CN106370838A - Activating fluorescent latex microsphere for D-dipolymer fluorescence immunochromatography test card - Google Patents
Activating fluorescent latex microsphere for D-dipolymer fluorescence immunochromatography test card Download PDFInfo
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Abstract
The invention discloses an activating fluorescent latex microsphere for D-dipolymer fluorescence immunochromatography test card and application thereof. The preparation method for the activating fluorescent latex microsphere for D-dipolymer fluorescence immunochromatography test card includes the following steps: 1, the surfactant is put into a buffer solution with pH 8-10, then dimethylformamide, N, N'dicyclohexylcarbodiimide and N-H-hydroxy succinimide are added in; 2, the dispersion liquid of fluorescent latex microsphere on the surface of amidogen is taked out, after regulate the pH to 8-10 with the buffer solution, the dispersion liquid is added into the liquid mixture obtained from step 1, stirring and reaction are carried out, after the reaction is finished, the core and the supernatant are removed, and the surface-activated fluorescent latex microsphere is obtained; wherein, the surface activator is a mixture of dodecyl amidogen propionate, N-dodecyl amidogen propionate and stearate, the weight proportion of the three is 0.5-3:0.5-3:1.
Description
Technical field
The invention belongs to fluorescence immune chromatography technical field is and in particular to a kind of detection of d- dimer fluorescence immune chromatography blocks
Activation fluorescent latex microsphere and its preparation and application.
Background technology
Fluorescence immune chromatography detection card (reagent card), because of its quickly and easily characteristic, is widely used in fast inspection field.Determinand
Antibody labeling fluorescent latex microsphere, latex beadses have surface electronic repulsion in the liquid phase and Van der Waals force makes in liquid phase
Latex particle keeps being uniformly distributed.But it is sprayed at glass fibre element film and evaporated dry, surface tension with solution after drying
Promote infinitely near leading to intermolecular repulsion to disappear between latex particle, Van der Waals force increases, and finally makes multiple latex micels
It is polymerized to big and small different granule.When adding sample to redissolve latex beadses when immunochromatography, latex beadses no longer uniformly divide
Dissipate and lead to chromatograph unsuccessfully.Adopting fluorescence immune chromatography test kit therefore this area, card containing detection adds sample buffer more.Increase
Detection complexity.Or due to latex beadses partial agglomeration so that detection sensitivity reduces, false negative in testing result.
In addition, latex beadses is unstable, the stability of fluoroscopic examination card also can be made poor, be not easy to preserve and transport.
After d- dimer (d-dimer) is the crosslinking of fibrin monomer activated factor xiii, then through fibrinolytic enzyme hydrolysiss institute
A kind of selective degradation product producing, is a specific fibrinolytic process markup thing.In fibrinolytic protein catabolite, only d-
Dimer crosslinking fragment can reflect the thrombolysis activity after thrombosiss.The dimeric level of d- raises, and shows to there is frequency in vivo
Numerous fibrin degradation process.D- dimer for Clinics and Practices fibrinolytic system disease (such as dic, various thrombosis) and with
Fibrinolytic system has related disorders (as pregnancy syndrome), and thromboembolism treatment monitoring, has great significance.
Content of the invention
The technical problem to be solved is: the fluorescent latex microsphere of d- dimer fluorescence immune chromatography detection card exists
After drying, easily reunite, dispersion is uneven, thus leading to chromatograph unsuccessfully or make detection sensitivity to reduce.
In order to solve above-mentioned technical problem, the invention provides a kind of work of d- dimer fluorescence immune chromatography detection card
Change fluorescent latex microsphere and its preparation, apply the d- dimer dry type fluorescence immune chromatography inspection of this activation fluorescent latex microsphere preparation
Survey card, this detection card can be used for Quantitative in vitro and measures the dimeric content of d- in human serum, blood plasma or whole blood.The detection of the present invention
Card can solve the phenomenon that fluorescent latex microsphere occurs to reunite in spraying, drying glass fibre element film, to prepare label
Pad, thus obtaining dry type fluorescence immune chromatography detection card, configures detection kit without diluent.
The d- dimer fluorescence immune chromatography detection card that the present invention the provides preparation method of activation fluorescent latex microsphere, bag
Include following steps:
1) take surfactant to add in the buffer solution that ph is 8 ~ 10, add dimethylformamide, n, n '-dicyclohexyl carbon
Diimine and n- N-Hydroxysuccinimide；
2) take the dispersion liquid of the fluorescent latex microsphere of amino surface, adjusted after ph to 8 ~ 10 with buffer solution, be added to step 1) institute
In the mixture obtaining, stirring reaction, after completion of the reaction, centrifugation removes supernatant, obtains activating fluorescent latex microsphere；
Wherein, described surfactant is the mixing of dodecyl alanine salt, n- dodecyl alanine and stearate
Thing, the weight of three is than for 0.5 ~ 3:0.5 ~ 3:1.
Preferably, described stearate is one of stearic potassium, sodium, ammonium salt and tri ethanol ammonium salt or its two kinds
Above combination.
Preferably, described surfactant is dodecyl alanine salt, n- dodecyl alanine and stearate
Mixture, the weight of three is than for 1.5:1.5:1.
Preferably, described surfactant and the mass ratio of the fluorescent latex microsphere of described amino surface are 500 ~ 2000:
1.
Preferably, step 1) and step 2) described in buffer solution be carbonic acid buffer；Step 2) in stirring reaction
Time is 2 ~ 4 hours, and temperature is 20 ~ 30 DEG C；The fluorescent latex microsphere of amino surface is aminopolystyrene fluorescent microsphere.
The present invention also provides the d- dimer fluorescence immune chromatography detection card activation fluorescence that above-mentioned preparation method obtains
Latex beadses.
The present invention also provides above-mentioned d- dimer fluorescence immune chromatography to detect that card activates the labelling of fluorescent latex microsphere
The preparation method of thing working solution: take d- dimer fluorescence immune chromatography detection card activation fluorescent latex microsphere to be scattered in microsphere and delay
Rush in liquid, obtain label working solution；The ratio that the fluorescent latex microsphere of surface active is shared in label working solution is 0.5
~2wt%；
Wherein, the preparation method of microsphere buffer is: by bsa(bovine serum albumin), biological preservative, Lei meter Bang a and glycine betaine
It is dissolved in the phosphate buffer of 0.01m, ph7.4.
Preferably, bsa, biological preservative, the Lei meter Bang a and glycine betaine concentration in described microsphere buffer is
0.1wt%.
The present invention also provides the preparation method of the labeling pad of d- dimer fluorescence immune chromatography detection card, walks including following
Rapid:
1) preparation of the rabbit igg of activation fluorescent latex microsphere labelling: take the label working solution that above-mentioned preparation method obtains, from
The heart, after abandoning supernatant, is redissolved with labelling buffer, and is simultaneously introduced carbodiimide and rabbit igg, stirring reaction, be then centrifuged for,
Abandon supernatant, finally use labelling diluent to redissolve；
2) labelling of the activation fluorescent latex microsphere labelling preparation method of d- dimer monoclonal antibody: take above-mentioned preparation method
The label working solution obtaining, centrifugation, redissolved with labelling buffer after abandoning supernatant, and be simultaneously introduced carbodiimide and labelling
With d- dimer monoclonal antibody, stirring reaction, it is then centrifuged for, abandons supernatant, finally use labelling diluent to redissolve；
3) step 1) gained dispersion liquid and step 2 are taken) gained dispersion liquid, mixing, it is sprayed on glass fibre, dry；
The formula of described labelling buffer is: sodium carbonate 4.33g, sodium bicarbonate 2.96g, is dissolved in 1000ml water；Described labelling
The formula of diluent is: trisodium citrate 7.33g, citric acid 4.44g, sodium hydroxide 1g, is dissolved in 1000ml water.
The present invention provides a kind of d- dimer fluorescence immune chromatography detection card, include above-mentioned labeling pad, be coated pad and
Absorption pad, wherein said labeling pad is overlapped on the one end being coated pad, and absorption pad is overlapped in and is coated on the other end of pad, is coated
Pad is provided with nature controlling line and detection line, and nature controlling line is coated with the antibody of goat-anti rabbit igg, detection line is coated with and is coated with d- bis-
Aggressiveness monoclonal antibody.
The present invention can reach following technique effect:
1st, present invention research finds that the fluorescent latex microsphere through the activation of specific surfactant keeps intergranular when drying
Relative distance is difficult to reunite.During chromatography process, sample is added can at once to redissolve and smoothly chromatograph, so that preparation dry type fluorescence immunoassay
Chromatography detection card (reagent card), is user-friendly to, and testing result is stable, accurate, reliable.
2nd, the present invention selected by testing sieve be suitable for d- dimer fluorescence immune chromatography detection card fluorescent latex micro-
The specific surfactant combination of ball.Process the fluorescent latex microsphere of chemical modification using specific surfactant in advance, place
Latex beadses labelling d- homodimeric antibody after reason, for preparing the labeling pad of d- dimer fluorescence immune chromatography detection card,
Realize latex beadses even application, soilless sticking phenomenon.And, after adding sample to be tested, the fluorescent latex microsphere of labelling is permissible
Quickly, smoothly realize chromatography, without special sample diluent or buffer processing detection card.
3rd, the d- dimer dry type fluorescence immune chromatography detection card prepared by the present invention, stability is strong, easy and simple to handle, user
Close friend, testing result is accurate, reliable, and sensitivity is high.
Brief description
Fig. 1 is the section decomposition texture schematic diagram of the d- dimer fluorescence immune chromatography reagent card of the present invention.
Fig. 2 is the overlooking the structure diagram of the d- dimer fluorescence immune chromatography reagent card of the present invention.
Fig. 3 is the foundation of d- dimer standard curve.
In Fig. 4 whole blood sample, d- dimer content measures curve.
Fig. 5 is the fluorescence signal curve of the detection card of embodiment 5.
Fig. 6 is the fluorescence signal curve of the detection card of embodiment 6.
Fig. 7 is the fluorescence signal curve of the detection card of embodiment 7.
Fig. 8 is the fluorescence signal curve of the detection card of embodiment 8.
Fig. 9 is the fluorescence signal curve of the detection card of comparative example.
Specific embodiment
The invention will be further described with specific embodiment below in conjunction with the accompanying drawings, so that those skilled in the art is permissible
It is better understood from the present invention and can be practiced, but illustrated embodiment is not as a limitation of the invention.
The invention provides a kind of d- dimer fluorescence immune chromatography detection card activation fluorescent latex microsphere, including as follows
Step:
1) take surfactant to add in the buffer solution that ph is 8 ~ 10, add dimethylformamide, n, n '-dicyclohexyl carbon two
Imines and n- N-Hydroxysuccinimide, stirring reaction；
2) take the dispersion liquid of the fluorescent latex microsphere of amino surface, adjusted after ph to 8 ~ 10 with buffer solution, be added to step 1) institute
In the mixture obtaining, stirring reaction, after completion of the reaction, centrifugation removes supernatant, obtains activating fluorescent latex microsphere；
Wherein, described surfactant is the mixing of dodecyl alanine salt, n- dodecyl alanine and stearate
Thing, the weight of three is than for 0.5 ~ 3:0.5 ~ 3:1.
The present invention adopts the mixture of dodecyl alanine salt, n- dodecyl alanine and stearate to pass through altogether
The method that valency is coupled modifies latex beadses outer surface so as to have hydrophobicity.Using the activation fluorescent latex microsphere mark after processing
Note d- homodimeric antibody.After the rabbit igg of the d- homodimeric antibody of fluorescent latex microsphere labelling and fluorescent latex microsphere labelling mixes
It is sprayed on and is prepared into labeling pad 1 on glass fibre element film, be coated pad 2(nitrocellulose filter) cover near one end of nature controlling line
Upper adsorptive pads, near the other end overlay marks thing pad of detection line, for preparing dry type fluorescence immune chromatography reagent card, detection line
On be coated and use d- homodimeric antibody.After adding sample to be tested, the d- dimer of d- dimer and fluorescent latex microsphere labelling
Antibody haptoreaction, latex beadses can keep dispersity not reunite, and can rapidly, uniformly divide after contact measured sample
Dissipate, and along nitrocellulose filter flash chromatography.
Fluorescent latex microsphere used in following examples of the present invention refers to the polyphenyl second of amino surface being modified by sulphation
Alkene fluorescent microsphere, diameter 100-500nm.
First, activate the preparation of fluorescent latex microsphere
Embodiment 1
1) surfactant (4.5mg sodium dodecyl aminopropionitrile, 4.5mg n- dodecyl alanine and 3mg stearic acid are taken
Sodium) it is initially dissolved in the carbonic acid buffer (h value to 9.6) of 0.5m ph9.6, add in the dmf of 0.1ml, the dmf of 0.1ml
In, add n, after n '-dicyclohexylcarbodiimide (dcc) 1mg and 0.6mg n- N-Hydroxysuccinimide (nhs), be stirred at room temperature 3
Hour.
2) 1ml is contained the fluorescent latex microspheres solution carbonic acid buffer of 0.5m ph9.6 of the amino surface of 1% solidss
Adjust ph value to be added in step 1) to after 9.6, continue stirring reaction 3 hours.Both the latex beadses that must activate.
3) with high speed centrifuge, fluorescent latex microsphere reactant liquor is centrifuged at a high speed, removes reaction solution supernatant, use
1ml microsphere buffer redissolves fluorescent latex microsphere, forms label working solution.
Microsphere phosphate buffer is prepared: the phosphate buffer of 0.01m ph7.4, and bsa containing 0.1wt% gives birth to containing 0.1 wt %
Thing preservative, containing 0.1 wt % Lei meter nation a, containing 0.1 wt % glycine betaine.Microsphere phosphate buffer provides certain Ionic strength
With ph value, make microsphere dispersed.
Embodiment 2
The operational approach of the present embodiment is similar to Example 1, and difference is, in step 1), surfactant is: 3 mg ten
Dialkyl amido sodium propionate, 3 mg n- dodecyl alanine and 6mg sodium stearate.
Embodiment 3
The operational approach of the present embodiment is similar to Example 1, and difference is, in step 1), surfactant is: 4.5mg ten
Dialkyl amido sodium propionate, 4.5 mg n- dodecyl alanine and 1.5mg sodium stearate.
Embodiment 4
The operational approach of the present embodiment is similar to Example 1, and difference is, in step 1), surfactant is: 4 mg ten
Dialkyl amido sodium propionate, 6 mg n- dodecyl alanine and 2 mg sodium stearates.
2nd, the activation fluorescent latex microsphere to embodiment 1 ~ 4 preparation taking d- dimer fluorescence immune chromatography detection card as a example
Application and effect illustrate.
Embodiment 5
The fluorescence immune chromatography detection card of the label working solution preparation being obtained using embodiment 1
1) the rabbit igg of fluorescent latex microsphere labelling: take the label working solution that 5ml embodiment 1 obtains, use centrifuge
30min(rotating speed is 10000r/min), abandon supernatant after being centrifuged, redissolved with 5ml labelling buffer, add the rabbit igg of 1mg
Mix, another addition 5mg carbodiimides, reaction 1h be stirred at room temperature, being then centrifuged for 15min(rotating speed is 10000r/min), abandon
Clear liquid, is redissolved standby after mixing with the label diluted of 10ml.
2) labelling of fluorescent latex microsphere labelling d- dimer monoclonal antibody
Take the label working solution that 10ml embodiment 1 obtains, be 10000r/min with centrifuge 30min(rotating speed), centrifugation
Abandon supernatant after complete, redissolved with 10ml labelling buffer, the labelling adding 2mg is mixed with d- dimer monoclonal antibody, separately
Add 10mg carbodiimides, reaction 1h be stirred at room temperature, being then centrifuged for 15min(rotating speed is 10000r/min), abandon supernatant, use
The label diluted of 20ml is redissolved standby after mixing.
Step 1) and 2) in the formula of labelling buffer be: sodium carbonate 4.33g, sodium bicarbonate 2.96g, be dissolved in 1000ml water
In；The formula of labelling diluent is: trisodium citrate 7.33g, citric acid 4.44g, sodium hydroxide 1g, is dissolved in 1000ml water.
3) labeling pad preparation
Step 2) fluorescence that obtained with step 1) with d- dimer monoclonal antibody of the labelling of fluorescent latex microsphere labelling that obtains
The rabbit igg of latex beadses labelling mixes, and the two volume ratio is 2:1.Then the fluorescent latex microsphere mixing is pressed 1 microlitre/li
The amount even application of rice drying baker of 45 DEG C of transposition on glass fibre element paper dries the preparation completing labeling pad for 2 hours.
4) it is coated liquid preparation: take and be coated with, in d- dimer monoclonal antibody addition phosphate buffer, making detection line
It is coated liquid；Take goat-anti rabbit igg to add in phosphate buffer, make nature controlling line and be coated liquid.The formula of this phosphate buffer is:
0.99g sodium dihydrogen phosphate, 5.16g disodium hydrogen phosphate is dissolved in 1000ml purified water.
5) it is coated the process of pad: detection line is coated liquid and is covered with line in bag and is coated formation detection line, and nature controlling line is coated liquid
It is coated formation nature controlling line being covered with line in bag, be then dried.
6) detection card assembling:
By dried labeling pad and the absorption pad (absorbent paper) cut out, by being coated the close Quality Control of pad (nitrocellulose filter)
One end of line covers upper absorption pad, and the requirement near the other end overlay marks thing pad of detection line carries out joint strip.By cutting cutter mark
Quasi- rule of operation is operated, and is cut into the wide detection card of 4mm ± 0.1mm.
As illustrated in fig. 1 and 2, detection card includes: is coated pad 2(nitrocellulose filter), it is respectively arranged at the two ends with a linkage section (such as
First linkage section 20, the second linkage section 22 shown in figure), for detection section 21 in the middle of two linkage sections, detection section 21 surface is provided with detection
Line 5 and nature controlling line 7；Labeling pad 1(glass fibre element film), its one end is provided with linkage section 10, and the linkage section 10 of labeling pad covers
Cover and be fixed on the first linkage section 20 being coated pad 2；Label 8 is coated with labeling pad 1, specifically in an embodiment
In, it is in (away from linkage section 2mm, label is 5mm along the length being coated pad length direction) spraying mark near linkage section 10
Note thing 8(contains the rabbit igg of fluorescent latex microsphere labelling and the labelling d- dimer monoclonal anti of fluorescent latex microsphere labelling
Body).
Absorption pad 3(absorbent paper), its one end is provided with linkage section 30, and the linkage section 30 of absorption pad covers and is fixed on and is coated pad
On 2 the second linkage section 22；
Base plate 4, is coated pad 2, labeling pad 1 and absorption pad 3 and is both secured on base plate.
This detection is stuck in and is existed with one plant of d- dimer monoclonal antibody (being coated with d- dimer monoclonal antibody) during use
On nitrocellulose filter, line is coated, and as p-wire, rules to be coated with goat-anti rabbit multi-resistance and make matter on nitrocellulose filter
Control line.By another plant of d- dimer monoclonal antibody (labelling d- dimer monoclonal antibody) of latex fluorescent microsphere labelling with
The rabbit igg of latex fluorescent microsphere labelling is sprayed on after mixing and is prepared into labeling pad on glass fibre element film.Nitrocellulose filter
Cover upper adsorptive pads near one end of nature controlling line, near the other end overlay marks thing pad of p-wire.Add toward in labeling pad
Standard substance or blood sample to be measured, antigen will be with label hybrid reaction and along nitrocellulose filter chromatography, respectively with p-wire
With nature controlling line reaction.When test result is effective, nature controlling line shows certain light intensity.At this moment the light signal strength ratio on p-wire
The ratio (t/c) of nature controlling line light signal strength becomes positive correlation with concentration of specimens, can be drawn by standard curve calculating and treat test sample
Product concentration.
(1) the present embodiment is obtained detection card calibration curve and the mensure of sample to be tested
50ul calibration object or sample to be tested are slowly added dropwise in labeling pad.Standing reaction in 15 minutes at ambient temperature,
Detection card is put into detection in savant-100 fluorescence immune chromatography analyser after terminating by reaction.Each concentration point of calibration object
Repeat to do 3 times, after taking the meansigma methodss of t/c area ratio, generate a standard curve (range of linearity 0.15-9 μ g/ml) with concentration.
Standard curve determination result is as shown in the table, and standard curve is as shown in Figure 3.
Linearly
| x1 | x2 | x3 | x4 | x5 | |
| 0.150 | 2.187 | 4.566 | 6.876 | 8.986 | |
| 0.160 | 2.217 | 4.068 | 6.414 | 8.372 | |
| 0.159 | 2.166 | 3.992 | 7.595 | 8.412 | |
| Meansigma methodss | 0.156 | 2.190 | 4.209 | 6.962 | 8.590 |
| sd | 0.006 | 0.026 | 0.312 | 0.595 | 0.344 |
| cv | 3.52% | 1.17% | 7.41% | 8.55% | 4.00% |
| Theoretical value | Meansigma methodss | ||||
| x1 | 0.150 | 0.156 | |||
| x2 | 2.363 | 2.190 | |||
| x3 | 4.575 | 4.209 | |||
| x4 | 6.788 | 6.962 | |||
| x5 | 9.000 | 8.590 |
3 sample duplicate detection in the range of line taking 15 times, its coefficient of variation (cv%) should be not higher than 15.0%, to verify detection
The repeatability of card:
Collect clinical sample 200, with German Roche (roche) medical diagnostic prods d- dimer detection kit (immunity ratio
Turbid method) carry out comparison and detection, pattern detection value is as shown in Figure 4 it is seen that as r > 0.975 when it was demonstrated that with German Roche (roche)
The testing result of medical diagnostic prods is equal to, and has same effectiveness.
(2) dispersibility with regard to fluorescent latex microsphere and springing up property
After the completion of the chromatography of detection card, it is coated the fluorescence signal in the detection section 21 of pad using instrument collection, it will detect section edge
Length direction is bisected into 300 sections, and every section is the unit output signal of telecommunication (fluorescence intensity).Amount to and export 300 signals of telecommunication, such as Fig. 5
Shown.
In Fig. 5, vertical coordinate: fluonescence intensity luminous intensity；Abscissa: the position of detection section 21 is long
Degree (detection section 21 is 0 near a side of the second linkage section 22)；What abscissa 50-100 surveyed is nature controlling line 7, and value is 50-
The peak area of 100 this sections.What abscissa 200-250 surveyed is detection line 5, and value is the peak area of this section of 200-250.
As can be seen from Figure 5, the fluorescent latex microsphere of surface active easily forms monodisperse status, improves springing up property of microsphere
Energy.When the detection line of blood movement to antigen, the complex of determinand and reagent just can sufficiently be specifically bound
(200-250 section peak value).Peak is clear, and in detection section 21, in addition to detection line with nature controlling line, unstressed configuration microsphere remains.
The present embodiment be obtained detection card, detection sensitivity height, high specificity, luminous efficiency height and being capable of quantitative response blood
The concentration of detectable substance in liquid.
Embodiment 6
The fluorescence immune chromatography detection card of the label working solution preparation being obtained using embodiment 2
The present embodiment fluorescence immune chromatography detection card similar to Example 5, difference be step 1) and 2) in use embodiment 2
The label working solution obtaining replaces the label working solution that embodiment 1 obtains.
The dispersibility and springing up property of fluorescent latex microsphere is detected, method is shown in Fig. 6, this enforcement with embodiment 5 result
The fluorescent latex microsphere having the effect that surface active of the fluorescence immune chromatography detection card that example is obtained easily forms single dispersing shape
State, improves microsphere and springs up performance.When the detection line of blood movement to antigen, the complex of determinand and reagent just can be carried out
Sufficiently specific binding (200-250 section peak value).Peak is clear, in detection section 21 in addition to detection line with nature controlling line unstressed configuration
Microsphere remains.
The present embodiment be obtained detection card, detection sensitivity height, high specificity, luminous efficiency height and being capable of quantitative response blood
The concentration of detectable substance in liquid.
Embodiment 7
The fluorescence immune chromatography detection card of the label working solution preparation being obtained using embodiment 3
The present embodiment fluorescence immune chromatography detection card similar to Example 5, difference be step 1) and 2) in use embodiment 3
The label working solution obtaining replaces the label working solution that embodiment 1 obtains.
The dispersibility and springing up property of fluorescent latex microsphere is detected, with embodiment 5, result is shown in Fig. 7, this enforcement to method
The fluorescent latex microsphere having the effect that surface active of the fluorescence immune chromatography detection card that example is obtained easily forms single dispersing shape
State, improves microsphere and springs up performance.When the detection line of blood movement to antigen, the complex of determinand and reagent just can be carried out
Sufficiently specific binding (200-250 section peak value).Peak is clear, in detection section 21 in addition to detection line with nature controlling line unstressed configuration
Microsphere remains.
The present embodiment be obtained detection card, detection sensitivity height, high specificity, luminous efficiency height and being capable of quantitative response blood
The concentration of detectable substance in liquid.
Embodiment 8
The fluorescence immune chromatography detection card of the label working solution preparation being obtained using embodiment 4
The present embodiment fluorescence immune chromatography detection card similar to Example 5, difference be step 1) and 2) in use embodiment 4
The label working solution obtaining replaces the label working solution that embodiment 1 obtains.
The dispersibility and springing up property of fluorescent latex microsphere is detected, with embodiment 5, result is shown in Fig. 8, this enforcement to method
The fluorescent latex microsphere having the effect that surface active of the fluorescence immune chromatography detection card that example is obtained easily forms single dispersing shape
State, improves microsphere and springs up performance.When the detection line of blood movement to antigen, the complex of determinand and reagent just can be carried out
Sufficiently specific binding (200-250 section peak value).Peak is clear, in detection section 21 in addition to detection line with nature controlling line unstressed configuration
Microsphere remains.
The present embodiment be obtained detection card, detection sensitivity height, high specificity, luminous efficiency height and being capable of quantitative response blood
The concentration of detectable substance in liquid.
Comparative example
The fluorescence immune chromatography detection card of this comparative example is similar to Example 5, and difference is step 1) and 2) in using commercially available
Aminopolystyrene fluorescent microsphere dispersion liquid replaces the label working solution that embodiment 1 obtains.
, with embodiment 1, testing result is shown in Fig. 9, as can be seen from Figure 9: 1. unactivated micro- for dispersibility and springing up property detection method
Ball, climbs film effect poor.Latex beadses easily condense, and simultaneously because of its hydrophobicity, easily non-specific adsorption occur, cause granule coalescence.
2., in the chromatography process in detection, the fluorescent microsphere of powerful adsorptivity conglomerate is difficult to re-form mono-dispersion microballoon layer
Analysis.3. it is porous network structure film it is thus possible to be unfavorable for chromatographing the non-of detection with larger particles because being coated pad (nc film)
Specific adsorption and lead to bulky grain fluorescent microsphere release property poor.4. before cause nc film (between detection line the 5 to the first linkage section 20
Detection section 21) there are substantially a large amount of fluorescent microspheres residuals at end.200-300 section in Fig. 9, test peak is difficult to differentiate.
Embodiment described above is only the preferred embodiment lifted for absolutely proving the present invention, the protection model of the present invention
Enclose not limited to this.Equivalent substitute or conversion that those skilled in the art are made on the basis of the present invention, all in the present invention
Protection domain within.Protection scope of the present invention is defined by claims.
Claims (10)
1. a kind of d- dimer fluorescence immune chromatography detection card with the preparation method of activation fluorescent latex microsphere it is characterised in that
Comprise the steps:
1) take surfactant to add in the buffer solution that ph is 8 ~ 10, add dimethylformamide, n, n '-dicyclohexyl carbon
Diimine and n- N-Hydroxysuccinimide；
2) take the dispersion liquid of the fluorescent latex microsphere of amino surface, adjusted after ph to 8 ~ 10 with buffer solution, be added to step 1) institute
In the mixture obtaining, stirring reaction, after completion of the reaction, centrifugation removes supernatant, obtains activating fluorescent latex microsphere；
Wherein, described surfactant is the mixing of dodecyl alanine salt, n- dodecyl alanine and stearate
Thing, the weight of three is than for 0.5 ~ 3:0.5 ~ 3:1.
2. preparation method according to claim 1 is it is characterised in that described stearate is stearic potassium, sodium, ammonium salt
And one of tri ethanol ammonium salt or its two or more combination.
3. preparation method according to claim 2 is it is characterised in that described surfactant is dodecyl alanine
The mixture of salt, n- dodecyl alanine and stearate, the weight of three is than for 1.5:1.5:1.
4. preparation method according to claim 1 it is characterised in that described surfactant and described amino surface glimmering
The mass ratio of light latex beadses is 500 ~ 2000:1.
5. preparation method according to claim 1 is it is characterised in that step 1) and step 2) described in buffer solution be
Carbonic acid buffer；Step 2) in the stirring reaction time be 2 ~ 4 hours, temperature be 20 ~ 30 DEG C；The fluorescent latex of amino surface is micro-
Ball is aminopolystyrene fluorescent microsphere.
6. the d- dimer fluorescence immune chromatography detection card activation that the preparation method described in any one of claim 1 ~ 5 obtains is glimmering
Light latex beadses.
7. the label work of activation fluorescent latex microsphere of the d- dimer fluorescence immune chromatography detection card described in claim 6
The preparation method of liquid is it is characterised in that take d- dimer fluorescence immune chromatography detection card activation fluorescent latex microsphere to be scattered in
In microsphere buffer, obtain label working solution；The shared ratio in label working solution of the fluorescent latex microsphere of surface active
Example is 0.5 ~ 2wt%；
Wherein, the preparation method of microsphere buffer is: by bsa, biological preservative, Lei meter Bang a and glycine betaine be dissolved in 0.01m,
In the phosphate buffer of ph7.4.
8. preparation method according to claim 7 is it is characterised in that bsa, biological preservative, Lei meter Bang a and glycine betaine
Concentration in described microsphere buffer is 0.1wt%.
The preparation method of the labeling pad of 9.d- dimer fluorescence immune chromatography detection card is it is characterised in that comprise the steps:
1) preparation of the rabbit igg of activation fluorescent latex microsphere labelling: take the labelling that preparation method described in claim 7 or 8 obtains
Thing working solution, centrifugation, after abandoning supernatant, redissolved with labelling buffer, and be simultaneously introduced carbodiimide and rabbit igg, stirring is anti-
Should, it is then centrifuged for, abandons supernatant, finally use labelling diluent to redissolve；
2) labelling of the activation fluorescent latex microsphere labelling preparation method of d- dimer monoclonal antibody: take claim 7 or 8
The label working solution that described preparation method obtains, centrifugation, redissolved with labelling buffer after abandoning supernatant, and be simultaneously introduced carbon
Diimine and labelling d- dimer monoclonal antibody, stirring reaction, are then centrifuged for, abandon supernatant, finally use labelling diluent
Redissolve；
3) step 1) gained dispersion liquid and step 2 are taken) gained dispersion liquid, mixing, it is sprayed on glass fibre, dry；
The formula of described labelling buffer is: sodium carbonate 4.33g, sodium bicarbonate 2.96g, is dissolved in 1000ml water；Described labelling
The formula of diluent is: trisodium citrate 7.33g, citric acid 4.44g, sodium hydroxide 1g, is dissolved in 1000ml water.
10. a kind of d- dimer fluorescence immune chromatography detection card it is characterised in that include the labeling pad described in claim 9,
It is coated pad and absorption pad, wherein said labeling pad is overlapped on the one end being coated pad, and absorption pad is overlapped in and is coated the another of pad
On end, bag is covered with and is provided with nature controlling line and detection line, nature controlling line is coated with the antibody of goat-anti rabbit igg, detection line is coated with
It is coated with d- dimer monoclonal antibody.
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Address after: 101111 Four Floor F4E of Building No. 2, Kechuang East Five Street, Tongzhou District, Beijing Patentee after: Beijing Huaketai Biotechnology Co., Ltd. Address before: 100070 Room 501, 2 building, 8 Haiying Road, Science City, Fengtai District, Beijing (Park) Patentee before: Beijing Elcoteq Bio-Technology Co., Ltd. |
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