CN105466920A - A rapid antithrombin III detecting kit based on interaction between thrombin and a chromogenic substrate and a detecting method thereof - Google Patents
A rapid antithrombin III detecting kit based on interaction between thrombin and a chromogenic substrate and a detecting method thereof Download PDFInfo
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- CN105466920A CN105466920A CN201510808327.6A CN201510808327A CN105466920A CN 105466920 A CN105466920 A CN 105466920A CN 201510808327 A CN201510808327 A CN 201510808327A CN 105466920 A CN105466920 A CN 105466920A
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- antithrombin iii
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
Abstract
The invention belongs to the field of biological medicine in-vitro diagnosis, and particularly relates to a rapid antithrombin III detecting kit based on interaction between thrombin and a chromogenic substrate and a detecting method thereof. The kit comprises R1 reagents, an R2 reagent, a stop buffer and a chromogenic measuring card. The R2 reagent comprises a chromogenic substrate reagent. The chromogenic measuring card is provided with a colorimetric reference belt and a reaction cell. The colorimetric reference belt is provided with six color zones which have colors from deep to light and are respectively corresponding to 20%, 40%, 60%, 80%, 100% and higher than 100% of activity of the antithrombin III. Naked-eye distinctiveness of a chromogenic reaction is improved by improving a formula of the chromogenic substrate and the activity of the antithrombin III and colors distinguishable with naked eyes are correlated to obtain the colorimetric reference belt distinguishable by human eyes, and therefore the activity of the antithrombin III can be rapidly and semiquantitatively detected only by naked-eye determination without the need of using matched instruments.
Description
Technical field
The invention belongs to biological medicine external diagnosis reagent field, be specifically related to a kind of based on fibrin ferment and the interactional Antithrombin III quick detection kit of chromogenic substrate and detection method thereof.
Background technology
The principle that Chromogenic assay detects is: Prof. Du Yucang is a kind of can by the compound of thromboplastin catalytic pyrolysis to be measured, this compound connects upper a kind of chromogenic substrate, in testing process, produce color substance can be dissociated, make in test sample, to occur that color changes, the activity of tested thromboplastin can be extrapolated according to color change.Produce color substance and generally select connection paranitroanilinum (pNA).
Current the method is usually used on the equipment such as Biochemical Analyzer and spectrophotometer, and free pNA is in yellow, and it measures wavelength and generally selects 405nm.Under this wavelength, the absorption of other material to light is less than pNA to 1% of light absorption.Concrete detection method both can adopt dynamic method, also can adopt end-point method.Namely dynamic method is the absorbance change of recording sample continuously, calculates the variable quantity of unit interval absorbance, and reports the result with the change of absorbance per minute.Namely end-point method refers to after organized enzyme is with product color substance effect a period of time, adds acetic acid cessation reaction, detects the change of this section of time internal absorbance, and then extrapolate the activity of enzyme to be checked.
Use product color substance roughly can be divided into Three models when detecting thrombus/hemostasis index.
(1) detection of enzyme: namely directly add product color substance in containing the sample of enzyme, because enzyme cleavable produces color substance release pNA, monitors due to pNA release and causes test sample in the change of 405nm place light absorption, just can calculate the activity of enzyme in sample.As the detection to fibrin ferment, fibrinolysin etc.
(2) to the detection of proenzyme: will measure certain proenzyme, first must be activated with activator, make it activate site and expose, just the pNA cracking of producing on color substance can be got off.The activator added must be excessive, because proenzyme only in this way just can be made all to be activated, the amount of proenzyme just becomes certain quantitative relation together with the activity of enzyme in sample.In sample, the activity of enzyme discharges by pNA, and namely the reacting condition of sample absorbance out, then can extrapolate the content of proenzyme in sample thus.
(3) detection of enzyme inhibitor: the enzyme first adding excessive correspondence in measuring samples neutralizes this mortifier, remaining enzyme cleavable produces color substance release pNA, monitor the change causing light absorption due to pNA release, just can measure the activity of enzyme, and then the content of mortifier in sample can be extrapolated.As to antithrombin Ⅲ (AT-III) etc.
Present diagnostic techniques is to large-scale integrated and simple and quickization both direction development, chromogenic assay diagnoses industry in vitro, especially the application of Thrombosis and hemostasis field is very extensive, and the accuracy of testing result is good, but the test of existing chromogenic assay must use supporting instrument could realize detecting.But many times, in situation as unallowed in condition, (do not have instrument or instrument do not have this function time) just can not carry out the test item of this aspect.When only need simple and quick judgement roughly content or only need qualitatively judge yin and yang attribute time, the detection method of existing chromogenic assay just compares shortcoming.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, object is to provide a kind of based on fibrin ferment and the interactional Antithrombin III quick detection kit of chromogenic substrate and detection method thereof.
For achieving the above object, the technical solution used in the present invention is:
A kind of based on fibrin ferment and the interactional Antithrombin III quick detection kit of chromogenic substrate, comprise R1 reagent, R2 reagent, stop buffer and chromogenic assay card, described R1 pack is containing heparin, fibrin ferment, described R2 pack is containing chromophoric substrate reagent, described chromogenic assay card there are colorimetric reference band and reaction tank, described colorimetric reference band divides for by being deep to 6 shallow yellow area, from be deep to the corresponding Antithrombin III of shallow difference activity 20%, 40%, 60%, 80%, 100%, > 100%.
In such scheme, described chromophoric substrate reagent the ratio of each component is: chromophoric substrate Su60.25mg/ml, Tris-HCl50mM, pH8.4, Tween-20 1wt%, PEG80001wt%, trehalose 3.5wt% and Sodium azide 0.01wt%.
In such scheme, described colorimetric reference band obtains by the following method: sample Antithrombin III standard items normal saline dilution being become a series of concentration known, described detection kit is adopted to detect respectively, testing result is the reactant liquor of the different colours of display in each reaction tank, use digital camera to take pictures into figure testing result and analyze with image processing software, identify the R value of picture under RGB pattern, G value and B value, by described R value, G value is integrated with B value mapping software and is drawn and obtains the reactant liquor color colorimetric reference band corresponding with the activity of Antithrombin III.
In such scheme, described R value and G value are 255, and the activity of described B value and Antithrombin III presents good linear relationship, and linear relationship function is defined as: Y=-0.318X+99.93, R
2=0.999.
In such scheme, described reaction tank adopts trapezium structure design.
In such scheme, described chromogenic assay card adopts PVC, PP, PE, LDPE or ABS material to prepare, and wherein, more preferably, adopts the PVC material of white to prepare.
In such scheme, the formula of described stop buffer is: acetic acid 50wt%, Sodium azide 0.01wt%.
In such scheme, the formula of described R1 reagent is: fibrin ferment 5000IU/L, heparin 3000IU/L, Tris-glycocoll 50mM, pH8.4, sodium chloride 175mM, sweet mellow wine 5wt%, Na
2eDTA0.2wt%, BSA2wt% and Sodium azide 0.1wt%.
A kind of based on fibrin ferment and the interactional Antithrombin III method for quick of chromogenic substrate, it is characterized in that, comprise the steps:
(1) join in reaction tank containing the sample blood plasma of anti-coagulants or sample serum;
(2) in reaction tank, add R1 reagent, mixing, makes excessive fibrin ferment and Antithrombin III fully react;
(3) add R2 reagent again, mixing, makes remaining fibrin ferment and chromophoric substrate reagent fully react;
(4) finally stop buffer is added, cessation reaction; The color of reactant liquor in reaction tank and colorimetric reference band are contrasted, according to the degree of closeness of 6 field color in reactant liquor color and colorimetric reference band, judges the activity of Antithrombin III.
In such scheme, described sample blood plasma or sample serum: R1 reagent: R2 reagent: the volume ratio of stop buffer is 1:3:1:0.5, the reaction time that the described excessive fibrin ferment of step (2) and Antithrombin III fully react is 1 minute; The reaction time that the described remaining fibrin ferment of step (3) and chromophoric substrate reagent fully react is 3 minutes.
Beneficial effect of the present invention is as follows: (1) to instant invention overcomes in prior art chromogenic assay and detects Antithrombin III activity and must use supporting instrument, cannot launch the deficiency of the detection of this project in the unallowed situation of instrument condition, provides a kind ofly to detect kit and the detection method thereof of Antithrombin III activity by rapid semi-quantitative; (2) the present invention is by improving chromophoric substrate formula, the bore hole improving chromogenic reaction can identification, successfully cognizable to the activity of Antithrombin III and bore hole color is associated effectively, prepare the distinguishable colorimetric reference band of human eye, the reading solving testing result in prior art must rely on instrument and equipment, cannot naked eyes judge technical matters; (3) detection method is simple to operate, does not need to use necessary instrument, only needs naked eyes to judge just semiquantitative detection to obtain Antithrombin III activity.
Accompanying drawing explanation
Fig. 1 is the structural representation of chromogenic assay card of the present invention, and wherein 1 is colorimetric reference band, and 2 is reaction tank.
Fig. 2 is vertical view and the front view of reaction tank of the present invention, and wherein 3 is vertical view, and 4 is front view.
Fig. 3 is the linear relationship chart of the B value of testing result of the present invention and the concentration of Antithrombin III.
Embodiment
In order to understand the present invention better, illustrate content of the present invention further below in conjunction with embodiment, but content of the present invention is not only confined to the following examples.
Embodiment 1 detection kit and detection method thereof
A kind of based on fibrin ferment and the interactional Antithrombin III quick detection kit of chromogenic substrate, comprise R1 reagent, R2 reagent, stop buffer and chromogenic assay card.
Described R1 reagent is: fibrin ferment 5000IU/L, heparin 3000IU/L, Tris-glycocoll 50mM, pH8.4, sodium chloride 175mM, sweet mellow wine 5wt% (mass concentration), Na
2eDTA0.2wt%, BSA2wt% and Sodium azide .0.1wt%.Packing freeze-drying, redissolves by equal-volume purified water before using.
Described R2 reagent is: chromogenic substrate Su60.25mg/ml, Tris-HCl50mM, pH8.4, Tween-20 1wt%, PEG80001wt%, trehalose 3.5wt%, Sodium azide 0.01wt%.
Described stop buffer is: acetic acid 50wt%, Sodium azide 0.01wt%.
Described chromogenic assay card (concrete structure is as Fig. 1) is prepared from by white PVC plastic, the volume of chromogenic assay card: long 7cm, wide 2cm, thick 0.5cm; Chromogenic assay card having colorimetric reference band and reaction tank, is 1cm × 0.5cm than the area of colour band, and reaction tank adopts trapezium structure design (Fig. 2 is shown in by vertical view and front view), and volume is 600 μ L.Described colorimetric reference band divides for by being deep to 6 shallow yellow area, from the concentration being deep to shallow corresponding Antithrombin III be respectively 20%, 40%, 60%, 80%, 100%, > 100%.
Described colorimetric reference band obtains by the following method: sample Antithrombin III standard items normal saline dilution being become a series of concentration known, described detection kit is adopted to detect respectively, testing result is the reactant liquor of the different colours of display in each reaction tank, use digital camera to take pictures into figure testing result and analyze with image processing software, identify the R value of picture under RGB pattern, G value and B value, described R value, G value and B value mapping software are integrated and draw obtain the reactant liquor color colorimetric reference band corresponding with the activity of Antithrombin III.
Described R value and G value are 255, and the activity of described B value and Antithrombin III presents good linear relationship, and linear relationship function is defined as: Y=-0.318X+99.93, R
2=0.999.
Adopt the above-mentioned method detecting Antithrombin III activity based on fibrin ferment and the interactional Antithrombin III quick detection kit of chromogenic substrate, it is characterized in that, comprise the steps:
(1) the sample blood plasma or the sample serum that 100 μ L are contained anti-coagulants join in reaction tank;
(2) in reaction tank, add 300 μ LR1 reagent, mixing, makes excessive fibrin ferment and Antithrombin III fully react 1 minute;
(3) add R2100 μ L reagent again, mixing, makes remaining fibrin ferment and chromophoric substrate reagent fully react 3 minutes;
(4) 50 μ L stop buffers are finally added, cessation reaction; The color of reactant liquor in reaction tank and colorimetric reference band are contrasted, according to the degree of closeness of 6 field color in reactant liquor color and colorimetric reference band, judges the activity of Antithrombin III.
Embodiment 2 kit accuracy in detection
Reference blood plasma normal saline dilution is become a series of concentration (100%, 75%, 50%, 25%, 0%) kit of the present invention, is then adopted to detect Antithrombin III active, use certain blood coagulation analyzer existing to detect, contrast testing result is as following table 1 simultaneously:
AT-III activity is differently detected after table 1 reference blood plasma dilutes in proportion:
Gather patient's blood plasma as detection sample, adopt kit base detection method of the present invention to detect, detect simultaneously with certain blood coagulation analyzer existing, contrast testing result is as following table 2:
It is active that table 2 patient blood plasma detects AT-III by different detection method:
The testing result of table 1 and table 2 shows: adopt kit of the present invention can the detection Antithrombin III of rapid semi-quantitative active, quantitatively detect Antithrombin III activity to adopt blood coagulation analyzer to be as the criterion, the present invention passes through the testing result of naked eyes identification color judgement accurately and reliably, be applicable to not allow at instrument condition, and only need the roughly activity of simple and quick judgement Antithrombin III or only need qualitatively judge the situation of yin and yang attribute.
Obviously, above-described embodiment is only for the example done clearly is described, and the restriction not to embodiment.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here exhaustive without the need to also giving all embodiments.And therefore amplified apparent change or variation are still within the protection domain of the invention.
Claims (10)
1. one kind based on fibrin ferment and the interactional Antithrombin III quick detection kit of chromogenic substrate, it is characterized in that, comprise R1 reagent, R2 reagent, stop buffer and chromogenic assay card, described R1 pack is containing heparin, fibrin ferment, described R2 pack is containing chromophoric substrate reagent, described chromogenic assay card there are colorimetric reference band and reaction tank, described colorimetric reference band divide for by being deep to 6 shallow yellow area, from the activity 20%, 40%, 60%, 80%, 100% and the > 100% that are deep to the corresponding Antithrombin III of shallow difference.
2. Antithrombin III quick detection kit according to claim 1, it is characterized in that, each component proportion of described chromophoric substrate reagent is: chromophoric substrate Su60.25mg/ml, Tris-HCl50mM, pH84, Tween-20 1wt%, PEG80001wt%, trehalose 3.5wt% and Sodium azide 0.01wt%.
3. Antithrombin III quick detection kit according to claim 1, it is characterized in that, described colorimetric reference band obtains by the following method: sample Antithrombin III standard items normal saline dilution being become a series of concentration known, described detection kit is adopted to detect respectively, testing result is the reactant liquor of the different colours of display in each reaction tank, digital camera is used to take pictures into figure and analyze with image processing software the reactant liquor of different colours, identify the R value of picture under RGB pattern, G value and B value, by described R value, G value is integrated with B value mapping software and is drawn and obtains the reactant liquor color colorimetric reference band corresponding with the activity of Antithrombin III.
4. Antithrombin III quick detection kit according to claim 3, is characterized in that, described R value and G value are 255, and described B value and Antithrombin III activity present good linear relationship, and linear relationship function is defined as: Y=
-0.318X+99.93，R
2=0.999。
5. Antithrombin III quick detection kit according to claim 1, is characterized in that, described reaction tank adopts trapezium structure design.
6. Antithrombin III quick detection kit according to claim 1, is characterized in that, described chromogenic assay card adopts PVC, PP, PE, LDPE or ABS material to prepare.
7. Antithrombin III quick detection kit according to claim 1, is characterized in that, the formula of described stop buffer is: acetic acid 50wt%, Sodium azide 0.01wt%.
8. Antithrombin III quick detection kit according to claim 1, is characterized in that, the formula of described R1 reagent
For: fibrin ferment 5000IU/L, heparin 3000IU/L, Tris-glycocoll 50mM, pH8.4, sodium chloride 175mM, sweet mellow wine 5wt%, Na
2eDTA0.2wt%, BSA2wt% and Sodium azide 0.1wt%.
9. claim 1 ~ 8 is arbitrary described based on fibrin ferment and the interactional Antithrombin III method for quick of chromogenic substrate, it is characterized in that, comprises the steps:
(1) join in reaction tank containing the sample blood plasma of anti-coagulants or sample serum;
(2) in reaction tank, add R1 reagent, mixing, makes excessive fibrin ferment and Antithrombin III fully react;
(3) add R2 reagent again, mixing, makes remaining fibrin ferment and chromophoric substrate reagent fully react;
(4) finally stop buffer is added, cessation reaction; The color of reactant liquor in reaction tank and colorimetric reference band are contrasted, according to the degree of closeness of 6 field color in reactant liquor color and colorimetric reference band, judges the activity of Antithrombin III.
10. detection method according to claim 9, it is characterized in that, described sample blood plasma or sample serum: R1 reagent: R2 reagent: the volume ratio of stop buffer is 1:3:1:0.5, the reaction time that the described excessive fibrin ferment of step (2) and Antithrombin III fully react is 1 minute; The reaction time that the described remaining fibrin ferment of step (3) and chromophoric substrate reagent fully react is 3 minutes.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107167439A (en) * | 2017-06-30 | 2017-09-15 | 上海贞元诊断用品科技有限公司 | A kind of kit that dabigatran content is detected based on Chromogenic assay |
| CN107255622A (en) * | 2017-06-30 | 2017-10-17 | 上海贞元诊断用品科技有限公司 | A kind of kit that dabigatran content is detected based on fibrin ferment Chromogenic assay |
| CN107727587A (en) * | 2017-09-22 | 2018-02-23 | 宁波瑞源生物科技有限公司 | A kind of Antithrombin III assay kit and its detection method |
| CN108037120A (en) * | 2017-12-14 | 2018-05-15 | 长春理工大学 | It is a kind of donate blood before hemoglobin detection method |
| CN109239061A (en) * | 2018-09-14 | 2019-01-18 | 迪瑞医疗科技股份有限公司 | A kind of liquid-type Antiprothrombin antibodies assay kit |
| CN111826418A (en) * | 2020-08-28 | 2020-10-27 | 保定天岳生物工程有限公司 | Antithrombin III determination kit |
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| CN107167439A (en) * | 2017-06-30 | 2017-09-15 | 上海贞元诊断用品科技有限公司 | A kind of kit that dabigatran content is detected based on Chromogenic assay |
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| CN107727587B (en) * | 2017-09-22 | 2020-06-19 | 宁波瑞源生物科技有限公司 | Antithrombin III determination kit and detection method thereof |
| CN108037120A (en) * | 2017-12-14 | 2018-05-15 | 长春理工大学 | It is a kind of donate blood before hemoglobin detection method |
| CN109239061A (en) * | 2018-09-14 | 2019-01-18 | 迪瑞医疗科技股份有限公司 | A kind of liquid-type Antiprothrombin antibodies assay kit |
| CN111826418A (en) * | 2020-08-28 | 2020-10-27 | 保定天岳生物工程有限公司 | Antithrombin III determination kit |
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Application publication date: 20160406 |