CN106405070B - Procalcitonin fluorescence immune chromatography detection card activation fluorescent latex microballoon - Google Patents
Procalcitonin fluorescence immune chromatography detection card activation fluorescent latex microballoon Download PDFInfo
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- CN106405070B CN106405070B CN201610804732.5A CN201610804732A CN106405070B CN 106405070 B CN106405070 B CN 106405070B CN 201610804732 A CN201610804732 A CN 201610804732A CN 106405070 B CN106405070 B CN 106405070B
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Abstract
The invention discloses a kind of Procalcitonin fluorescence immune chromatography to detect card activation fluorescent latex microballoon and application.A kind of Procalcitonin fluorescence immune chromatography detection card preparation method of activation fluorescent latex microballoon of the present invention, comprises the following steps：1）Take surfactant to add in the cushioning liquid that pH is 8 ~ 10, add dimethylformamide, N, N ' dicyclohexylcarbodiimides and N HOSu NHSs；2）The dispersion liquid of the fluorescent latex microballoon of amino surface is taken, after adjusting pH to 8 ~ 10 with cushioning liquid, is added to step 1）In the mixture of gained, stirring reaction, after completion of the reaction, centrifugation remove supernatant, obtain the fluorescent latex microballoon of surface active；Wherein, the surfactant is the mixture of cithrol and stearate, and the weight ratio of the two is 0.5 ~ 5:1.
Description
Technical field
The invention belongs to fluorescence immune chromatography technical field, and in particular to a kind of Procalcitonin fluorescence immune chromatography detection card
Activation fluorescent latex microballoon and its preparation and application.
Background technology
Fluorescence immune chromatography detection card（Reagent card）Because of its quickly and easily characteristic, fast inspection field is widely used in.Determinand
Antibody labeling fluorescent latex microballoon, there is surface electronic repulsion and Van der Waals force in the liquid phase to cause in liquid phase for latex beads
Latex particle keeps being uniformly distributed.But as solution is evaporated dry, surface tension after being sprayed at glass fibre element film and drying
It is unlimited close to causing intermolecular repulsion to disappear between promotion latex particle, Van der Waals force increase, finally cause multiple latex micels
It is polymerized to big and small different particle.When sample redissolution latex beads is added when immunochromatography, latex beads no longer uniformly divides
Dissipating causes chromatography to fail.Therefore this area uses fluorescence immune chromatography kit more, and card containing detection adds sample buffer.Increase
Detection complexity.Or due to latex beads partial agglomeration so that detection sensitivity reduces, and false negative occurs in testing result.
In addition, latex beads is unstable, it can also make it that the stability of fluoroscopic examination card is poor, be not easy to preserve and transport.
Procalcitonin（PCT）The prohormone being made up of 116 amino acid, molecular weight are about 12.7kD.PCT is by nerve
Endocrine cell（C cells including thyroid gland, lung and pancreatic tissue）Expression, is decomposed into through digestion（Prematurity）Calcitonin, carboxylic
Cardinal extremity peptide and amino terminal peptide.Only contain a small amount of PCT in Healthy People blood.PCT can be significantly raised after bacterium infection.Animal model tries
When testing display body generation septicopyemia, more tissues can express PCT.
The horizontal rises of PCT see bacillary septicopyemia, especially severe septicopyemia and infectious shock.PCT can make
For the prognostic indicator of septicopyemia, and the reliability index of acute critical pancreatitis and its major complications.Obtained for community
Property respiratory tract infection and air-conditioning inductivity patients with pneumonia, the index that PCT can select as antibiotic and curative effect judges.
The content of the invention
The technical problems to be solved by the invention are：Procalcitonin（PCT）The fluorescent latex of fluorescence immune chromatography detection card
Microballoon after the drying, is easily reunited, scattered uneven, so as to cause chromatography failure or cause detection sensitivity to reduce.
In order to solve the above-mentioned technical problem, the invention provides a kind of work of Procalcitonin fluorescence immune chromatography detection card
Change fluorescent latex microballoon and its preparation, the Procalcitonin dry type fluorescence immune chromatography prepared using the activation fluorescent latex microballoon is examined
Survey card, content of this detection card available for Procalcitonin in Quantitative in vitro measure human serum, blood plasma or whole blood.The detection of the present invention
Card can solve the phenomenon that fluorescent latex microballoon is reunited in spraying, drying glass fibre element film, to prepare label
Pad, so as to obtain dry type fluorescence immune chromatography detection card, detection kit is configured without dilution.
The preparation method of Procalcitonin fluorescence immune chromatography detection card activation fluorescent latex microballoon provided by the invention, bag
Include following steps：
1）Take surfactant to add in the cushioning liquid that pH is 8 ~ 10, add dimethylformamide, N, the hexamethylenes of N '-two
Base carbodiimide and n-hydroxysuccinimide；
2）The dispersion liquid of the fluorescent latex microballoon of amino surface is taken, after adjusting pH to 8 ~ 10 with cushioning liquid, is added to step
1）In the mixture of gained, stirring reaction, after completion of the reaction, centrifugation remove supernatant, obtain activating fluorescent latex microballoon；
Wherein, the surfactant is the mixture of cithrol and stearate, the weight ratio of the two
For 0.5 ~ 5:1.
Preferably, cithrol is polyethylene glycol stearate；The stearate be stearic potassium,
One kind or its two or more combination in sodium, ammonium salt and tri ethanol ammonium salt.
It is highly preferred that the surfactant is polyethylene glycol stearate and the mixture of odium stearate, the weight of the two
Amount is than being 3:1.
Preferably, the mass ratio of the surfactant and the fluorescent latex microballoon of the amino surface is 500 ~ 2000:
1。
Preferably, step 1）With step 2）Described in cushioning liquid be carbonic acid buffer；Step 2）In stirring reaction
Time is 2 ~ 4 hours, and temperature is 20 ~ 30 DEG C；The fluorescent latex microballoon 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 beads.
The present invention also provides the mark of above-mentioned Procalcitonin fluorescence immune chromatography detection card activation fluorescent latex microballoon
The preparation method of thing working solution：Take Procalcitonin fluorescence immune chromatography detection card to be scattered in microballoon with activation fluorescent latex microballoon to delay
In fliud flushing, label working solution is obtained；The fluorescent latex microballoon of surface active ratio shared in label working solution is 0.5
~2wt%；
Wherein, the preparation method of microballoon buffer solution is：BSA, biological preservative, Lamepon A and glycine betaine are dissolved in
In 0.01M, pH7.4 phosphate buffer.
Preferably, BSA（Bovine serum albumin）, biological preservative, Lamepon A and glycine betaine be in the microballoon buffer solution
Concentration be 0.1wt%.
The present invention also provides the preparation method of the labeling pad of Procalcitonin fluorescence immune chromatography detection card, including following step
Suddenly:
1）Activate the preparation of the rabbit igg of fluorescent latex microballoon mark：The label working solution that above-mentioned preparation method obtains is taken,
Centrifugation, after abandoning supernatant, redissolved with mark buffer solution, while adding carbodiimide and rabbit igg, stirring reaction, Ran Houli
The heart, supernatant is abandoned, finally redissolved with mark dilution；
2）Activate the preparation method of the mark Procalcitonin monoclonal antibody of fluorescent latex microballoon mark：Take above-mentioned preparation
The label working solution that method obtains, centrifugation, abandon after supernatant and redissolved with mark buffer solution, while add carbodiimide and
Mark Procalcitonin monoclonal antibody, stirring reaction, is then centrifuged for, and abandons supernatant, is finally redissolved with mark dilution；
3）Take step 1）Gained dispersion liquid and step 2）Gained dispersion liquid, mixing, is sprayed on glass fibre, dries；
It is described mark buffer solution formula be：Sodium carbonate 4.33g, sodium acid carbonate 2.96g, are dissolved in 1000mL water；It is described
Mark dilution formula be：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, including above-mentioned labeling pad, coating pad and
Absorption pad, wherein the labeling pad is overlapped on one end of coating pad, absorption pad is overlapped on the other end of coating pad, coating
Pad is provided with nature controlling line and detection line, and the antibody of goat anti-rabbit igg is coated with nature controlling line, and coating drop calcium is coated with detection line
The former monoclonal antibody of element.
The present invention can reach following technique effect：
1st, present invention research finds that the fluorescent latex microballoon through the activation of specific surfactant keeps particle in drying
Between relative distance be not easy to reunite.During chromatography process, adding sample can at once redissolve and smoothly chromatograph, to prepare dry type fluorescence
Immunochromatographydetection detection card（Reagent card）, it is user-friendly, 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 it is micro-
The specific surfactant combination of ball.Handle the fluorescent latex microballoon of chemical modification in advance using specific surfactant, locate
Latex beads mark Procalcitonin antibody after reason, for preparing the labeling pad of Procalcitonin fluorescence immune chromatography detection card,
Realize latex beads even application, soilless sticking phenomenon.Also, after sample to be tested is added, the fluorescent latex microballoon of mark can be with
Quickly, chromatography is smoothly realized, without special sample dilution or buffer solution processing detection card.
3rd, the Procalcitonin dry type fluorescence immune chromatography detection card prepared by the present invention, stability is strong, easy to operate, user
Close friend, testing result is accurate, reliable, high sensitivity.
Brief description of the drawings
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 samples.
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.
Embodiment
The invention will be further described with specific embodiment below in conjunction with the accompanying drawings, so that those skilled in the art can be with
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 to detect card activation fluorescent latex microballoon, including as follows
Step：
1）Take surfactant to add in the cushioning liquid that pH is 8 ~ 10, add dimethylformamide, N, N '-dicyclohexyl
Carbodiimide and n-hydroxysuccinimide, stirring reaction；
2）The dispersion liquid of the fluorescent latex microballoon of amino surface is taken, after adjusting pH to 8 ~ 10 with cushioning liquid, is added to step
1）In the mixture of gained, stirring reaction, after completion of the reaction, centrifugation remove supernatant, obtain activating fluorescent latex microballoon；
Wherein, the surfactant is the mixture of cithrol and stearate, the weight ratio of the two
For 0.5 ~ 5:1.
The present invention modifies breast using the mixture of cithrol and stearate by the method for covalent coupling
Glue microballoon outer surface, makes it have hydrophobicity.Procalcitonin antibody is marked using the activation fluorescent latex microballoon after processing.Fluorescence
The rabbit igg of the Procalcitonin antibody of latex beads mark and fluorescent latex microballoon mark is sprayed on glass fibre element film after mixing
Labeling pad 1 is prepared into, is coated with pad 2（Nitrocellulose filter）Close to the upper adsorptive pads of one end of nature controlling line covering, close to detection line
Other end overlay marks thing pad, for preparing dry type fluorescence immune chromatography reagent card, coating calcitonin is coated with detection line
Original antibody.After adding sample to be tested, the Procalcitonin antibody haptoreaction of Procalcitonin and fluorescent latex microballoon mark, 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 microballoon used refers to the polyphenyl second for the amino surface being modified by sulphation in following examples of the present invention
Alkene fluorescent microsphere, diameter 100-500nm.
First, the preparation of fluorescent latex microballoon is activated
Embodiment 1
, the amino acid type surfactant is sodium lauroyl glutamate；The stearate is odium stearate；It is described poly-
Glycol fatty acid ester is polyethylene glycol monolaurate.
1）Take surfactant（4 mg sodium lauroyl glutamates, 4mg polyethylene glycol monolaurates, 2mg odium stearate）
It is initially dissolved in 0.5M pH9.6 carbonic acid buffer（H values are to 9.6）, add in 0.1ml DMF, in 0.1ml DMF,
Add N, N '-dicyclohexylcarbodiimide (DCC) 1mg and 0.6mg n-hydroxysuccinimides（NHS）After to be stirred at room temperature 3 small
When.
2）By the fluorescent latex microspheres solution 0.5M pH9.6 of amino surfaces of the 1ml containing 1% solids carbonic acid buffer
PH value is adjusted to being added to step 1 after 9.6）In, continue stirring reaction 3 hours.Both the latex beads that must have been activated.
3）Fluorescent latex microballoon reaction solution is centrifuged at a high speed with supercentrifuge, removes reaction solution supernatant, is used
1mL microballoons buffer solution redissolves fluorescent latex microballoon, forms label working solution.
Microballoon phosphate buffer is prepared：0.01M pH7.4 phosphate buffer, BSA containing 0.1wt%, given birth to containing 0.1 wt %
Thing preservative, containing 0.1 wt % Lamepon As, containing 0.1 wt % glycine betaines.Microballoon phosphate buffer provides certain Ionic strength
And pH value, make microballoon dispersed.
Embodiment 2
The operating method of the present embodiment is similar to Example 1, and difference is, step 1）Middle surfactant is：5
Mg sodium lauroyl glutamates, 1mg polyethylene glycol monolaurates, 3mg odium stearate.
Embodiment 3
The operating method 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 monolaurates, 0.5mg potassium stearates.
Embodiment 4
The operating method of the present embodiment is similar to Example 1, and difference is, step 1）Middle surfactant is：4
Mg dodecyl dimethylene amino sodium diformates, 4mg polyethylene glycol monolaurates, 2mg odium stearate.
2nd, the activation fluorescent latex microballoon prepared by taking Procalcitonin fluorescence immune chromatography detection card as an example to embodiment 1 ~ 3
Application and effect illustrate.
Embodiment 5
Fluorescence immune chromatography prepared by the label working solution obtained using embodiment 1 detects card
1）The rabbit igg of fluorescent latex microballoon mark：Take the label working solution that 5mL embodiments 1 obtain, with centrifuge from
Heart 30min（Rotating speed is 10000r/min）, supernatant is abandoned after having centrifuged, is redissolved with 5mL mark buffer solutions, adds 1mg rabbit
IgG is mixed, another to add 5mg carbodiimides, and reaction 1h is stirred at room temperature, is then centrifuged for 15min（Rotating speed is 10000r/min）, abandon
Supernatant, redissolved with 10mL label diluted standby after mixing.
2）The mark Procalcitonin monoclonal antibody of fluorescent latex microballoon mark
The label working solution that 10mL embodiments 1 obtain is taken, with centrifuge 30min（Rotating speed is 10000r/min）,
Supernatant is abandoned after having centrifuged, is redissolved with 10mL mark buffer solutions, the mark for adding 2mg is mixed with Procalcitonin monoclonal antibody
It is even, it is another to add 10mg carbodiimides, reaction 1h is stirred at room temperature, is then centrifuged for 15min（Rotating speed is 10000r/min）, abandon supernatant
Liquid, redissolved with 20mL label diluted standby after mixing.
Step 1）With 2）It is middle mark buffer solution formula be：Sodium carbonate 4.33g, sodium acid carbonate 2.96g, are dissolved in 1000mL water
In；Mark dilution formula be：Trisodium citrate 7.33g, citric acid 4.44g, sodium hydroxide 1g, are dissolved in 1000mL water.
3）It is prepared by labeling pad
Step 2）Mark the determinand antibody and step 1 of obtained fluorescent latex microballoon mark）Obtained fluorescent latex
The rabbit igg of microballoon mark mixes, and the two volume ratio is 2:1.Then by the fluorescent latex microballoon of mixing by 1 microlitre/centimetre
The drying baker for measuring even application 45 DEG C of transposition on glass fibre element paper dries the preparation for completing labeling pad in 2 hours.
4）It is prepared by coating buffer：Take coating Procalcitonin monoclonal antibody to add in phosphate buffer, detection line is made
Coating buffer；Take goat anti-rabbit igg to add in phosphate buffer, nature controlling line coating buffer is made.The formula of the phosphate buffer is：
0.99g sodium dihydrogen phosphates, 5.16g disodium hydrogen phosphates are dissolved in 1000mL purified waters.
5）It is coated with the processing of pad：Detection line coating buffer is covered with line coating in bag and forms detection line, nature controlling line coating buffer
Nature controlling line is formed being covered with line coating in bag, is then dried.
6）Detection card assembling：
By dried labeling pad and the absorption pad cut out（Blotting paper）, padded by coating（Nitrocellulose filter）It is close
The upper absorption pad of one end covering of nature controlling line, the requirement close to the other end overlay marks thing pad of detection line carry out joint strip.By slitting
Machine standard practice instructions is operated, and is cut into the wide detection cards of 4mm ± 0.1mm.
As illustrated in fig. 1 and 2, detection card includes：It is coated with pad 2（Nitrocellulose filter）, its both ends is respectively provided with a linkage section（Such as
First linkage section 20 shown in figure, the second linkage section 22）, two linkage sections centre is detection section 21, and detection section 21 surface, which is provided with, to be detected
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 of coating 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, length of the label along coating pad length direction is 5mm）Spraying mark
Remember thing 8（The mark Procalcitonin monoclonal of rabbit igg containing fluorescent latex microballoon mark and fluorescent latex microballoon mark resists
Body）.
Absorption pad 3（Blotting paper）, its one end is provided with linkage section 30, and the linkage section 30 of absorption pad covers and is fixed on coating pad
On 2 the second linkage section 22；
Bottom plate 4, coating pad 2, labeling pad 1 and absorption pad 3 are both secured on bottom plate.
This detection blocks when in use with one plant of Procalcitonin monoclonal antibody（Coating Procalcitonin monoclonal antibody）
Rule and be coated with nitrocellulose filter, as p-wire, resist coating of being rule on nitrocellulose filter that matter is made with goat-anti rabbit more
Control line.Another plant of Procalcitonin monoclonal antibody that latex fluorescent microsphere is marked（Mark Procalcitonin monoclonal antibody）With
The rabbit igg of latex fluorescent microsphere mark is sprayed on glass fibre element film after mixing and is prepared into labeling pad.Nitrocellulose filter
Close to the upper adsorptive pads of one end of nature controlling line covering, close to the other end overlay marks thing pad of p-wire.Added on toward labeling pad
Standard items or blood sample to be measured, antigen will with label hybrid reaction and being chromatographed along nitrocellulose filter, respectively with p-wire
Reacted with nature controlling line.When test result is effective, nature controlling line shows certain luminous intensity.At this moment the light signal strength ratio on p-wire
The ratio of nature controlling line light signal strength（T/C) it can be drawn by standard curve calculating into positive correlation with concentration of specimens and treat test sample
Product concentration.
（One）The measure of the obtained detection card calibration curve of the present embodiment and sample to be tested
50ul calibration objects or sample to be tested are slowly added dropwise in labeling pad.Stand 15 minutes instead at ambient temperature
Should, detection card is put into Savant-100 fluorescence immune chromatography analyzers and detected by reaction after terminating.Calibration object it is each dense
Degree point repeat does 3 times, take T/C areas than average value after with concentration generate a standard curve（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 | |
| Average value | 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 | Average value | ||||
| x1 | 0.17 | 0.17 | |||
| x2 | 0.55 | 0.57 | |||
| x3 | 2.5 | 2.62 | |||
| x4 | 10 | 9.30 | |||
| x5 | 50 | 52.75 |
3 samples in the range of line taking repeat detection 15 times, its coefficient of variation（CV%）15.0% should be not higher than, to verify
Detect the repeatability of card：
Clinical sample 200 is collected, with German Roche（ROCHE）Medical diagnostic prods Procalcitonin detection kit（Electricity
Chemoluminescence method）Contrasting detection is carried out, pattern detection value is as shown in Figure 4, it is seen that works as r>When 0.975, it was demonstrated that with German Roche
（ROCHE）The testing result of medical diagnostic prods is equal, and has same validity.
（Two）On the dispersiveness of fluorescent latex microballoon and springing up property
After the completion of the chromatography for detecting card, the fluorescence signal in the detection section 21 of coating pad is gathered using instrument, it will detection
Section is bisected into 300 sections along its length, and every section is that unit exports electric signal（Fluorescence intensity）.300 electric signals are exported altogether, such as
Shown in Fig. 5.
In Fig. 5, ordinate：Fluonescence Intensity luminous intensities；Abscissa：Detect the position length of section 21
Degree（It is 0 that section 21, which is detected, close to a side of the second linkage section 22）；What abscissa 50-100 was surveyed is nature controlling line 7, and value is 50-
The peak area of 100 this sections.What abscissa 200-250 was surveyed is detection line 5, and value is the peak area of this sections of 200-250.
As can be seen from Figure 5, the fluorescent latex microballoon of surface active easily forms monodisperse status, improves springing up property of microballoon
Energy.When blood is moved to the detection line of antigen, the compound of determinand and reagent can be sufficiently to be specifically bound
（200-250 section peak values）.Peak is clear, and unstressed configuration microballoon remains in addition to detection line and nature controlling line in detection section 21.
The obtained detection card of the present embodiment, detection sensitivity height, high specificity, luminous efficiency are high and being capable of quantitative reaction blood
The concentration of detectable substance in liquid.
Embodiment 6
Fluorescence immune chromatography prepared by the label working solution obtained using embodiment 2 detects card
The fluorescence immune chromatography detection card of the present embodiment is similar to Example 5, and difference is step 1）With 2）It is middle to use in fact
Apply the label working solution that the label working solution substitution embodiment 1 that example 2 obtains obtains.
The dispersiveness of fluorescent latex microballoon and springing up property are detected, method is shown in Fig. 6, this implementation with the result of embodiment 5
Fluorescence immune chromatography detection card has the effect that made from example：The fluorescent latex microballoon of surface active easily forms single dispersing shape
State, improve microballoon and spring up performance.When blood is moved to the detection line of antigen, the compound of determinand and reagent can be with progress
Sufficiently specific binding（200-250 section peak values）.Peak is clear, detects in section 21 unstressed configuration in addition to detection line and nature controlling line
Microballoon remains.
The obtained detection card of the present embodiment, detection sensitivity height, high specificity, luminous efficiency are high and being capable of quantitative reaction blood
The concentration of detectable substance in liquid.
Embodiment 7
Fluorescence immune chromatography prepared by the label working solution obtained using embodiment 3 detects card
The fluorescence immune chromatography detection card of the present embodiment is similar to Example 5, and difference is step 1）With 2）It is middle to use in fact
Apply the label working solution that the label working solution substitution embodiment 1 that example 3 obtains obtains.
The dispersiveness of fluorescent latex microballoon and springing up property are detected, as a result method is shown in Fig. 7, this implementation with embodiment 5
Fluorescence immune chromatography detection card has the effect that made from example：The fluorescent latex microballoon of surface active easily forms single dispersing shape
State, improve microballoon and spring up performance.When blood is moved to the detection line of antigen, the compound of determinand and reagent can be with progress
Sufficiently specific binding（200-250 section peak values）.Peak is clear, detects in section 21 unstressed configuration in addition to detection line and nature controlling line
Microballoon remains.
The obtained detection card of the present embodiment, detection sensitivity height, high specificity, luminous efficiency are high and being capable of quantitative reaction blood
The concentration of detectable substance in liquid.
Embodiment 8
Fluorescence immune chromatography prepared by the label working solution obtained using embodiment 4 detects card
The fluorescence immune chromatography detection card of the present embodiment is similar to Example 5, and difference is step 1）With 2）It is middle to use in fact
Apply the label working solution that the label working solution substitution embodiment 1 that example 4 obtains obtains.
The dispersiveness of fluorescent latex microballoon and springing up property are detected, as a result method is shown in Fig. 8, this implementation with embodiment 5
Fluorescence immune chromatography detection card has the effect that made from example：The fluorescent latex microballoon of surface active easily forms single dispersing shape
State, improve microballoon and spring up performance.When blood is moved to the detection line of antigen, the compound of determinand and reagent can be with progress
Sufficiently specific binding（200-250 section peak values）.Peak is clear, detects in section 21 unstressed configuration in addition to detection line and nature controlling line
Microballoon remains.
The obtained detection card of the present embodiment, detection sensitivity height, high specificity, luminous efficiency are high and being capable of quantitative reaction 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）It is middle to use city
The label working solution that the aminopolystyrene fluorescent microsphere dispersion liquid substitution embodiment 1 sold obtains.
Dispersiveness and springing up property detection method are with embodiment 1, and testing result is shown in Fig. 9, as can be seen from Figure 9：It is 1. unactivated micro-
Ball, it is poor to climb film effect.Latex beads easily condenses, while because of its hydrophobicity, non-specific adsorption easily occurs, causes particle 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 coating pads（NC films）It is porous network structure film, it is thus possible to be unfavorable for the non-of chromatography detection with larger particles
Specific adsorption and cause bulky grain fluorescent microsphere release property poor.4. before causing NC films（Between the linkage section of detection line 5 to the first 20
Detection section 21）There are obvious a large amount of fluorescent microsphere residuals at end.200-300 sections in Fig. 9, test peak are difficult to differentiate.
Embodiment described above is only to absolutely prove preferred embodiment that is of the invention and being lifted, protection model of the invention
Enclose not limited to this.The equivalent substitute or conversion that those skilled in the art are made on the basis of the present invention, in the present invention
Protection domain within.Protection scope of the present invention is defined by claims.
Claims (7)
- A kind of 1. preparation method of Procalcitonin fluorescence immune chromatography detection card activation fluorescent latex microballoon, it is characterised in that Comprise the following steps：1）Take surfactant to add in the carbonic acid buffer that pH is 8 ~ 10, add dimethylformamide, N, N '-dicyclohexyl Carbodiimide and n-hydroxysuccinimide；2）The dispersion liquid of the fluorescent latex microballoon of amino surface is taken, after adjusting pH to 8 ~ 10 with carbonic acid buffer, is added to step 1） In the mixture of gained, stirring reaction 2 ~ 4 hours at 20 ~ 30 DEG C, after completion of the reaction, centrifugation remove supernatant, are activated Fluorescent latex microballoon, the fluorescent latex microballoon of amino surface is aminopolystyrene fluorescent microsphere；Wherein, the surfactant is the mixture of cithrol and stearate, and the weight ratio of the two is 0.5 ~5:1；The mass ratio of the surfactant and the fluorescent latex microballoon of the amino surface is 500 ~ 2000:1.
- 2. preparation method according to claim 1, it is characterised in that cithrol is polyethylene glycol stearic acid Ester；The stearate is one kind or its two or more combination in stearic potassium, sodium, ammonium salt and tri ethanol ammonium salt.
- 3. preparation method according to claim 2, it is characterised in that the surfactant is polyethylene glycol stearate With the mixture of odium stearate, the weight ratio of the two is 3:1.
- 4. the Procalcitonin fluorescence immune chromatography detection card activation that the preparation method described in any one of claim 1 ~ 3 obtains is glimmering Light latex beads.
- 5. the Procalcitonin fluorescence immune chromatography detection card described in claim 4 is worked with the label of activation fluorescent latex microballoon The preparation method of liquid, it is characterised in that take Procalcitonin fluorescence immune chromatography detection card to be scattered in activation fluorescent latex microballoon In microballoon buffer solution, label working solution is obtained；The fluorescent latex microballoon of surface active ratio shared in label working solution Example is 0.5 ~ 2wt%；Wherein, the preparation method of microballoon buffer solution is：By BSA, biological preservative, Lamepon A and glycine betaine be dissolved in 0.01M, In pH7.4 phosphate buffer；The BSA, biological preservative, Lamepon A and glycine betaine are dense in the microballoon buffer solution Degree is 0.1wt%.
- 6. the preparation method of the labeling pad of Procalcitonin fluorescence immune chromatography detection card, it is characterised in that comprise the following steps:1）Activate the preparation of the rabbit igg of fluorescent latex microballoon mark：Take the label work that preparation method described in claim 5 obtains Make liquid, centrifuge, after abandoning supernatant, redissolved with mark buffer solution, while carbodiimide and rabbit igg are added, stirring reaction, so After centrifuge, abandon supernatant, finally with mark dilution redissolve；2）Activate the preparation method of the mark Procalcitonin monoclonal antibody of fluorescent latex microballoon mark：Take claim 5 institute The label working solution that preparation method obtains is stated, is centrifuged, is redissolved after abandoning supernatant with mark buffer solution, while adding carbon two Imines and mark Procalcitonin monoclonal antibody, stirring reaction, are then centrifuged for, abandon supernatant, finally answered with mark dilution It is molten；3）Take step 1）Gained dispersion liquid and step 2）Gained dispersion liquid, mixing, is sprayed on glass fibre, dries；It is described mark buffer solution formula be：Sodium carbonate 4.33g, sodium acid carbonate 2.96g, are dissolved in 1000mL water；The mark The formula of dilution is：Trisodium citrate 7.33g, citric acid 4.44g, sodium hydroxide 1g, are dissolved in 1000mL water.
- 7. a kind of Procalcitonin fluorescence immune chromatography detection card, it is characterised in that be prepared into including the method described in claim 6 Labeling pad, coating pad and the absorption pad arrived, wherein the labeling pad is overlapped on one end of coating pad, absorption pad is overlapped in It is coated with the other end of pad, bag is covered with provided with nature controlling line and detection line, and the antibody of goat anti-rabbit igg, inspection are coated with nature controlling line Procalcitonin monoclonal antibody is coated with survey line.
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| 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 |
| 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 |
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| 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 |
| 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 |
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| 降钙素原荧光免疫层析定量检测方法的建立及性能评估;方琦 等;《中华检验医学杂志》;20121231;第35卷(第12期);摘要，第1103-1104页"材料与方法" * |
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