CN106405070A - Activated fluorescent latex microsphere for procalcitonin fluorescent immunochromatographic test card - Google Patents
Activated fluorescent latex microsphere for procalcitonin fluorescent immunochromatographic test card Download PDFInfo
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Abstract
The invention discloses an activated fluorescent latex microsphere for a procalcitonin fluorescent immunochromatographic test card, and an application thereof. The preparation method of the activated fluorescent latex microsphere for the procalcitonin fluorescent immunochromatographic test card comprises the steps of 1) adding a surfactant into a buffer solution that is 8-10 in pH value, and then adding dimethylformamide, adding N, N'-dicyclohexylcarbodiimide and N-hydroxysuccinimide; 2) adjusting the dispersion liquid of a fluorescent latex microsphere on the amino surface to be 8-10 in pH value, adding the dispersion liquid into a mixture obtained in the step 1), stirring for reaction, centrifuging to remove a supernatant after the completion of the reaction, and obtaining a surface-activated fluorescent latex microsphere. The surfactant is a mixture of polyethylene glycol fatty acid ester and stearate, wherein the weight ratio of polyethylene glycol fatty acid ester to stearate is 0.5-5:1.
Description
Technical field
The invention belongs to fluorescence immune chromatography technical field is and in particular to a kind of detection of Procalcitonin. 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, it 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.
Procalcitonin.（PCT）The prohormone being made up of 116 aminoacid, molecular weight is about 12.7kD.PCT is by nerve
Endocrine cell（C cell including thyroid, lung and pancreatic tissue）Expression, is decomposed into through enzyme action（Immaturity）Calcitonin, carboxylic
Cardinal extremity peptide and amino terminal peptide.A small amount of PCT is contained only in healthy human blood.After bacterium infection, PCT can be significantly raised.Animal model tries
When testing display body generation pyemia, organize more and all can express PCT.
PCT level raises and sees bacillary pyemia, especially serious symptom pyemia and septic shock.PCT can make
For the prognostic indicator of pyemia, it is also the reliability index of acute critical pancreatitis and its major complications.Community is obtained
Property respiratory tract infection and air-conditioning inductivity patients with pneumonia, PCT can select as antibiotic and Outcome measure index.
Content of the invention
The technical problem to be solved is：Procalcitonin.（PCT）The fluorescent latex of fluorescence immune chromatography detection card
Microsphere after the drying, is easily reunited, and 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 Procalcitonin. fluorescence immune chromatography detection card
Change fluorescent latex microsphere and its preparation, apply the Procalcitonin. dry type fluorescence immune chromatography inspection of this activation fluorescent latex microsphere preparation
Survey card, this detection card can be used for the content that Quantitative in vitro measures Procalcitonin. 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 Procalcitonin. 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-hydroxy-succinamide；
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 mixture of cithrol and stearate, and the weight of the two ratio is for 0.5
~5:1.
Preferably, cithrol is polyglycol distearate；Described stearate be stearic potassium,
One of sodium, ammonium salt and tri ethanol ammonium salt or its two or more combination.
It is highly preferred that described surfactant is the mixture of polyglycol distearate and sodium stearate, the weight of the two
Amount ratio is 3:1.
Preferably, described surfactant and the mass ratio of the fluorescent latex microsphere of described amino surface are 500 ~ 2000:
1.
Preferably, step 1）With 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 Procalcitonin. fluorescence immune chromatography detection card activation fluorescence that above-mentioned preparation method obtains
Latex beadses.
The present invention also provides above-mentioned Procalcitonin. fluorescence immune chromatography to detect that card activates the labelling of fluorescent latex microsphere
The preparation method of thing working solution：Take Procalcitonin. fluorescence immune chromatography detection card activation fluorescent latex microsphere to be scattered in microsphere to 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, biological preservative, Lamepon A and glycine betaine be dissolved in 0.01M,
In the phosphate buffer of pH7.4.
Preferably, BSA（Bovine serum albumin）, biological preservative, Lamepon A and glycine betaine be in described microsphere buffer
Concentration be 0.1wt%.
The present invention also provides the preparation method of the labeling pad of Procalcitonin. fluorescence immune chromatography detection card, walks including following
Suddenly:
1）The 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）The preparation method of the labelling Procalcitonin monoclonal antibody of activation fluorescent latex microsphere labelling：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 Procalcitonin monoclonal antibody, stirring reaction, it is then centrifuged for, abandons supernatant, finally use labelling diluent to redissolve；
3）Take step 1）Gained dispersion liquid and step 2）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, are dissolved in 1000mL water；Described labelling
The formula of diluent is：Trisodium citrate 7.33g, citric acid 4.44g, sodium hydroxide 1g, are dissolved in 1000mL water.
The present invention provides a kind of Procalcitonin. 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 fall calcium
The former monoclonal antibody of element.
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）, it is user-friendly to, and testing result is stable, accurate, reliable.
2nd, the present invention selected by testing sieve be suitable for Procalcitonin. 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 Procalcitonin. antibody after reason, for preparing the labeling pad of Procalcitonin. 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 Procalcitonin. 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 Procalcitonin. fluorescence immune chromatography reagent card of the present invention.
Fig. 2 is the overlooking the structure diagram of the Procalcitonin. fluorescence immune chromatography reagent card of the present invention.
Fig. 3 is the foundation of Procalcitonin. standard curve.
Procalcitonin. assay curve in Fig. 4 whole blood sample.
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 Procalcitonin. 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-hydroxy-succinamide, 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 mixture of cithrol and stearate, and the weight of the two ratio is for 0.5
~5:1.
The present invention adopts cithrol and the mixture of stearate to modify breast by the method for covalent coupling
Glue microsphere outer surface is so as to have hydrophobicity.Using the activation fluorescent latex microsphere labelling Procalcitonin. antibody after processing.Fluorescence
It is sprayed on glass fibre element film after the Procalcitonin. antibody of latex beadses labelling and the rabbit igg mixing of fluorescent latex microsphere labelling
It is prepared into labeling pad 1, be coated pad 2（Nitrocellulose filter）Cover upper adsorptive pads near one end of nature controlling line, near detection line
Other end overlay marks thing pad, for preparing dry type fluorescence immune chromatography reagent card, detection line is coated and uses calcitonin
Original antibody.After adding sample to be tested, the Procalcitonin. antibody haptoreaction of Procalcitonin. and fluorescent latex microsphere labelling, latex is micro-
Ball can keep dispersity not reunite, and can be rapid, dispersed after contact measured sample, 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
, described amino acid type surfactant is sodium lauroyl glutamate；Described stearate is sodium stearate；Described poly- second two
Alcohol fatty acid ester is polyethylene glycol monolaurate.
1）Take surfactant（4 mg sodium lauroyl glutamates, 4mg polyethylene glycol monolaurate, 2mg sodium stearate）
It is initially dissolved in the carbonic acid buffer of 0.5M pH9.6（H-number is to 9.6）, add in the DMF of 0.1ml, in the DMF of 0.1ml,
Add N, N '-dicyclohexylcarbodiimide (DCC) 1mg and 0.6mg N-hydroxy-succinamide（NHS）After to be stirred at room temperature 3 little
When.
2）1ml is contained the fluorescent latex microspheres solution carbonic acid buffer of 0.5M pH9.6 of the amino surface of 1% solidss
PH value is adjusted to be added to step 1 to after 9.6）In, 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, BSA containing 0.1wt%, gives birth to containing 0.1 wt %
Thing preservative, containing 0.1 wt % Lamepon A, containing 0.1 wt % glycine betaine.Microsphere phosphate buffer provides certain Ionic strength
And pH value, make microsphere dispersed.
Embodiment 2
The operational approach of the present embodiment is similar to Example 1, and difference is, step 1）Middle surfactant is：The 5 mg months
Osmanthus acyl sodium glutamate, 1mg polyethylene glycol monolaurate, 3mg sodium stearate.
Embodiment 3
The operational approach of the present embodiment is similar to Example 1, and difference is, step 1）Middle surfactant is：6 mg N-
Acyl glutamic acid sodium, 4mg polyethylene glycol monolaurate, 0.5mg potassium stearate.
Embodiment 4
The operational approach of the present embodiment is similar to Example 1, and difference is, step 1）Middle surfactant is：4 mg ten
Dialkyl group dimethylene amino sodium diformate, 4mg polyethylene glycol monolaurate, 2mg sodium stearate.
2nd, the activation fluorescent latex microsphere to embodiment 1 ~ 3 preparation taking Procalcitonin. 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 is stirred at room temperature, is then centrifuged for 15min（Rotating speed is 10000r/min）, abandon
Clear liquid, is redissolved standby after mixing with the label diluted of 10mL.
2）The labelling Procalcitonin monoclonal antibody of fluorescent latex microsphere labelling
Take the label working solution that 10mL embodiment 1 obtains, use centrifuge 30min（Rotating speed is 10000r/min）, centrifugation
Abandon supernatant after complete, redissolved with 10mL labelling buffer, the labelling adding 2mg is mixed with Procalcitonin monoclonal antibody, separately
Add 10mg carbodiimides, reaction 1h is stirred at room temperature, is then centrifuged for 15min（Rotating speed is 10000r/min）, abandon supernatant, use
The label diluted of 20mL is redissolved standby after mixing.
Step 1）With 2）The formula of middle labelling buffer is：Sodium carbonate 4.33g, sodium bicarbonate 2.96g, are dissolved in 1000mL water
In；The formula of labelling diluent is：Trisodium citrate 7.33g, citric acid 4.44g, sodium hydroxide 1g, are dissolved in 1000mL water.
3）Prepared by labeling pad
Step 2）The labelling of the fluorescent latex microsphere labelling obtaining determinand antibody and step 1）The fluorescent latex microsphere obtaining
The rabbit igg of labelling mixes, and the two volume ratio is 2:1.Then will be equal by the amount of 1 microlitre/centimetre for the fluorescent latex microsphere mixing
The even drying baker being sprayed on 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 to be coated and added in phosphate buffer with Procalcitonin monoclonal antibody, make 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 cut out（Absorbent paper）, by being coated pad（Nitrocellulose filter）Near Quality Control
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：It is coated pad 2（Nitrocellulose filter）, it is respectively arranged at the two ends with a linkage section（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 near linkage section 10（Away from linkage section 2mm, label is 5mm along the length being coated pad length direction）Spraying mark
Note thing 8（The labelling Procalcitonin. monoclonal anti of the rabbit igg containing fluorescent latex microsphere labelling and 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 during use with one plant of Procalcitonin monoclonal antibody（It is coated and use Procalcitonin monoclonal antibody）?
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.Another plant of Procalcitonin monoclonal antibody by latex fluorescent microsphere labelling（Labelling Procalcitonin monoclonal antibody）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 of nature controlling line light signal strength（T/C) become positive correlation with concentration of specimens, can be drawn by standard curve calculating and treat test sample
Product concentration.
（One）Detection card calibration curve and the mensure of sample to be tested that the present embodiment is obtained
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 with concentration（Range of linearity 0.1-50 ng/mL）.
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 | |
| 0.17 | 0.57 | 2.53 | 9.03 | 54.98 | |
| 0.18 | 0.61 | 2.57 | 9.33 | 54.56 | |
| 0.17 | 0.54 | 2.76 | 9.55 | 48.71 | |
| Meansigma methodss | 0.17 | 0.57 | 2.62 | 9.30 | 52.75 |
| SD | 0.01 | 0.03 | 0.12 | 0.26 | 3.51 |
| CV | 3.00% | 5.82% | 4.71% | 2.81% | 6.65% |
| Theoretical value | Meansigma methodss | ||||
| x1 | 0.17 | 0.17 | |||
| x2 | 0.55 | 0.57 | |||
| x3 | 2.5 | 2.62 | |||
| x4 | 10 | 9.30 | |||
| x5 | 50 | 52.75 |
3 sample duplicate detection in the range of line taking 15 times, its coefficient of variation（CV%）15.0% should be not higher than, to verify detection
The repeatability of card：
Collect clinical sample 200, with German Roche（ROCHE）Medical diagnostic prods Procalcitonin. detection kit（Electrochemistry
Luminescence method）Carry out comparison and detection, pattern detection value is as shown in Figure 4 it is seen that work as r>When 0.975 it was demonstrated that with German Roche
（ROCHE）The testing result of medical diagnostic prods is equal to, and has same effectiveness.
（Two）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 exports the signal of telecommunication for unit（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 fluorescence immune chromatography detection card of the present embodiment is similar to Example 5, and difference is step 1）With 2）Middle 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
What the fluorescence immune chromatography detection that example is obtained blocked has the effect that：The fluorescent latex microsphere of surface active 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 specifically bind（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 fluorescence immune chromatography detection card of the present embodiment is similar to Example 5, and difference is step 1）With 2）Middle 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
What the fluorescence immune chromatography detection that example is obtained blocked has the effect that：The fluorescent latex microsphere of surface active 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 specifically bind（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 fluorescence immune chromatography detection card of the present embodiment is similar to Example 5, and difference is step 1）With 2）Middle 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
What the fluorescence immune chromatography detection that example is obtained blocked has the effect that：The fluorescent latex microsphere of surface active 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 specifically bind（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）With 2）Middle 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 for dispersibility and springing up property detection method：1. unactivated micro-
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. because being coated pad（NC film）It is porous network structure film it is thus possible to be unfavorable for chromatographing the non-of detection with larger particles
Specific adsorption and lead to bulky grain fluorescent microsphere release property poor.4. before causing NC film（Between detection line the 5 to the first linkage section 20
Detection section 21）There are substantially a large amount of fluorescent microsphere 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 Procalcitonin. 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-hydroxy-succinamide；
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 mixture of cithrol and stearate, and the weight of the two ratio is for 0.5
~5:1.
2. preparation method according to claim 1 is it is characterised in that cithrol is Polyethylene Glycol stearic acid
Ester；Described stearate is one of stearic potassium, sodium, ammonium salt and 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 polyglycol distearate
With the mixture of sodium stearate, the weight of the two is than for 3:1.
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）With 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 Procalcitonin. 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 Procalcitonin. fluorescence immune chromatography detection card described in claim 6
The preparation method of liquid is it is characterised in that take Procalcitonin. 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, Lamepon 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, Lamepon A and glycine betaine
Concentration in described microsphere buffer is 0.1wt%.
9. the preparation method of the labeling pad of Procalcitonin. fluorescence immune chromatography detection card is it is characterised in that comprise the steps:
1）The 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）The preparation method of the labelling Procalcitonin monoclonal antibody of activation fluorescent latex microsphere labelling：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 Procalcitonin monoclonal antibody, stirring reaction, are then centrifuged for, and abandon supernatant, finally use labelling diluent
Redissolve；
3）Take step 1）Gained dispersion liquid and step 2）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, are dissolved in 1000mL water；Described labelling
The formula of diluent is：Trisodium citrate 7.33g, citric acid 4.44g, sodium hydroxide 1g, are dissolved in 1000mL water.
10. a kind of Procalcitonin. 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 and use Procalcitonin monoclonal antibody.
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| CN107037206A (en) * | 2017-03-31 | 2017-08-11 | 深圳市在田翊方科技有限公司 | A kind of time-resolved fluoroimmunoassay chromatography |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1576842A (en) * | 2003-06-30 | 2005-02-09 | 希森美康株式会社 | Sample pretreatment solution for immunological test and method for using the same |
| US20060240438A1 (en) * | 2003-07-28 | 2006-10-26 | Yukio Nagasaki | Surface of base material being inhibited in non-specific adsorption |
| CN102023211A (en) * | 2010-11-19 | 2011-04-20 | 广州万孚生物技术有限公司 | Immunochromatographic test strip for full quantitative detection of C-reactive protein and preparation method thereof |
| CN105693906A (en) * | 2015-01-20 | 2016-06-22 | 于乐 | Zwitterionic polymer microspheres and preparing method thereof |
-
2016
- 2016-09-06 CN CN201610804732.5A patent/CN106405070B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1576842A (en) * | 2003-06-30 | 2005-02-09 | 希森美康株式会社 | Sample pretreatment solution for immunological test and method for using the same |
| US20060240438A1 (en) * | 2003-07-28 | 2006-10-26 | Yukio Nagasaki | Surface of base material being inhibited in non-specific adsorption |
| CN102023211A (en) * | 2010-11-19 | 2011-04-20 | 广州万孚生物技术有限公司 | Immunochromatographic test strip for full quantitative detection of C-reactive protein and preparation method thereof |
| CN105693906A (en) * | 2015-01-20 | 2016-06-22 | 于乐 | Zwitterionic polymer microspheres and preparing method thereof |
Non-Patent Citations (2)
| Title |
|---|
| 张改平: "《免疫层析试纸快速检测技术》", 31 August 2015, 河南科学技术出版社 * |
| 方琦 等: "降钙素原荧光免疫层析定量检测方法的建立及性能评估", 《中华检验医学杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107037206A (en) * | 2017-03-31 | 2017-08-11 | 深圳市在田翊方科技有限公司 | A kind of time-resolved fluoroimmunoassay chromatography |
| CN107037206B (en) * | 2017-03-31 | 2019-03-05 | 深圳市在田翊方科技有限公司 | A kind of time-resolved fluoroimmunoassay chromatography |
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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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