CN103235130A - High-sensitivity high-linearity D-D dimer detection kit and application method thereof - Google Patents
High-sensitivity high-linearity D-D dimer detection kit and application method thereof Download PDFInfo
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- CN103235130A CN103235130A CN2013101350718A CN201310135071A CN103235130A CN 103235130 A CN103235130 A CN 103235130A CN 2013101350718 A CN2013101350718 A CN 2013101350718A CN 201310135071 A CN201310135071 A CN 201310135071A CN 103235130 A CN103235130 A CN 103235130A
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Abstract
The invention discloses a high-sensitivity high-linearity D-D dimer detection kit and an application method thereof. A reagent one is a 30mmol/L trihydroxymethyl aminomethane buffer solution. A reagent two comprises a mixed liquid of large-diameter mouse-anti-human D-D dimer monoclonal antibody latex particles with a concentration of 0.01-0.04% (w/v) and small-diameter mouse-anti-human D-D dimer monoclonal antibody latex particles with a concentration of 0.05-0.2% (w/v). Specific antibody content in the small-diameter mouse-anti-human D-D dimer monoclonal antibody latex particles is 0.05-10% of that in the large-diameter mouse-anti-human D-D dimer monoclonal antibody latex particles. Through optimizing the average diameter of large and small particles and concentrations thereof in the reaction system, D-D dimer high-sensitivity detection under low-concentration situation can be realized, and detection range under high-concentration situation can be expanded.
Description
Technical field
The present invention relates to the detection of biological samples field, particularly relate to a kind of kit and using method thereof of utilizing two kinds of different emulsion particles of mean diameter to improve detection of biological samples sensitivity and sensing range, specifically a kind of highly sensitive, high linear D-D dimer detection kit and using method thereof.
Background technology
D dimer (D-D dimer) derives from the crosslinked fibrin grumeleuse of fibrinolysin dissolving.Be in the blood clotting fibrinolytic system, because the effect of solidifying factor Xlll, stable crosslinked fibrin is a kind of by the zymolytic FDP's of fibrinolytic, after the activated factor XI, plasma thromboplastin antecedent II of fibrin monomer is crosslinked, a species specificity catabolite that produces through the fibrinolysin hydrolysis is a specific fibrinolytic process label again.
In the dimeric increase proof of the D body thrombosis is arranged in the blood, start fibrinolytic system, in malignant tumour, obstetrics' illness, vascular lesion, DIC(Disseminated Intravascular Coagulation Syndrome; The disseminated intravascular coagulation syndrome) etc. show high value in the disease.The dimeric level of fibrin degradation product (FDP) D-raises, and shows to exist fibrin degradation process frequently in the body.Therefore, fiber D-dimer is DVT (DVT), pulmonary embolism (PE), the key index of disseminated intravascular coagulation (DIC).The diagnostic method that this indication adopts is that the D dimer is measured.The method that the dimeric mensuration of serum D detects at present is the latex immunoturbidimetry, and the D-D dimer in the sample and mouse-anti people D-D dimer monoclonal antibody latex strengthen particle generation antigen-antibody reaction, produces aggegation so that turbidity and rises.By to turbidity, measure D dimer concentration.
Its course of reaction is as follows:
D-D dimer in the serum+mouse-anti people D-D dimer monoclonal antibody → antigen antibody complex.
Yet existing method is all not wide on detection sensitivity and linearity, also usually is subjected to the linear deficiency of high concentration pattern detection when being used for clinical detection D-D dimer or the low restriction of low concentration sample detection sensitivity.
Summary of the invention
Technical matters to be solved by this invention is at above-mentioned prior art present situation, and provide a kind of two kinds of different emulsion particles of mean diameter that use simultaneously that the D-D dimer is detected, and mean diameter and their concentration in reaction system by optimizing large and small particulate, can realize the high-sensitivity detection of low concentration occasion, the sensing range of high concentration field under closing widened, thereby and the accuracy in detection that promotes the low concentration occasion obtain highly sensitive, high linear D-D dimer detection kit and the using method thereof of clinical judgement accurately.
The present invention solves the problems of the technologies described above the technical scheme that adopts:
A kind of highly sensitive; high linear D-D dimer detection kit; wherein: include reagent one; reagent two; D-D dimer standard items and the contrast unusual serum of D-D dimer; reagent one is TRIS buffer; reagent two includes concentration be 0.01% (w/v) to the major diameter mouse-anti people D-D dimer monoclonal antibody latex particle of 0.04% (w/v) and concentration be 0.05 (w/v) to the minor diameter mouse-anti people D-D dimer monoclonal antibody latex particle mixed liquor of 0.2% (w/v), the specific antibody content on the minor diameter mouse-anti people D-D dimer monoclonal antibody latex particle be on the major diameter mouse-anti people D-D dimer monoclonal antibody latex particle specific antibody content 0.5% to 10%.
The present invention solves the problems of the technologies described above the technical scheme that adopts:
Above-mentioned major diameter mouse-anti people D-D dimer monoclonal antibody latex particle diameter is between 0.15 micron to 0.29 micron, and minor diameter mouse-anti people D-D dimer monoclonal antibody latex particle diameter is between 0.05 micron to 0.10 micron.
The protein quality of the antibody on above-mentioned major diameter mouse-anti people D-D dimer monoclonal antibody latex particle and the minor diameter mouse-anti people D-D dimer monoclonal antibody latex particle is higher than 14% of two kinds of latex particle total protein concentration quality.
The D-D dimer monoclonal antibody that contains in the above-mentioned reagent two is 85% to 120% of major diameter mouse-anti people D-D dimer monoclonal antibody latex particle and the adsorbable D-D dimer of minor diameter mouse-anti people D-D dimer monoclonal antibody latex particle monoclonal antibody maximum.
The D-D dimer monoclonal antibody that contains in the above-mentioned reagent two is 95% to 120% of major diameter mouse-anti people D-D dimer monoclonal antibody latex particle and the adsorbable D-D dimer of minor diameter mouse-anti people D-D dimer monoclonal antibody latex particle monoclonal antibody maximum.
Above-mentioned reagent one is the TRIS buffer of 30mmol/L.
A kind of using method of highly sensitive, high linear D-D dimer detection kit may further comprise the steps:
Step 1, in reagent one, add D-D dimer trial target, place 37 ℃ of water-baths 5 minutes, add reagent two then, place 37 ℃ of water-baths, make D-D dimer and the major diameter mouse-anti people D-D dimer monoclonal antibody latex particle in the reagent two and minor diameter mouse-anti people D-D dimer monoclonal antibody latex particle generation antigen-antibody reaction in the D-D dimer trial target, add reagent after 2 10 seconds, read absorbance A 0 by spectrophotometer at the 570nm place, after 3 minutes, read absorbance A 1 by spectrophotometer at the 570nm place again, draw △ A
Trial target=A1-A0;
Step 2, by known D-D dimer standard items absorbance △ A
StandardCalculate D-D dimer trial target concentration with D-D dimer standard items concentration; Formula is: D-D dimer trial target concentration=D-D dimer standard items concentration * △ A
Trial target/ △ A
Standard
Above-mentioned D-D dimer trial target concentration is at 0mg/L to 20.8mg/L.
Compared with prior art; reagent two of the present invention includes concentration be 0.01% (w/v) to the major diameter mouse-anti people D-D dimer monoclonal antibody latex particle of 0.04% (w/v) and concentration be 0.05 (w/v) to the minor diameter mouse-anti people D-D dimer monoclonal antibody latex particle mixed liquor of 0.2% (w/v), the specific antibody content on the minor diameter mouse-anti people D-D dimer monoclonal antibody latex particle be on the major diameter mouse-anti people D-D dimer monoclonal antibody latex particle specific antibody content 0.5% to 10%.These two kinds of emulsion particles all belong to the sensitization emulsion particle.Use the detection sensitivity that can improve the low concentration occasion and the sensing range of widening the high concentration occasion of two kinds of different emulsion particles of mean diameter; Carry out immune response behind the antibody of the coupled some of the emulsion particle that mean diameter is big (major diameter mouse-anti people D-D dimer monoclonal antibody latex particle), can reach or near immunoreactive maximum rate, and carry out immune response behind the coupled antibody that is less than certain amount of the little emulsion particle (minor diameter mouse-anti people D-D dimer monoclonal antibody latex particle) of mean diameter, can reduce reaction rate; By mean diameter and their concentration in reaction system of optimizing large and small particulate, can realize the high-sensitivity detection of low concentration occasion, the sensing range of high concentration field under closing widened, thereby and the accuracy in detection that promotes the low concentration occasion obtain clinical judgement accurately.The method that detects D-D dimer concentration is to strengthen particle generation antigen-antibody reaction by the D-D dimer in the sample and mouse-anti people D-D dimer monoclonal antibody latex, produces aggegation so that turbidity and rises.By the rate of change of spectrophotometric determination turbidity, obtain the dimeric concentration of D-D with the standard items contrast at the 570nm place again.Mouse-anti people D-D dimer monoclonal antibody in non-specific ground of the latex particle adsorption sample that the advanced nanometer technology of D-D dimer detection reagent employing is made makes its immobilization, is easy to disperse.In the preparation of D-D dimer monoclonal antibody latex particle, will be with the different big or small microballoons of antibody difference mark of two kinds of different affinity, with the big microballoon of high-affinity antibody mark, its sensing range is narrow but highly sensitive, be fit to low concentration sample is detected, and with low-affinity antibody mark minimicrosphere, be fit to quantitatively detect the high concentration sample.So namely overcome the hook effect in the serology, also improved the sensitivity that reagent detects simultaneously, this be other detection side's science of law can not possess.With the antibody of the high-affinity of big microballoon bag quilt easily and the antigen generation antigen-antibody reaction of low concentration, the high concentration antigen in the easy and sample of the antibody of the low-affinity of minimicrosphere bag quilt reacts.We can say that this reagent can take into account the sample of high-concentration and low-concentration simultaneously and measure.
Embodiment
Below embodiments of the invention are described in further detail.
A kind of highly sensitive, high linear D-D dimer detection kit of the present invention, wherein: include reagent one, reagent two, D-D dimer standard items and the contrast unusual serum of D-D dimer, reagent one is the TRIS buffer of 30mmol/L.Reagent two includes concentration be 0.01% (w/v) to the major diameter mouse-anti people D-D dimer monoclonal antibody latex particle of 0.04% (w/v) and concentration be 0.05 (w/v) to the minor diameter mouse-anti people D-D dimer monoclonal antibody latex particle mixed liquor of 0.2% (w/v), the specific antibody content on the minor diameter mouse-anti people D-D dimer monoclonal antibody latex particle be on the major diameter mouse-anti people D-D dimer monoclonal antibody latex particle specific antibody content 0.5% to 10%.
Major diameter mouse-anti people D-D dimer monoclonal antibody latex particle diameter is between 0.15 micron to 0.29 micron, and minor diameter mouse-anti people D-D dimer monoclonal antibody latex particle diameter is between 0.05 micron to 0.10 micron.
The protein quality of the antibody on major diameter mouse-anti people D-D dimer monoclonal antibody latex particle and the minor diameter mouse-anti people D-D dimer monoclonal antibody latex particle is higher than 14% of two kinds of latex particle total protein concentration quality.
The D-D dimer monoclonal antibody that contains in the reagent two is 95% to 120% of major diameter mouse-anti people D-D dimer monoclonal antibody latex particle and the adsorbable D-D dimer of minor diameter mouse-anti people D-D dimer monoclonal antibody latex particle monoclonal antibody maximum.
A kind of using method of highly sensitive, high linear D-D dimer detection kit may further comprise the steps:
Step 1, in reagent one, add D-D dimer trial target, place 37 ℃ of water-baths 5 minutes, add reagent II then, place 37 ℃ of water-baths, make D-D dimer and the major diameter mouse-anti people D-D dimer monoclonal antibody latex particle in the reagent two and minor diameter mouse-anti people D-D dimer monoclonal antibody latex particle generation antigen-antibody reaction in the D-D dimer trial target, add reagent after 2 10 seconds, read absorbance A 0 by spectrophotometer at the 570nm place, after 3 minutes, read absorbance A 1 by spectrophotometer at the 570nm place again, draw △ A
Trial target=A1-A0;
Step 2, by known D-D dimer standard items absorbance △ A
StandardCalculate D-D dimer trial target concentration with D-D dimer standard items concentration; Formula is: D-D dimer trial target concentration=D-D dimer standard items concentration * △ A
Trial target/ △ A
Standard
Above-mentioned D-D dimer trial target concentration is at 0mg/L to 20.8mg/L.
Because the antibody content that reduces on the latex particle can slow down immunoreactive speed, therefore can pass through calculating reacting time, select the suitableeest antibody content.In the detection occasion of high concentration, use typical curve that measurement range is extended to the upper limit direction of detectability.In addition, the amount of coupled specific antibody on particulate can be diluted and free adjustment the solution that contains heterogenetic antibody or antigen compatibility fragment by using damping fluid.In addition, should be according to the type of antigen to be checked, and testing goal, sensing range, antibody titer etc., can appropriateness select better suitable coupled dosage in the above-mentioned limit that defines with interior.On the other hand, the antibody that is adsorbed on the emulsion particle is more many, and reaction rate is more fast; The emulsion particle diameter is more big, and reaction rate is also more fast.Coupled specific antibody on big particulate is more many, and then the detection sensitivity in the low concentration occasion is more high.Therefore, coupled specific antibody on big particulate needs many as much as possible.So, in 100% adsorption isothermal, preferably can arrive 85% ~ 120% of maximal absorptive capacity, can reach 95% ~ 120% better.According to the concept of sorption isotherm on the surface chemistry, be easy to decide at the antibody maximal absorptive capacity of 100% adsorption isothermal.Among the present invention, immunoassay and corresponding reagent are all set up.Use the present invention can realize the high-sensitivity detection of low concentration occasion, also can make high concentration field close sensing range down and be widened, thereby and the accuracy in detection of lifting low concentration occasion obtain clinical judgement accurately.
D-D dimer detection kit
Model: biochemical reagents
Production code member: 9010-717
Specification:
Each D-D dimer detection kit (400 test) contains:
1,1 one bottles of 60ml of reagent
2,2 one bottles of 20ml of reagent
3, one bottle of 1ml of D-D dimer standard items
4, one bottle of 1ml of D-D dimer standard items dilution
5, a operation instructions
The reagent configuration:
This kit is liquid double reagent, can directly use on the machine.If be subject to analyser cuvette capacity limitation, can adjust reagent R1, sample/standard items and reagent R2 use amount to scale.
Stable and the storage period of reagent
Reagent and standard items keep in Dark Place 2～8 ℃ 1 year, forbid freezing.
Sample process:
1) blood sampling and separation of serum on an empty stomach as early as possible;
2) week is stablized in 2～8 ℃ of preservations of serum specimen, and-20 ℃ of freezing preservations can be stablized 3 months.
Test parameters and operation steps:
37 ℃ of temperature
The method of testing end-point method
Start reaction 14 microlitre sample/standard items+150 microlitre reagent R1 (or mixing) to scale
Incubation time 5min
Cessation reaction adds 50 microlitre reagent R2 (or mixing) to scale
Time delay 10min
Tester Hitachi 7100
Detect medium: serum or blood plasma
Be applicable to in-vitro diagnosis
Purposes
D-D dimer detection kit is used for measuring the D-D dimer content of serum or plasma specimen, thereby being used for judging has or not thrombosis, simultaneously in malignant tumour, and obstetrics' illness, vascular lesion, DIC (Disseminated Intravascular Coagulation Syndrome; The disseminated intravascular coagulation syndrome) etc. show high value in the disease.Therefore, fiber D-dimer is DVT (DVT), pulmonary embolism (PE), the key index of disseminated intravascular coagulation (DIC).
Course of reaction:
In 0.150ml reagent I(30mmol/L TRIS buffer) in add the D-D dimer of 14 microlitres, place 37 ℃ of water-bath 5min, add 0.050ml reagent II (mouse-anti people D dimer monoclonal antibody latex particle), behind 37 ℃ of water-bath 10S, read absorbance A 0, after 3 minutes, read absorbance A 1, △ A
Trial target=A1-A0.
2) calculating and result:
Computing formula: D-D dimer concentration (mg/L)=D-D dimer trial target concentration=D-D dimer standard items concentration * △ A
Trial target/ △ A
Standard
Result: D-D dimer range of linearity 0.5mg/L to 20mg/L.
Points for attention
1. reagent can be different because of instrument with amount of samples, in proportion increase and decrease.
In the test the normal or rising serum of D-D dimer to correlating as negative or positive reference.
3. if sample D-D dimer concentration greater than 20mg/L, must be used the physiological saline dilute sample, the result multiply by extension rate.
4. improper as preserving, when reagent is found muddiness is arranged, should abandon.
5. haemolysis has interference to mensuration, will avoid haemolysis in the operating process as far as possible.
Title about present patent application is explained, high sensitivity and high linear concept are at the IVD(external diagnosis reagent in fact), it is the technical term of reagent index, sensitivity refers to a kind of ability that biochemical diagnosis reagent detects at low value, and is linear then refer to a kind of ability that biochemical diagnosis reagent detects in the high value.Be 200-1200ug/L as the disclosed D-D dimer of prior art sensing range, and the sensing range of present patent application is 0-20000ug/L, compares lowly at low value, compares height on the high value, just say present patent application sensitivity and linear high, be better than the disclosed index of prior art.
Claims (8)
- One kind highly sensitive; high linear D-D dimer detection kit; it is characterized in that: include reagent one; reagent two; D-D dimer standard items and the contrast unusual serum of D-D dimer; described reagent one is TRIS buffer; described reagent two includes concentration be 0.01% (w/v) to the major diameter mouse-anti people D-D dimer monoclonal antibody latex particle of 0.04% (w/v) and concentration be 0.05 (w/v) to the minor diameter mouse-anti people D-D dimer monoclonal antibody latex particle mixed liquor of 0.2% (w/v), the specific antibody content on the described minor diameter mouse-anti people D-D dimer monoclonal antibody latex particle be on the major diameter mouse-anti people D-D dimer monoclonal antibody latex particle specific antibody content 0.5% to 10%.
- 2. a kind of highly sensitive, high linear D-D dimer detection kit according to claim 1, it is characterized in that: described major diameter mouse-anti people D-D dimer monoclonal antibody latex particle diameter is between 0.15 micron to 0.29 micron, and described minor diameter mouse-anti people D-D dimer monoclonal antibody latex particle diameter is between 0.05 micron to 0.10 micron.
- 3. a kind of highly sensitive, high linear D-D dimer detection kit according to claim 2, it is characterized in that: the protein quality of the antibody on described major diameter mouse-anti people D-D dimer monoclonal antibody latex particle and the minor diameter mouse-anti people D-D dimer monoclonal antibody latex particle is higher than 14% of two kinds of latex particle total protein concentration quality.
- 4. a kind of highly sensitive, high linear D-D dimer detection kit according to claim 3, it is characterized in that: the D-D dimer monoclonal antibody that contains in the described reagent two is 85% to 120% of described major diameter mouse-anti people D-D dimer monoclonal antibody latex particle and the adsorbable D-D dimer of minor diameter mouse-anti people D-D dimer monoclonal antibody latex particle monoclonal antibody maximum.
- 5. a kind of highly sensitive, high linear D-D dimer detection kit according to claim 4, it is characterized in that: the D-D dimer monoclonal antibody that contains in the described reagent two is 95% to 120% of described major diameter mouse-anti people D-D dimer monoclonal antibody latex particle and the adsorbable D-D dimer of minor diameter mouse-anti people D-D dimer monoclonal antibody latex particle monoclonal antibody maximum.
- 6. a kind of highly sensitive, high linear D-D dimer detection kit according to claim 5, it is characterized in that: described reagent one is the TRIS buffer of 30mmol/L.
- 7. the using method of highly sensitive, a high linear D-D dimer detection kit as claimed in claim 1 is characterized in that: may further comprise the steps:Step 1, in reagent one, add D-D dimer trial target, place 37 ℃ of water-baths 5 minutes, add reagent two then, place 37 ℃ of water-baths, make D-D dimer and the major diameter mouse-anti people D-D dimer monoclonal antibody latex particle in the reagent two and minor diameter mouse-anti people D-D dimer monoclonal antibody latex particle generation antigen-antibody reaction in the D-D dimer trial target, add reagent after 2 10 seconds, read absorbance A 0 by spectrophotometer at the 570nm place, after 3 minutes, read absorbance A 1 by spectrophotometer at the 570nm place again, draw △ A Trial target=A1-A0;Step 2, by known D-D dimer standard items absorbance △ A StandardCalculate D-D dimer trial target concentration with D-D dimer standard items concentration; Formula is: D-D dimer trial target concentration=D-D dimer standard items concentration * △ A Trial target/ △ A Standard
- 8. the using method of a kind of highly sensitive, high linear D-D dimer detection kit according to claim 7, it is characterized in that: described D-D dimer trial target concentration is at 0mg/L to 20.8mg/L.
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| CN106405076A (en) * | 2016-08-31 | 2017-02-15 | 上海科华生物工程股份有限公司 | D-dimer detection kit and preparation method thereof |
| CN107942048A (en) * | 2016-10-13 | 2018-04-20 | 北京众驰伟业科技发展有限公司 | A kind of detection reagent, its preparation method and the application of D dimers |
| CN107942051A (en) * | 2017-11-15 | 2018-04-20 | 南通伊仕生物技术股份有限公司 | A kind of D dimer detection kits and its application method |
| CN109212204A (en) * | 2018-09-14 | 2019-01-15 | 山东艾科达生物科技有限公司 | A kind of kit of latex immunoturbidimetry measurement d-dimer content |
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| CN115948342A (en) * | 2022-12-19 | 2023-04-11 | 山东大学 | CHO cell strain, D-dimer monoclonal antibody and application thereof |
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| CN106405076A (en) * | 2016-08-31 | 2017-02-15 | 上海科华生物工程股份有限公司 | D-dimer detection kit and preparation method thereof |
| CN107942048A (en) * | 2016-10-13 | 2018-04-20 | 北京众驰伟业科技发展有限公司 | A kind of detection reagent, its preparation method and the application of D dimers |
| CN107942051A (en) * | 2017-11-15 | 2018-04-20 | 南通伊仕生物技术股份有限公司 | A kind of D dimer detection kits and its application method |
| CN109212204A (en) * | 2018-09-14 | 2019-01-15 | 山东艾科达生物科技有限公司 | A kind of kit of latex immunoturbidimetry measurement d-dimer content |
| CN109813910A (en) * | 2018-12-29 | 2019-05-28 | 宁波普瑞柏生物技术股份有限公司 | The method and kit for eliminating hook effect are reacted using competitive immunization |
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| CN110658336A (en) * | 2019-11-19 | 2020-01-07 | 北京赛科希德科技股份有限公司 | D-dimer latex immunoturbidimetry detection reagent |
| CN111999506A (en) * | 2020-08-20 | 2020-11-27 | 安徽伊普诺康生物技术股份有限公司 | D-dimer detection kit and preparation method thereof |
| CN111999506B (en) * | 2020-08-20 | 2023-03-31 | 安徽伊普诺康生物技术股份有限公司 | D-dimer detection kit and preparation method thereof |
| CN112213498A (en) * | 2020-09-28 | 2021-01-12 | 河南迈达斯实业有限公司 | D-dimer determination kit and application thereof |
| CN115948342A (en) * | 2022-12-19 | 2023-04-11 | 山东大学 | CHO cell strain, D-dimer monoclonal antibody and application thereof |
| CN115948342B (en) * | 2022-12-19 | 2023-10-03 | 山东大学 | CHO cell strain, D-dimer monoclonal antibody and application thereof |
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Application publication date: 20130807 |