CN105021822A - Quantitative determination method, reagent and kit for troponin I - Google Patents
Quantitative determination method, reagent and kit for troponin I Download PDFInfo
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- CN105021822A CN105021822A CN201510069194.5A CN201510069194A CN105021822A CN 105021822 A CN105021822 A CN 105021822A CN 201510069194 A CN201510069194 A CN 201510069194A CN 105021822 A CN105021822 A CN 105021822A
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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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
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
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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
- G01N2800/324—Coronary artery diseases, e.g. angina pectoris, myocardial infarction
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Abstract
The invention relates to a reagent for quantitative determination of the content of troponin I in human serum. The reagent is composed of a reagent I and a reagent II which are separately placed, wherein the reagent I contains glycine buffer and polyethylene glycol 6000, and the reagent II contains glycine buffer, sheep antibody against human troponin I, latex, Tween-20, a protein protective agent 2 and calf alhumin. The invention further provides a quantitative determination kit and method for troponin I. The kit and method only need dozens of ml of serum, do not need separation treatment like centrifugation or electrophoresis, are simple to operate, can meet requirements of automatic analysis and are applicable to timely and accurate detection of large-scale samples.
Description
Technical field
The application relates to the method for quantitatively determining of Troponin I in a kind of human serum, reagent and kit.
Background technology
Troponin I, be made up of TnT (cTnT), Troponin I (cTnI) and TnC (cTnC) three kinds of subunits, by regulating the activity of calcium ion to striated muscle filamentous actin ATP enzyme to come modulate actin and myosin interaction together with tropomyosin.When after myocardial damage, cardiac muscle troponin I compound is discharged in blood, and after 4-6 hour, start to raise in blood, the Troponin I of rising can keep 6-10 days for a long time in blood.Troponin I has height Cardiac-specific and sensitivity, so Troponin I has become current optimal myocardial infarction mark.
The method of mensuration troponin known at present has electrochemiluminescence immunodetection; Enzyme connection fluorometry.These methods have the shortcoming detecting the range of linearity little, complex operation, length consuming time, expensive, excess waste resource, are unsuitable for routine inspection, detect while especially extensive epidemic investigation or all entries of clinical sample in enormous quantities.
Summary of the invention
The object of the application is to provide a kind of method for quantitatively determining, reagent and the kit that can be applied to Troponin I in the human serum of clinical trial.
The application have employed following technical scheme:
The one side of the application discloses a kind of reagent of quantitatively determining human serum cTnT I content, and this reagent is made up of the reagent I placed respectively and reagent II, and wherein, described reagent I contains glycine buffer, Macrogol 6000; Described reagent II contains glycine buffer, goat-anti human troponin I antibody, latex, Tween-20, protein protective agent 2, bovine albumin.
Further, described protein protective agent 2 is glycerine.
Further, in described reagent I, glycine buffer liquid hold-up is 100-200mmol/L, and the concentration of Macrogol 6000 is 1.8-2.6%.
Further, in described reagent I, glycine buffer liquid hold-up is 120mmol/L, and the concentration of Macrogol 6000 is 2.4%.
Further; in described reagent II, glycine buffer liquid hold-up is 100-200mmol/L; goat-anti human troponin I antibody content is 0.1-0.5g/L; content of latex is 1-5g/L; Tween-20 content is 0.1%-0.5% (V/V), and protein protective agent 2 content is 0.5%-1.5%, bovine albumin content is 20-100g/L.
Further, in described reagent II, glycine buffer liquid hold-up is 120mmol/L, and goat-anti human troponin I antibody content is 0.2g/L; content of latex is 2g/L; Tween-20 content is 0.3% (V/V), and protein protective agent 2 content is 1%, and bovine albumin content is 50g/L.
The another aspect of the application provides a kind of kit of quantitatively determining human serum cTnT I content, and the reagent of above-mentioned quantitatively determining human serum cTnT I content is wherein housed, and this reagent is made up of the reagent I placed respectively and reagent II.
The another aspect of the application provides a kind of method of quantitatively determining human serum cTnT I content, and the method comprises and add described reagent I in blood serum sample, hatches 5 minutes for 37 DEG C, working sample absorbance A 1 under certain wavelength; Then in sample, add reagent II, after continuing to hatch 5 minutes, under above-mentioned wavelength, measure absorbance A 2, calculate △ A sample by following formula (1); Use the same method and measure the absorbance △ A standard of calibration solution; The Troponin I content of blood serum sample is calculated again by following formula (2):
△A＝A2-A1 (1)
Serum cTnT I content (ng/mL)=△ A sample/△ A standard × concentration of standard solution (2)
We's ratio juris is: adopt the calibration modes such as multiple spot non-linear LOGIT-LOG4P or LOGIT-LOG5P or SPLINE to carry out multiple spot calibration (blank without water), automatically after generating calibration curve by instrument according to calibration object concentration and corresponding △ A, calculation sample concentration.
In the application, specific antibody is incorporated into latex particle surface, sample mixes in damping fluid with latex particle, cTnl in sample is combined with the antibody on latex particle surface, adjacent latex particle is cross-linked to each other, near 500nm, measure solution turbidity increase, its degree increased is relevant to the cTnl content in sample.
Direct quantitative of the present invention measures the kit of Troponin I content in human serum sample, is to be loaded in kit package with different specifications by the mentioned reagent I placed respectively and reagent II.This kit has multiple different specification, can be applicable to the automated chemical analyser of the various domestic and international brand generally used in clinical labororatory at present respectively.
The kit that the present invention adopts and detection method only need tens HL serum, without the need to separating treatment such as centrifugal or electrophoresis, easy and simple to handle, can meet the full-automatic requirement analyzed, be applicable to the promptly and accurately detection of extensive sample.
Embodiment
Embodiment one
According to following compositions and the following reagent I of the present invention and reagent II of proportional arrangement:
Reagent I:
Glycine buffer 120mmol/L
Macrogol 6000 2.4%
Reagent II:
By 225 μ l reagent I and the mixing of 25 μ l blood serum samples in sample hose, 5 minutes are hatched at 37 DEG C, use Abbott Laboratories C16000 automatic clinical chemistry analyzer, at predominant wavelength 500nm, measure absorbance A 1, in sample, then add 70 μ l reagent II, mixing, at 37 DEG C, hatch 5 minutes, measure the absorbance A 2 under phase co-wavelength.△ A sample is calculated according to following formula (1).Use the same method and the absorbance of condition bioassay standard liquid, and calculate △ A standard.Then according to Troponin I content in the blood serum sample of formula (2) calculation sample:
△A＝A2-A1 (1)
Serum cTnT I content (ng/mL)=△ A sample/△ A standard × concentration of standard solution (2)
The application's calibration solution used is Bole's calibration object.
Embodiment two
According to following compositions and the following reagent I of the present invention and reagent II of proportional arrangement:
Reagent I:
Glycine buffer 100mmol/L
Macrogol 6000 1.8%
Reagent II:
By 225 μ l reagent I and the mixing of 25 μ l blood serum samples in sample hose, 5 minutes are hatched at 37 DEG C, use Hitachi 7180 type automatic clinical chemistry analyzer, at predominant wavelength 500nm, measure absorbance A 1, in sample, then add 70 μ l reagent II, mixing, at 37 DEG C, hatch 5 minutes, measure the absorbance A 2 under phase co-wavelength.△ A sample is calculated according to following formula (1).Use the same method and the absorbance of condition bioassay standard liquid, and calculate △ A standard.Then according to Troponin I content in the blood serum sample of formula (2) calculation sample:
△A＝A2-A1 (1)
Serum cTnT I content (ng/mL)=△ A sample/△ A standard × concentration of standard solution (2)
Embodiment three
According to following compositions and the following reagent I of the present invention and reagent II of proportional arrangement:
Reagent I:
Glycine buffer 200mmol/L
Macrogol 6000 2.6%
Reagent II:
By 225 μ l reagent I and the mixing of 25 μ l blood serum samples in sample hose, 5 minutes are hatched at 37 DEG C, use Olympus AU400 type automatic clinical chemistry analyzer, at predominant wavelength 500nm, measure absorbance A 1, in sample, then add 70 μ l reagent II, mixing, at 37 DEG C, hatch 5 minutes, measure the absorbance A 2 under phase co-wavelength.△ A sample is calculated according to following formula (1).Use the same method and the absorbance of condition bioassay standard liquid, and calculate △ A standard.Then according to Troponin I content in the blood serum sample of formula (2) calculation sample:
△A＝A2-A1 (1)
Serum cTnT I content (ng/mL)=△ A sample/△ A standard × concentration of standard solution (2)
Embodiment four
According to following compositions and the following reagent I of the present invention and reagent II of proportional arrangement:
Reagent I:
Glycine buffer 120mmol/L
Macrogol 6000 1.8%
Reagent II:
By 225 μ l reagent I and the mixing of 25 μ l blood serum samples in sample hose, 5 minutes are hatched at 37 DEG C, use gloomy imperial 8020 type automatic clinical chemistry analyzers, at predominant wavelength 500nm, measure absorbance A 1, in sample, then add 70 μ l reagent II, mixing, at 37 DEG C, hatch 5 minutes, measure the absorbance A 2 under phase co-wavelength.△ A sample is calculated according to following formula (1).Use the same method and the absorbance of condition bioassay standard liquid, and calculate △ A standard.Then according to Troponin I content in the blood serum sample of formula (2) calculation sample:
△A＝A2-A1 (1)
Serum cTnT I content (ng/mL)=△ A sample/△ A standard × concentration of standard solution (2)
Embodiment five
Use reagent listed in the present embodiment 1, according to the method described in embodiment 1 and condition, the serum cTnT I content to 100 routine blood serum samples measures, and every part of blood serum sample carries out contrastive mensuration with commercially available serum cTnT I assay kit (river, Taiyuan is to Bioisystech Co., Ltd) simultaneously.
1, SPSS quantitative statistical analysis
1.1 statistics describe
Table 1 descriptive statistics amount
Table 2 descriptive statistics amount
1.2 paired-samples T-test
Table 3 paired samples related coefficient
| N | Related coefficient | Sig. | |
| To 1 data & contrasting data of the present invention | 100 | .992 | .000 |
Table 4 paired samples is checked
Table 5 paired samples is checked
| t | df | Sig. (bilateral) | |
| To 1 data-contrasting data of the present invention | -1.728 | 99 | .087 |
Wherein, P value=0.087, > 0.05, shows that two groups of data are separate, is the result of two different kits
1.3 bivariate correlativitys
Table 6 correlativity
*. at the upper significant correlation of .01 level (bilateral).
R＝0.992
1.4 novel regretional analyses and loose some table
Table 7 model gathers
A. predictive variable: (constant), VAR00019.
B. dependent variable: data of the present invention.
Table 8 coefficient
a
A. dependent variable: data of the present invention
Equation: Y=0.994*X-0.002, r=0.992, X-axis: contrasting data, Y-axis: data of the present invention
2, the qualitative statistical study of clinical effectiveness
Clinical research Troponin I measures kit and contrast product measurement result is analyzed:
Cross tabulating is analyzed
Table 9 clinical definite and control group reagent
With contrast product kit for reference group:
Positive coincidence rate=[29/ (29+1)] × 100%=96.66%
Negative match-rate=[69/ (69+1)] × 100%=98.57%
Total consistance=[(69+29)/100] × 100%=98%
3, Performance Analysis
3.1 accuracy
Carry out three horizontal surveies with known Troponin I definite value serum to measure, get average, draw:
Table 10
3.2 repeated
Same batch of reagent carries out 10 replications to same sample, calculates sample measurement result mean value, draws batch interior imprecision: CV≤5.0%.
Table 10
3.3 the range of linearity
Choose the serum of Troponin I 24.9ng/ml, doubling dilution is carried out with physiological saline, get five gradient concentrations, each concentration replication is averaged for 3 times, calculate theoretical concentration and corresponding measured value average carries out linear regression analysis, calculate correlation coefficient r, straight-line regression statistical study is carried out to theoretical value and measured value, regression equation is Y=25.675X+0.316, and has statistical significance (p<0.0001).
Table 11
Show that this law is in 0.3-25.0ng/ml serum cTnI concentration range, measured value and theoretical concentration related coefficient are r:0.994.
4. discuss and conclusion
In above-mentioned 100 routine samples, Troponin I monstrosity 30 example, Troponin I normal specimen 70 example is by cross tabulating analysis, SPSS software statistics, data statistics result shows: accuracy of the present invention is high, reproducible, the range of linearity is wide, and meets in positive coincidence rate, negative match-rate, total consistance and correlativity etc. better with contrast product.Therefore, Troponin I of the present invention measures kit can meet clinical performance requirement, and validity is strong, and security is high, can meet the full-automatic requirement analyzed, be applicable to the promptly and accurately detection of extensive sample.
Above content is the further description done the application in conjunction with concrete embodiment, can not assert that the concrete enforcement of the application is confined to these explanations.For the application person of ordinary skill in the field, under the prerequisite not departing from the application's design, some simple deduction or replace can also be made, all should be considered as the protection domain belonging to the application.
Claims (8)
1. a reagent for quantitatively determining human serum cTnT I content, this reagent is made up of the reagent I placed respectively and reagent II, and wherein, described reagent I contains glycine buffer, Macrogol 6000; Described reagent II contains glycine buffer, goat-anti human troponin I antibody, latex, Tween-20, protein protective agent 2, bovine albumin.
2. reagent according to claim 1, is characterized in that, described protein protective agent 2 is glycerine.
3. the reagent according to any one of claim 1-2, is characterized in that, in described reagent I, glycine buffer liquid hold-up is 100-200mmol/L, and the concentration of Macrogol 6000 is 1.8-2.6%/L.
4. reagent according to claim 3, is characterized in that, in described reagent I, glycine buffer liquid hold-up is 120mmol/L, and the concentration of Macrogol 6000 is 2.4%/L.
5. the reagent according to any one of claim 1-2; it is characterized in that; in described reagent II, glycine buffer liquid hold-up is 100-200mmol/L; goat-anti human troponin I antibody content is 0.1-0.5g/L; content of latex is 1-5g/L; Tween-20 content is 0.1%-0.5%, and protein protective agent 2 content is 0.5%-1.5%, and bovine albumin content is 20-100g/L.
6. reagent according to claim 5; it is characterized in that; in described reagent II, glycine buffer liquid hold-up is 120mmol/L; goat-anti human troponin I antibody content is 0.2g/L; content of latex is 2g/L; Tween-20 content is 0.3%, and protein protective agent 2 content is 1%, and bovine albumin content is 50g/L.
7. the kit of a quantitatively determining human serum cTnT I content, it is characterized in that, the reagent of arbitrary described quantitatively determining human serum cTnT I content in claim 1 to 6 is wherein housed, and this reagent is made up of the reagent I placed respectively and reagent II.
8. a method for quantitatively determining human serum cTnT I content, the method comprises and add described reagent I in blood serum sample, hatches 5 minutes for 37 DEG C, working sample absorbance A 1 under certain wavelength; Then in sample, add reagent II, after continuing to hatch 5 minutes, under above-mentioned wavelength, measure absorbance A 2, calculate △ A sample by following formula (1); Use the same method and measure the absorbance △ A standard of calibration solution; The Troponin I content of blood serum sample is calculated again by following formula (2):
△A＝A2-A1 (1)
Serum cTnT I content (ng/mL)=△ A sample/△ A standard × concentration of standard solution (2).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201510069194.5A CN105021822A (en) | 2015-02-10 | 2015-02-10 | Quantitative determination method, reagent and kit for troponin I |
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| CN201510069194.5A CN105021822A (en) | 2015-02-10 | 2015-02-10 | Quantitative determination method, reagent and kit for troponin I |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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 |
-
2015
- 2015-02-10 CN CN201510069194.5A patent/CN105021822A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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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