CN202631543U - D-dimer fluorescent immune quantitative measurement test strip - Google Patents
D-dimer fluorescent immune quantitative measurement test strip Download PDFInfo
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- CN202631543U CN202631543U CN 201220301065 CN201220301065U CN202631543U CN 202631543 U CN202631543 U CN 202631543U CN 201220301065 CN201220301065 CN 201220301065 CN 201220301065 U CN201220301065 U CN 201220301065U CN 202631543 U CN202631543 U CN 202631543U
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Abstract
The utility model relates to a D-dimer fluorescent immune quantitative measurement test strip. Two sample pads, a combined pad, a nitrocellulose film and a piece of absorbent paper are sequentially overlapped and attached on a bottom lining; the combined pad is wrapped by anti-D-dimer antibody A marked by fluorescent rubber latex microspheres; and the nitrocellulose film is wrapped by an anti-D-dimer antibody B as a detection line and a rabbit anti-mouse IgG antibody as a quality control line. The D-dimer fluorescent immune quantitative measurement test strip can accurately and quantitatively check the D-dimer content in human plasma, and has the characteristics of simplicity and convenience in operation, high accuracy, high sensitivity, low cost and the like.
Description
Technical field
The utility model belongs to medical immunology in-vitro diagnosis field, is specifically related to a kind of D-dimer fluorescence immunoassay quantitative measurement test strips.
Background technology
The D-dimer is that the fibrinogen in the blood receives effects such as fibrin ferment and solidifies the polymkeric substance of formation, is the specificity catabolite of crosslinked fibrin.Under physiological status, generally below 200 μ g/L, body is keeping blood coagulation and fibrinolytic mobile equilibrium to the dimeric level of the normal D-of human body, in time forms and removes to guarantee fibrin.If this balance is destroyed, the intravascular coagulation tendency strengthens, and fibrin is assembled, and fibrin degradation product (FDP) increases, and the D-dimer content increases.Therefore, the rising antimer intravascular coagulation of D-Dimer levels and the dual activation of fibrinolytic system can be used as one of molecular marker of interior hypercoagulative state of body and hyperfibrinolysis.The D-dimer content raises and can detect the course of disease of multiple disease, like DVT (DVT), disseminated intravascular coagulation (DIC), myocardial infarction, serious hepatitis, pulmonary embolism diseases such as (PE).Therefore the D-dimer content detects the diagnosis that helps preceding state of thrombus and thrombosis disease in the blood, and observation of curative effect and prognosis are judged.
At present, D-dimer detection method mainly contains: latex agglutination, enzyme linked immunological absorption (ELISA) method, fluorescence antibody detection method, immune-gold labeled method and latex immunoturbidimetry etc.The latex agglutination method can only qualitative detection, can not measure the variation of D-dimer content in the blood plasma.The ELISA processing ease receives enzyme activity change, and the result is not too accurate.The immune colloid gold method receives the influence of rheumatoid factor, heparin and blood fat, poor accuracy as a result easily.Latex turbidimetry atopic is bad, required reagent more complicated.
Based on the accurate dimeric content of D-in the detection by quantitative sample of the immunochromatography of fluorescence labeling technology, the while is highly sensitive.Simultaneously, the utility model is transformed traditional test strips structure, on original sample brace plinth, has increased one deck sample pad, has effectively filtered the erythrocyte of remnants in the sample, makes testing result more accurately and reliably.
Summary of the invention
The purpose of the utility model provide a kind of easy and simple to handle, accuracy is high, highly sensitive, cost is low and can carry out the dimeric fluorescence immunoassay quantitative measurement of detection by quantitative D-test strips.
The technical scheme of the utility model is: a kind of D-dimer fluorescence immunoassay quantitative measurement test strips; Two-layer sample pad is pasted on overlap joint ground successively on end liner; Pad; Nitrocellulose filter and thieving paper are coated with the anti-D-homodimeric antibody A of fluorescent latex microballoon mark on the described pad, be coated with anti-D-homodimeric antibody B on the described nitrocellulose filter and be detection line and rabbit anti-mouse igg antibody as nature controlling line.
Said latex microsphere particle size range is 50nm-500nm.
Described fluorescent latex microballoon is to adsorb fluorescently-labeled Streptavidin by latex microsphere to prepare.
Said fluorescence is direct fluorescent material, a kind of among preferred fluorescein isothiocynate, RB 200, TRITC, the luciferin Cy5.
Said pad material is plain film of spun glass or polyester film.
It is the whole blood filter membrane that the material of said two-layer sample pad has at least a kind of.
Experimental result confirms, the test strips of the utility model is D-dimer content in the detection by quantitative human plasma accurately, have easy and simple to handle, accuracy is high, highly sensitive, characteristics such as cost is low.
Description of drawings
Fig. 1 is the utility model D-dimer fluorescence immunoassay quantitative measurement test strips structural representation;
Fig. 2 is the calibration curve of the prepared test strips of the utility model embodiment 1;
Fig. 3 is the range of linearity correlativity of the prepared test strips of the utility model embodiment 1;
Fig. 4 is that the utility model embodiment 1 prepared test strips and the German Siemens D-of company dimer kit testing result correlativity compares;
Wherein, Fig. 1: 1, end liner; 2, ground floor sample pad; 3, second layer sample pad; 4, pad; 5, nitrocellulose filter; 6, thieving paper; 7, detection line; 8, nature controlling line.
Embodiment
Below in conjunction with accompanying drawing and embodiment the utility model is done further detailed explanation.
D-dimer fluorescence immunoassay quantitative measurement test strips; On end liner, overlap ground sample pad 2,3 successively; Pad 4; Nitrocellulose filter 5 and thieving paper 6 are coated with the anti-D-dimer monoclonal antibody M1 of fluorescent latex microballoon mark on the said pad 4, encapsulate anti-D-dimer monoclonal antibody M2 on the said nitrocellulose filter 5 and be detection line 7 and rabbit anti-mouse igg antibody as nature controlling line 8.Above antibody is all available from Roche Holding Ag.
Detect 7 lines on the above-mentioned nitrocellulose filter and can also be and encapsulate the dimeric polyclonal antibody of anti-D-, said polyclonal antibody be that the conventional method immunized mice that adopts those skilled in the art to know obtains Antiserum Preparation and gets.
Further described latex microsphere particle size range is 50nm-500nm.
Said fluorescence is the autofluorescence material, a kind of among preferred fluorescein isothiocynate, RB 200, TRITC, the luciferin Cy5.
The preparation method is following for D-dimer fluorescence immunoassay quantitative measurement test strips:
1) preparation of fluorescent latex microballoon
The preparation of fluorescent latex microballoon: with adsorption-buffering liquid (citrate buffer of 50mM, pH5.8) dilution particle diameter be the 100nm latex microsphere to final concentration 20mg/ml, volume is 5ml, makes latex microsphere suspending liquid; Add an amount of luciferin Cy5 labelled streptavidin (grinding brilliant bio tech ltd available from Shanghai) in adsorption-buffering liquid, final volume is 5ml; Above-mentioned latex microsphere suspending liquid is joined in the above-mentioned adsorption-buffering liquid that contains fluorescein-labelled Streptavidin, make mixed liquor; With gained mixed liquor temperature bath at room temperature 1-2 hour, and constantly stir, centrifugal then, collecting precipitation, deposition is placed 4 ℃ of preservations with store buffer liquid (the adsorption-buffering liquid that contains 0.05%BSA) dissolving, and is subsequent use.
2) preparation of the anti-D-homodimeric antibody of biotinylation A
To resist D-dimer monoclonal antibody M1 to be diluted to 1mg/ml, with 0.1mol/L, pH8.0 sodium bicarbonate buffer liquid D-homodimeric antibody M1 fully dialysed alternately with 0.1mol/L, pH8.0 sodium bicarbonate buffer liquid; NHSB with 1ml DMSO dissolving 1mg obtains NHSB solution; Add 20 μ l NHSB solution to above-mentioned 1mlD-dimer monoclonal antibody M1 solution, stirring at room 2-4 hour, continued stirring at room 10 minutes, with 20mM, the dialysis of pH7.2 PBS damping fluid, promptly get the anti-D-dimer of biotinylation monoclonal antibody M1 then.
3) preparation of fluorescent latex microballoon mark D-homodimeric antibody A
Fluorescent latex microballoon and step 2 that step 1) is made) the biotinylation D-homodimeric antibody M1 that makes mixes, and reacts centrifugal after 30 minutes, and deposition returns to original volume with dissolving with store buffer liquid.
4) encapsulate the preparation of fluorescent latex microballoon mark D-homodimeric antibody A pad 4
The anti-D-homodimeric antibody of fluorescent latex microballoon mark M1 with step 3) makes presses 3.0 μ l/cm
3Consumption be sprayed on the plain film 4 of spun glass.
5) preparation of nitrocellulose filter 5
A) encapsulate the preparation of damping fluid: with the PBS of 0.025M, pH7.4, with 0.22 μ membrane filtration, place 4 ℃ subsequent use, the term of validity 7 days.
B) preparation of confining liquid: will contain 1%BSA, 1% sucrose, the PBS of 0.025M, pH7.5, with 0.22 μ membrane filtration, place 4 ℃ subsequent use, the term of validity 3 days.
C) preparation of D-homodimeric antibody detection line 7: will resist the concentration of D-dimer monoclonal antibody M2 by 2mg/ml, peristaltic pump is awarded liquid measure 0.4ml/min, line speed 50m/20min, and 20 ℃ of forced air dryings are 12 hours in drying box.
D) preparation of nature controlling line 8: the concentration of rabbit anti-mouse igg antibody being pressed 8mg/ml; Peristaltic pump is awarded liquid measure 0.4ml/min, line speed 50m/20min, line on nitrocellulose filter 5; This line is parallel with detection line 7, puts into 20 ℃ of forced air dryings of drying box 12 hours.
The nitrocellulose filter 5 that e) will contain detection line 7 and nature controlling line 8 with above-mentioned confining liquid is in 37 ℃ of sealings 60 minutes, take out rearmounted 37 ℃ down oven dry handled two hours, envelope is subsequent use.
6) test strips assembling
On end liner 1, paste sample pad 2,3 in order each other overlap joint, the plain film 4 of spun glass, nitrocellulose filter 5 obtains test paper plate with thieving paper 6, cuts into the test strips of proper width as requested.
Another kind of D-dimer fluorescence immunoassay quantitative measurement test strips preparation method is following:
1) preparation of fluorescent latex microballoon
The preparation of fluorescent latex microballoon: with adsorption-buffering liquid (citrate buffer of 50mM, pH5.8) dilution particle diameter be the 400nm latex microsphere to final concentration 30mg/ml, volume is 6ml, makes latex microsphere suspending liquid; In adsorption-buffering liquid, final volume is 6ml to add the plain rhodamine labelled streptavidin of an amount of red fluorescence (joining bio tech ltd available from the Shanghai enzyme); Above-mentioned latex microsphere suspending liquid is joined in the above-mentioned adsorption-buffering liquid that contains the plain rhodamine labelled streptavidin of red fluorescence, make mixed liquor; With gained mixed liquor temperature bath at room temperature 1-2 hour, and constantly stir, centrifugal then, collecting precipitation, deposition is placed 4 ℃ of preservations with store buffer liquid (the adsorption-buffering liquid that contains 0.06%BSA) dissolving, and is subsequent use.
2) preparation of the anti-D-homodimeric antibody of biotinylation A
To resist D-dimer monoclonal antibody M1 to be diluted to 1mg/ml, with 0.1mol/L, pH8.0 sodium bicarbonate buffer liquid D-homodimeric antibody M1 fully dialysed alternately with 0.1mol/L, pH8.0 sodium bicarbonate buffer liquid; NHSB with 1ml DMSO dissolving 1mg obtains NHSB solution; Add 25 μ l NHSB solution to above-mentioned 1mlD-dimer monoclonal antibody M1 solution, stirring at room 2-4 hour, continued stirring at room 10 minutes, with 20mM, the dialysis of pH7.2 PBS damping fluid, promptly get the anti-D-dimer of biotinylation monoclonal antibody M1 then.
3) preparation of fluorescent latex microballoon mark D-homodimeric antibody
Fluorescent latex microballoon and step 2 that step 1) is made) the biotinylation D-homodimeric antibody M1 that makes mixes, and reacts centrifugal after 30 minutes, and deposition returns to original volume with dissolving with store buffer liquid.
4) preparation of coating fluorescent latex microballoon mark D-homodimeric antibody A pad 4
The anti-D-homodimeric antibody of fluorescent latex microballoon mark M1 with step 3) makes presses 2 μ l/cm
3Consumption be sprayed on the polyester film 4.
5) preparation of nitrocellulose filter 5
A. encapsulate the preparation of damping fluid: with the PBS of 0.025M, pH7.4, with 0.22 μ membrane filtration, place 4 ℃ subsequent use, the term of validity 7 days;
B. the preparation of confining liquid: will contain 1%BSA, 1% sucrose, the PBS of 0.025M, pH7.5, with 0.22 μ membrane filtration, place 4 ℃ subsequent use, the term of validity 3 days;
The preparation of C.D-homodimeric antibody detection line 7: will resist the concentration of D-dimer polyclonal antibody by 3mg/ml, peristaltic pump is awarded liquid measure 0.4ml/min, line speed 50m/20min, and 20 ℃ of forced air dryings are 12 hours in drying box;
D. the preparation of nature controlling line 8: the concentration of rabbit anti-mouse igg antibody being pressed 8mg/ml; Peristaltic pump is awarded liquid measure 0.4ml/min, line speed 50m/20min, line on nitrocellulose filter 5; This line is parallel with detection line 7, puts into 20 ℃ of forced air dryings of drying box 12 hours;
The nitrocellulose filter 5 that E. will contain detection line 7 and nature controlling line 8 with above-mentioned confining liquid is in 37 ℃ of sealings 60 minutes, take out rearmounted 37 ℃ down oven dry handled two hours, envelope is subsequent use.
6) test strips assembling
On end liner 1, paste sample pad 2,3 each other in order overlap joint, polyester film 4, nitrocellulose filter 5 obtains test paper plate with thieving paper 6, cuts into the test strips of proper width as requested.
Embodiment 3 D-dimer fluorescence immunoassay quantitative measurement test strips detection by quantitative
3.1 drawing standard curve
On the D-dimer fluorescence immunoassay quantitative measurement test strips sample pad for preparing by embodiment 1, add variable concentrations D-dimer antigen standard items and (get seven variable concentrations; Be respectively 0,0.1,0.5,1,2,5,10mg/L; Each concentration is established 3 repetitions); After 10 minutes, the fluorescence immunoassay quantitative analysis instrument Getein1100 through Nanjing base egg bio tech ltd reads detection line 7 and nature controlling line 8 signals, experimental result and analysis in table 1:
Table 1 D-dimer standard items testing result
With the signal averaging drawing standard curve of D-dimer antigen standard items concentration and mensuration, as shown in Figure 2.
3.2 the detection range of linearity
Adopt the test strips of the utility model embodiment 1 preparation, do and detect range of linearity experiment.Get D-dimer standard items; Be diluted to 8 concentration with physiological saline, its concentration range is 0.1mg/L-10mg/L, each concentration replication 3 times; Draw value and the theoretical concentration of measuring concentration are carried out linear regression analysis; Calculate regression equation y=1.0183x+0.0313, correlation coefficient r=0.99995 shows the utility model test strips correlativity in the 0.1mg/L-10mg/L range of linearity fine (seeing accompanying drawing 3).
3.3 sensitivity (LDL)
With 5% human serum albumins is dummy; Test strips with the utility model embodiment 1 preparation is measured; Repeat 20 times; The result of calculation average is 0.021, and standard deviation SD is 0.036, is 0.093 according to add twice standard deviation method for reporting calculating fluorescence immunoassay quantitative analysis instrument Getein1100 mensuration reading variable quantity with blank average.Be respectively 0.05mg/L, 0.1mg/L and 0.15mg/L D-dimer standard solution mensuration fluorescence immunoassay quantitative analysis instrument Getein1100 mensuration reading changing value with concentration after diluting and be respectively 0.045,0.098,0.112, so the test strips detection D-dimer sensitivity of the utility model embodiment 1 preparation is 0.1mg/L.
3.4 repeatability and accuracy
The D-dimer standard solution of preparation 0.5mg/L and 1.0mg/L adopts the test strips of the utility model embodiment 1 preparation to measure, and each concentration difference replication 5 times calculates respectively and measures average and standard deviation.Calculate the coefficient of variation and carry out the repeatability investigation, the result shows that the coefficient of variation is respectively 4.10% and 3.97%; Calculate relative deviation with (1-average/standard value) * 100% and carry out the accuracy investigation, relative deviation is respectively 1.2% and 4%.
Table 2 repeatability and accuracy experiment
| Sequence number | Standard value 0.5mg/L | Standard value 1.0mg/ |
| 1 | 0.48 | 0.98 |
| 2 | 0.52 | 0.97 |
| 3 | 0.49 | 1.03 |
| 4 | 0.53 | 1.05 |
| 5 | 0.51 | 0.96 |
| Mean value | 0.506 | 0.998 |
| Standard deviation | 0.0207 | 0.0396 |
| The coefficient of variation (%) | 4.10% | 3.97% |
| Relative deviation (%) | 1.2% | 4% |
3.5 compare with the German Siemens D-of company dimer kit correlativity
Adopt the test strips of the utility model embodiment 1 preparation to measure; To contain the dimeric serum five equilibrium of D-; Get on the sample pad of sample pipetting volume to test strips of 100 μ l, the fluorescence immunoassay quantitative analysis instrument Getein1100 through Nanjing base egg bio tech ltd reads detection line 7 and nature controlling line 8 signals.From the centrifugal acquisition blood plasma of same aliquot sample, adopt the German Siemens D-of company dimer kit (latex turbidimetry) to detect at Sysmex CA-7000 automatic coagulation analyzer.Adopt this mode to prepare 75 duplicate samples and adopt two detection systems, Fig. 4 has shown the measured value of two systems, the fine r=0.9684 of its correlativity, no difference of science of statistics between kind of the method for P＞0.05, two.
Claims (6)
1. D-dimer fluorescence immunoassay quantitative measurement test strips; Sample pad (2,3) is pasted on overlap joint ground successively on end liner (1); Pad (4); Nitrocellulose filter (5) and thieving paper (6) is characterized in that being coated with on the described pad (4) the anti-D-homodimeric antibody A of fluorescent latex microballoon mark, are coated with anti-D-homodimeric antibody B on the described nitrocellulose filter (5) and are detection line (7) and rabbit anti-mouse igg antibody as nature controlling line (8).
2. D-dimer fluorescence immunoassay quantitative measurement test strips according to claim 1 is characterized in that said latex microsphere particle size range is 50nm-500nm.
3. D-dimer fluorescence immunoassay quantitative measurement test strips according to claim 1 is characterized in that described fluorescent latex microballoon is to adsorb fluorescently-labeled Streptavidin by latex microsphere to prepare.
4. D-dimer fluorescence immunoassay quantitative measurement test strips according to claim 3; It is characterized in that described fluorescence is direct fluorescent material, a kind of among fluorescein isothiocynate, RB 200, TRITC, the luciferin Cy5.
5. D-dimer fluorescence immunoassay quantitative measurement test strips according to claim 1 is characterized in that said pad (4) material is plain film of spun glass or polyester film.
6. D-dimer fluorescence immunoassay quantitative measurement test strips according to claim 1, it is the whole blood filter membrane that the material that it is characterized in that said sample pad (2) and sample pad (3) has at least a kind of.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102692504A (en) * | 2012-06-26 | 2012-09-26 | 南京基蛋生物科技有限公司 | D-dimer quantitative fluorescence immunoassay test strip and preparation method thereof |
| CN105759050A (en) * | 2016-01-29 | 2016-07-13 | 苏州联辰生物技术有限公司 | Immunofluorescence kit for quantitatively detecting content of troponin I and preparation method |
| CN106370838A (en) * | 2016-09-06 | 2017-02-01 | 北京华科泰生物技术有限公司 | Activating fluorescent latex microsphere for D-dipolymer fluorescence immunochromatography test card |
| CN106405078A (en) * | 2016-08-31 | 2017-02-15 | 中山市创艺生化工程有限公司 | D-dimer immunofluorescent quantitative test strip and preparation method thereof |
| CN110007094A (en) * | 2019-04-29 | 2019-07-12 | 江苏众红生物工程创药研究院有限公司 | People's d-dimer quantitative test card and its clinical application |
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2012
- 2012-06-26 CN CN 201220301065 patent/CN202631543U/en not_active Expired - Lifetime
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102692504A (en) * | 2012-06-26 | 2012-09-26 | 南京基蛋生物科技有限公司 | D-dimer quantitative fluorescence immunoassay test strip and preparation method thereof |
| CN105759050A (en) * | 2016-01-29 | 2016-07-13 | 苏州联辰生物技术有限公司 | Immunofluorescence kit for quantitatively detecting content of troponin I and preparation method |
| CN106405078A (en) * | 2016-08-31 | 2017-02-15 | 中山市创艺生化工程有限公司 | D-dimer immunofluorescent quantitative test strip and preparation method thereof |
| CN106370838A (en) * | 2016-09-06 | 2017-02-01 | 北京华科泰生物技术有限公司 | Activating fluorescent latex microsphere for D-dipolymer fluorescence immunochromatography test card |
| CN106370838B (en) * | 2016-09-06 | 2018-01-05 | 北京华科泰生物技术有限公司 | D dimers fluorescence immune chromatography detection card activation fluorescent latex microballoon |
| CN110007094A (en) * | 2019-04-29 | 2019-07-12 | 江苏众红生物工程创药研究院有限公司 | People's d-dimer quantitative test card and its clinical application |
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| GR01 | Patent grant | ||
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Owner name: GETEIN BIOTECH, INC. Free format text: FORMER NAME: NANJING EGG-BASED BIOTECHNOLOGY CO., LTD. |
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Address after: Liuhe District of Nanjing City, Jiangsu province 211505 Yangtze River Industrial Development Zone Bo Fu Road No. 9 Patentee after: GETEIN BIOTECH, Inc. Address before: Liuhe District of Nanjing City, Jiangsu province 211505 Yangtze River Industrial Development Zone Bo Fu Road No. 9 Patentee before: Nanjing Getein Biotechnology Co.,Ltd. |
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