CN102183652A - Kit for time resolved fluoroimmunoassay of D-dimer and preparation method thereof - Google Patents
Kit for time resolved fluoroimmunoassay of D-dimer and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a kit for time resolved fluoroimmunoassay of D-dimer and a preparation method thereof. The kit comprises the following ingredients: (1) a D-dimer calibrator; (2) a micropored plate coated with a D-dimer monoclonal antibody; (3) another D-dimer monoclonal antibody labeled with a europium element; (4) a cleaning solution; (5) a reinforcing solution; and (6) an analysis buffer solution. The kit disclosed by the invention can be used for quantitatively detecting the content of the D-dimer in human blood serum and has the characteristics of high sensitivity, high specificity, high throughout, high accuracy, stable property, lower cost and the like.
Description
Technical field
The invention belongs to the immunoassay medical domain, the invention discloses a kind of D-dimer time resolved fluoro-immunoassay kit and preparation method thereof.
Background technology
D-dimer (D-dimer) is a kind of degrade specifically product that the activated factor XIIIa of fibrin monomer (SFM) produces through the fibrinolysin hydrolysis after crosslinked again, D-dimer concentration rising antimer inner fibrin dissolubility strengthens, help the diagnosis of preceding state of thrombus and thrombosis disease, observation of curative effect and prognosis are judged.
Detecting the dimeric immune analysis method of D-at present mainly contains: latex agglutination, euzymelinked immunosorbent assay (ELISA), immunofiltration collaurum chromogenic reaction method.Said method all has shortcoming separately: latex agglutination can only qualitative detection, can not measure the variation of D-dimer content in the blood plasma.The euzymelinked immunosorbent assay (ELISA) processing ease is subjected to enzyme activity change, and the result is not too accurate.Immunofiltration collaurum chromogenic reaction method specificity is bad.
Summary of the invention
The objective of the invention is to overcome deficiency of the prior art, a kind of D-dimer time resolved fluoro-immunoassay kit is provided.
Second purpose of the present invention provides a kind of preparation method of D-dimer time resolved fluoro-immunoassay kit.
Technical scheme of the present invention is summarized as follows:
D-dimer time resolved fluoro-immunoassay kit, form by following compositions:
1) D-dimer calibration object is made with following method: with human plasma or people's red blood cell the pure product of D-dimer antigen are diluted to series concentration, every bottled 0.5 milliliter of each concentration point, vacuum drying, described D-dimer antigen purity 〉=90%;
2) microwell plate of D-dimer monoclonal antibody bag quilt;
3) another strain D-dimer monoclonal antibody of europium rubidium marking;
4) cleansing solution, make with following method: getting the 1.212g trishydroxymethylaminomethane, be dissolved in the 900mL deionized water, is 7.8 with the salt acid for adjusting pH value, adds deionized water to 1000mL, adds sodium chloride 9g again, adds Tween20 0.1mL, fully dissolving;
5) strengthen liquid, make: get the 1000mL distilled water, add the 3.6mL glacial acetic acid, 0.5g sodium acetate, 0.002～0.01g β-naphthoyltrifluoroacetone, 0.01～0.05g trioctyl phosphine oxide, 1～2mL Triton X-100, mixing with following method;
6) analysis buffer, make with following method: getting 6.06 trishydroxymethylaminomethanes, be dissolved in the 900mL deionized water, is 7.8 with the salt acid for adjusting pH value, adds deionized water to 1000mL, adds the 10g bovine serum albumin(BSA) again, fully dissolving.
The microwell plate of described D-dimer monoclonal antibody bag quilt is to make with following method: D-dimer monoclonal antibody is dissolved in the diluted liquid of bag, making its concentration is 5ug/mL, add in the microwell plate, make 200 μ l/ holes, placed 24 hours for 37 ℃, wash plate 1 time with bag diluted liquid 250 microlitres/hole, be the sealing of 2%BSA solution with mass percentage concentration again, drying at room temperature, the diluted liquid of described bag is to make with following method: get 6.06 trishydroxymethylaminomethanes, be dissolved in the 900mL deionized water, with the salt acid for adjusting pH value is 7.8, add deionized water to 1000mL, add the 10g bovine serum albumin(BSA) again, fully dissolving.
Another strain D-dimer monoclonal antibody of described europium rubidium marking is to make with following method: 50～100 μ g D-dimer monoclonal antibodies are dissolved in the carbonate buffer solution of 50 μ l 0.01M pH=8.5, the isothiocyanic acid benzyl diethylene triamine tetraacethyl europium sodium that adds the 40mg that uses the dissolving of 100 μ l distilled waters, 4 ℃ of reactions 12-18 hour, reactant liquor is transferred to chromatography on the Sephadex G50 post of using 0.05M pH=7.8Tris-HCl damping fluid balance in advance, with 0.05M pH=7.8Tris-HCl equilibrium liquid wash-out, automatically collect, use the UV spectrophotometer measuring protein concentration, collect first big peak.
The preparation method of D-dimer time resolved fluoro-immunoassay kit comprises the steps:
1) preparation D-dimer calibration object, compound method is: with human plasma or people's red blood cell the pure product of D-dimer antigen are diluted to series concentration, every bottled 0.5 milliliter of each concentration point, vacuum drying, described D-dimer antigen purity 〉=90%;
2) microwell plate of preparation D-dimer monoclonal antibody bag quilt;
3) another strain D-dimer monoclonal antibody of preparation europium rubidium marking;
4) preparation cleansing solution, compound method is: getting the 1.212g trishydroxymethylaminomethane, be dissolved in the 900mL deionized water, is 7.8 with the salt acid for adjusting pH value, adds deionized water to 1000mL, adds sodium chloride 9g again, adds Tween20 0.1mL, fully dissolving;
5) preparation strengthens liquid, and compound method is: get the 1000mL distilled water, add the 3.6mL glacial acetic acid, 0.5g sodium acetate, 0.002～0.01g β-naphthoyltrifluoroacetone, 0.01～0.05g trioctyl phosphine oxide, 1～2mL Triton X-100, mixing;
6) preparation analysis buffer, compound method is: getting 6.06 trishydroxymethylaminomethanes, be dissolved in the 900mL deionized water, is 7.8 with the salt acid for adjusting pH value, adds deionized water to 1000mL, adds the 10g bovine serum albumin(BSA) again, fully dissolving.
The microwell plate of described D-dimer monoclonal antibody bag quilt is to make with following method: D-dimer monoclonal antibody is dissolved in the diluted liquid of bag, making its concentration is 5ug/mL, add in the microwell plate, make 200 μ l/ holes, placed 24 hours for 37 ℃, wash plate 1 time with bag diluted liquid 250 microlitres/hole, be the sealing of 2%BSA solution with mass percentage concentration again, drying at room temperature, the diluted liquid of described bag is to make with following method: get 6.06 trishydroxymethylaminomethanes, be dissolved in the 900mL deionized water, with the salt acid for adjusting pH value is 7.8, add deionized water to 1000mL, add the 10g bovine serum albumin(BSA) again, fully dissolving.
Another strain D-dimer monoclonal antibody of described europium rubidium marking is to make with following method: 50～100 μ g D-dimer monoclonal antibodies are dissolved in the carbonate buffer solution of 50 μ l 0.01M pH=8.5, the isothiocyanic acid benzyl diethylene triamine tetraacethyl europium sodium that adds the 40mg that uses the dissolving of 100 μ l distilled waters, 4 ℃ of reactions 12-18 hour, reactant liquor is transferred to chromatography on the Sephadex G50 post of using 0.05M pH=7.8Tris-HCl damping fluid balance in advance, with 0.05M pH=7.8Tris-HCl equilibrium liquid wash-out, automatically collect, use the UV spectrophotometer measuring protein concentration, collect first big peak.
With kit of the present invention can the detection by quantitative human plasma in the dimeric content of D-, have highly sensitive, high special, high flux, accuracy height, stable performance, characteristics such as cost is lower.
Description of drawings
Fig. 1 is the calibration object Line Chart (double-log typical curve) in the prepared kit of embodiment 1.
Embodiment
The following examples can make those skilled in the art more fully understand the present invention, but and do not limit the present invention in any way.
Differentiate fluoroimmunoassay service time of the present invention, its ultimate principle is: use trivalent La rear earth ion and chelate thereof as tracer, replace fluorescent material, isotope, enzyme and chemiluminescent substance, materials such as labelled antigen, antibody, nucleic acid probe; After immune response takes place, according to the characteristics of the fluorescence spectrum of La rear earth ion chelate (the Stokes displacement of high specificity, fluorescence spectrum, life-span long), differentiate fluorescence analyser with the time, measure the fluorescence intensity of immune response end product.According to fluorescence intensity and relative intensity of fluorescence ratio, judge the concentration of analyte in the reaction system, reach the purpose of quantitative test.
The present invention is further illustrated below in conjunction with embodiment.
The preparation of embodiment 1 D-dimer calibration object
Personnel selection red blood cell (from blood station, Tanggu, Tianjin) is diluted to series concentration with D-dimer antigen (purity＞90%), and concentration is respectively: 0ng/mL, 10ng/mL, 50ng/mL, 200ng/mL, 600ng/mL, 1000ng/mL.Totally 6 bottles, every bottled 0.5mL, vacuum drying, standby.
People's red blood cell in the present embodiment also can substitute with human plasma.
Embodiment 2 D-dimer monoclonal antibody bags are by the method for microwell plate
The diluted liquid of preparation bag: getting 6.06 trishydroxymethylaminomethanes, be dissolved in the 900mL deionized water, is 7.8 with 0.1M salt acid for adjusting pH value, adds deionized water to 1000mL, adds the 10g bovine serum albumin(BSA) again, and dissolving is standby fully.
D-dimer monoclonal antibody is dissolved in the diluted liquid of bag, and making its concentration is 5ug/mL, adds in the microwell plate, make 200 μ l/ holes, placed 24 hours for 37 ℃, wash plate 1 time with bag diluted liquid 250 microlitres/hole, be the sealing of 2%BSA solution, drying at room temperature after 6 hours with mass percentage concentration again.
Another strain D-dimer monoclonal antibody method of embodiment 3 europium rubidium markings
80 another strain of μ g D-dimer monoclonal antibodies are dissolved in the carbonate buffer solution of 50 μ l 0.01M pH=8.5, the isothiocyanic acid benzyl diethylene triamine tetraacethyl europium sodium that adds the 40mg that uses the dissolving of 100 μ l distilled waters, 4 ℃ of reactions 14 hours, reactant liquor is transferred to chromatography on the Sephadex G50 post of using 0.05M pH=7.8Tris-HCl damping fluid balance in advance, with 0.05M pH=7.8Tris-HCl equilibrium liquid wash-out, automatically collect, use the UV spectrophotometer measuring protein concentration, collect first big peak, it is standby to merge back adding antiseptic.
Another strain D-dimer monoclonal antibody also can substitute 80 μ g of present embodiment with 50 μ g or 100 μ g, and other step is used for another strain D-dimer MONOCLONAL ANTIBODIES SPECIFIC FOR of europium rubidium marking with embodiment 3.
Also can be with 12 hours or 18 hours alternative present embodiments 14 hours in time of 4 ℃ of reactions, other with embodiment 3, is used for another strain D-dimer MONOCLONAL ANTIBODIES SPECIFIC FOR of europium rubidium marking with step.
Embodiment 4 D-dimer time resolved fluoro-immunoassay kits, form by following compositions:
1) D-dimer calibration object is prepared by embodiment 1 disclosed method;
2) microwell plate of D-dimer monoclonal antibody bag quilt is prepared by embodiment 2 disclosed methods;
3) another strain D-dimer monoclonal antibody of europium rubidium marking is prepared by embodiment 3 disclosed methods;
4) cleansing solution, make with following method: getting the 1.212g trishydroxymethylaminomethane, be dissolved in the 900mL deionized water, is 7.8 with the salt acid for adjusting pH value, adds deionized water to 1000mL, adds sodium chloride 9g again, adds Tween20 0.1mL, fully dissolving;
5) strengthen liquid, make: get the 1000mL distilled water, add the 3.6mL glacial acetic acid, 0.5g sodium acetate, 0.002～0.01g β-naphthoyltrifluoroacetone, 0.01～0.05g trioctyl phosphine oxide, 1～2mL Triton X-100, mixing with following method;
6) analysis buffer, make with following method: getting 6.06 trishydroxymethylaminomethanes, be dissolved in the 900mL deionized water, is 7.8 with the salt acid for adjusting pH value, adds deionized water to 1000mL, adds the 10g bovine serum albumin(BSA) again, fully dissolving.
The using method of embodiment 5 D-dimer time resolved fluoro-immunoassay kits of the present invention
1, sample requirement
Each detection needs 120 microlitre human plasma samples at least.Gather venous blood with the venipuncture method, add heparin anti-coagulating, isolated blood plasma in centrifugal 15 minutes with 2000g.Sample can be preserved 8 hours at 2 ℃-8 ℃.The sample of significant hemolysis is influential to measurement result.May contain the dimeric blood plasma of high concentration D-and answer the dilution of operational analysis damping fluid.
2, detection method
2.1 the preparation of reagent
(1) dilution of another strain D-dimer monoclonal antibody (label) of europium rubidium marking
Preparation half an hour before use.Every reacting hole needs the analysis buffer mixing of 15ul label and 1.5mL (according to analysis buffer: label=dilution in 100: 1), standby.Label after the dilution should use in 1 hour and once use up.
(2) dissolving of D-dimer calibration object
To add the 0.5mL deionized water in the calibration object bottle, the dissolving back is standby fully.
2.2 from sealing bag, take out the microwell plate of D-dimer monoclonal antibody bag quilt, take off the capillary strip of requirement, be fixed on the support, remaining capillary strip is put into sealing bag.
2.3 the application of sample step is as follows:
(1) every hole adds D-dimer calibration object or the sample of 100 μ l, and the room temperature vibration was hatched 1 hour.
(2) with cleansing solution 300 μ L/ holes flushing 4 times, pat dry gently.
(3) add the label of 100 μ l with the analysis buffer dilution, the room temperature vibration was hatched 1 hour.
(4) with cleansing solution 300 μ L/ holes flushing 6 times, pat dry gently.
(5) every hole adds enhancing liquid 200 μ l.(add before the enhancing liquid, suction nozzle should use enhancing liquid to wash twice, should avoid running into little bore edges or its bottom in the adition process, pollutes in order to avoid produce).
(6) after the room temperature vibration is hatched 5 minutes, differentiate luminoscope with the time and measure, guarantee that each sample lath is put in the measurement bay reposefully before measuring.
2.4 the Log value with calibration object concentration is a horizontal ordinate, the Log value of time-resolved fluorescence instrument reading is an ordinate drawing standard curve, finds the dimeric concentration of D-in this blood plasma with the reading of blood plasma to be measured on typical curve.See accompanying drawing 1.
The methodology of embodiment 6 kits of the present invention is identified
1, sensitivity
The sensitivity of kit is not higher than 1ng/mL.
2, specificity
There is not obvious cross reaction with other related substances.
3, measurement range
The measurement range 0-1000ng/mL of kit.
4, linearly dependent coefficient
In the measurement range of kit, dose-response curve linearly dependent coefficient (r) should be not less than 0.99.
5, measure accuracy
With D-dimer national standard product is reference substance, and the actual measurement of kit calibration object is tired and demarcated the ratio of tiring should be between 0.90～1.10.
6, precision of measurement
Batch interior imprecision (CV%) of kit should be no more than 10.0%; Imprecision (CV%) should be no more than 15.0% between batch.
7, quality-control product measured value
The quality-control product measured value should be in allowed band.
8, stability
Kit of the present invention is placed 37 ℃ ± 0.5 ℃ detect after 7 days above-mentioned 1～7 everyly, the result should meet the requirement of projects regulation.
Claims (6)
1.D-dimer time resolved fluoro-immunoassay kit is characterized in that being made up of following compositions:
1) D-dimer calibration object is made with following method: with human plasma or people's red blood cell the pure product of D-dimer antigen are diluted to series concentration, every bottled 0.5 milliliter of each concentration point, vacuum drying, described D-dimer antigen purity 〉=90%;
2) microwell plate of D-dimer monoclonal antibody bag quilt;
3) another strain D-dimer monoclonal antibody of europium rubidium marking;
4) cleansing solution, make with following method: getting the 1.212g trishydroxymethylaminomethane, be dissolved in the 900mL deionized water, is 7.8 with the salt acid for adjusting pH value, adds deionized water to 1000mL, adds sodium chloride 9g again, adds Tween200.1mL, fully dissolving;
5) strengthen liquid, make: get the 1000mL distilled water, add the 3.6mL glacial acetic acid, 0.5g sodium acetate, 0.002～0.01g β-naphthoyltrifluoroacetone, 0.01～0.05g trioctyl phosphine oxide, 1～2mL Triton X-100, mixing with following method;
6) analysis buffer, make with following method: getting 6.06 trishydroxymethylaminomethanes, be dissolved in the 900mL deionized water, is 7.8 with the salt acid for adjusting pH value, adds deionized water to 1000mL, adds the 10g bovine serum albumin(BSA) again, fully dissolving.
2. kit according to claim 1, the microwell plate that it is characterized in that described D-dimer monoclonal antibody bag quilt is to make with following method: D-dimer monoclonal antibody is dissolved in the diluted liquid of bag, making its concentration is 5ug/mL, add in the microwell plate, make 200 μ l/ holes, placed 24 hours for 37 ℃, wash plate 1 time with bag diluted liquid 250 microlitres/hole, be the sealing of 2%BSA solution with mass percentage concentration again, drying at room temperature, the diluted liquid of described bag is to make with following method: get 6.06 trishydroxymethylaminomethanes, being dissolved in the 900mL deionized water, is 7.8 with the salt acid for adjusting pH value, adds deionized water to 1000mL, add the 10g bovine serum albumin(BSA) again, fully dissolving.
3. kit according to claim 1, another strain D-dimer monoclonal antibody that it is characterized in that described europium rubidium marking is to make with following method: 50～100 μ g D-dimer monoclonal antibodies are dissolved in the carbonate buffer solution of 50 μ l 0.01M pH=8.5, the isothiocyanic acid benzyl diethylene triamine tetraacethyl europium sodium that adds the 40mg that uses the dissolving of 100 μ l distilled waters, 4 ℃ of reactions 12-18 hour, reactant liquor is transferred to chromatography on the Sephadex G50 post of using 0.05M pH=7.8 Tris-HCl damping fluid balance in advance, with 0.05M pH=7.8 Tris-HCl equilibrium liquid wash-out, automatically collect, use the UV spectrophotometer measuring protein concentration, collect first big peak.
4.D-the preparation method of dimer time resolved fluoro-immunoassay kit is characterized in that comprising the steps:
1) preparation D-dimer calibration object, compound method is: with human plasma or people's red blood cell the pure product of D-dimer antigen are diluted to series concentration, every bottled 0.5 milliliter of each concentration point, vacuum drying, described D-dimer antigen purity 〉=90%;
2) microwell plate of preparation D-dimer monoclonal antibody bag quilt;
3) another strain D-dimer monoclonal antibody of preparation europium rubidium marking;
4) preparation cleansing solution, compound method is: getting the 1.212g trishydroxymethylaminomethane, be dissolved in the 900mL deionized water, is 7.8 with the salt acid for adjusting pH value, adds deionized water to 1000mL, adds sodium chloride 9g again, adds Tween200.1mL, fully dissolving;
5) preparation strengthens liquid, and compound method is: get the 1000mL distilled water, add the 3.6mL glacial acetic acid, 0.5g sodium acetate, 0.002～0.01g β-naphthoyltrifluoroacetone, 0.01～0.05g trioctyl phosphine oxide, 1～2mL Triton X-100, mixing;
6) preparation analysis buffer, compound method is: getting 6.06 trishydroxymethylaminomethanes, be dissolved in the 900mL deionized water, is 7.8 with the salt acid for adjusting pH value, adds deionized water to 1000mL, adds the 10g bovine serum albumin(BSA) again, fully dissolving.
5. according to claims 4 described preparation methods, the microwell plate that it is characterized in that described D-dimer monoclonal antibody bag quilt is to make with following method: D-dimer monoclonal antibody is dissolved in the diluted liquid of bag, making its concentration is 5ug/mL, add in the microwell plate, make 200 μ l/ holes, placed 24 hours for 37 ℃, wash plate 1 time with bag diluted liquid 250 microlitres/hole, be the sealing of 2%BSA solution with mass percentage concentration again, drying at room temperature, the diluted liquid of described bag is to make with following method: get 6.06 trishydroxymethylaminomethanes, being dissolved in the 900mL deionized water, is 7.8 with the salt acid for adjusting pH value, adds deionized water to 1000mL, add the 10g bovine serum albumin(BSA) again, fully dissolving.
6. according to claims 4 described preparation methods, another strain D-dimer monoclonal antibody that it is characterized in that described europium rubidium marking is to make with following method: 50～100 μ g D-dimer monoclonal antibodies are dissolved in the carbonate buffer solution of 50 μ l 0.01M pH=8.5, the isothiocyanic acid benzyl diethylene triamine tetraacethyl europium sodium that adds the 40mg that uses the dissolving of 100 μ l distilled waters, 4 ℃ of reactions 12-18 hour, reactant liquor is transferred to chromatography on the Sephadex G50 post of using 0.05M pH=7.8 Tris-HCl damping fluid balance in advance, with 0.05M pH=7.8 Tris-HCl equilibrium liquid wash-out, automatically collect, use the UV spectrophotometer measuring protein concentration, collect first big peak.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102590529A (en) * | 2012-02-16 | 2012-07-18 | 邵伟 | Kit for detecting PCDH10 (Protocadherin10) in serum by TRFIA (Time Resolved Fluorescence Immunoassay method) |
| CN102608335A (en) * | 2012-04-19 | 2012-07-25 | 协和生物制药（天津）有限公司 | Method for preparing NT-proBNP time-resolved fluoroimmunoassay kit |
| CN105891176A (en) * | 2016-04-06 | 2016-08-24 | 上海奥普生物医药有限公司 | Cup type time-resolved fluorescence D-dimer analysis method and reagent kit based on microspheres |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102590529A (en) * | 2012-02-16 | 2012-07-18 | 邵伟 | Kit for detecting PCDH10 (Protocadherin10) in serum by TRFIA (Time Resolved Fluorescence Immunoassay method) |
| CN102608335A (en) * | 2012-04-19 | 2012-07-25 | 协和生物制药（天津）有限公司 | Method for preparing NT-proBNP time-resolved fluoroimmunoassay kit |
| CN105891176A (en) * | 2016-04-06 | 2016-08-24 | 上海奥普生物医药有限公司 | Cup type time-resolved fluorescence D-dimer analysis method and reagent kit based on microspheres |
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Application publication date: 20110914 |