stable troponin I detection reagent |
| 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 |
| CN108593929A (en) * | 2018-05-02 | 2018-09-28 | 广州市伊川生物科技有限公司 | A kind of Troponin I assay kit and its detection method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102636653A (en) * | 2012-04-19 | 2012-08-15 | 上海蓝怡科技有限公司 | Compounded latex particle-enveloped cystatin C detection kit |
| CN102841206A (en) * | 2011-06-22 | 2012-12-26 | 南京诺尔曼生物技术有限公司 | Troponin-T determination kit |
| CN103185798A (en) * | 2011-12-27 | 2013-07-03 | 苏州德沃生物技术有限公司 | Turbidimetric rapid detection kit for myocardial infarction nano-immunoenhancement and use method thereof |
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2013
- 2013-08-07 CN CN2013103447737A patent/CN103389385A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102841206A (en) * | 2011-06-22 | 2012-12-26 | 南京诺尔曼生物技术有限公司 | Troponin-T determination kit |
| CN103185798A (en) * | 2011-12-27 | 2013-07-03 | 苏州德沃生物技术有限公司 | Turbidimetric rapid detection kit for myocardial infarction nano-immunoenhancement and use method thereof |
| CN102636653A (en) * | 2012-04-19 | 2012-08-15 | 上海蓝怡科技有限公司 | Compounded latex particle-enveloped cystatin C detection kit |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103940986A (en) * | 2014-03-24 | 2014-07-23 | 安徽省煦棠医疗科技有限公司 | Preparation of troponin I specific locus antibody and detection kit thereof |
| CN104142406A (en) * | 2014-08-22 | 2014-11-12 | 山东博科生物产业有限公司 | Stable troponin I detection kit |
| CN104181308A (en) * | 2014-08-22 | 2014-12-03 | 山东博科生物产业有限公司 | Stable troponin I detection reagent |
| CN104142406B (en) * | 2014-08-22 | 2016-03-30 | 山东博科生物产业有限公司 | A kind of stable Troponin I detection kit |
| CN104181308B (en) * | 2014-08-22 | 2016-07-06 | 山东博科生物产业有限公司 | A kind of stable Troponin I detection reagent |
| 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 |
| CN108593929A (en) * | 2018-05-02 | 2018-09-28 | 广州市伊川生物科技有限公司 | A kind of Troponin I assay kit and its detection method |
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Application publication date: 20131113 |