CN106405109B - Ferritin fluorescence immune chromatography detection card activation fluorescent latex microballoon - Google Patents
Ferritin fluorescence immune chromatography detection card activation fluorescent latex microballoon Download PDFInfo
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- CN106405109B CN106405109B CN201610804765.XA CN201610804765A CN106405109B CN 106405109 B CN106405109 B CN 106405109B CN 201610804765 A CN201610804765 A CN 201610804765A CN 106405109 B CN106405109 B CN 106405109B
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Abstract
The invention discloses a kind of ferritin fluorescence immune chromatography to detect card activation fluorescent latex microballoon and application.A kind of ferritin 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 amino acid type surfactant, polyethylene glycol monolaurate and stearate, and the weight ratio of three is 1 ~ 10:1~5:1~10.
Description
Technical field
The invention belongs to fluorescence immune chromatography technical field, and in particular to a kind of ferritin fluorescence immune chromatography detection card is used
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.
Ferritin（Fer）It is a kind of high molecular weight protein, molecular weight is at least up to 440kD（Depending on its interior iron content is how many）.
Serum ferritin is the iron of bonding state in blood.It is the most sensitive index for checking internal asiderosis.The measure of ferritin is
Specify the usability methods of iron metabolism situation.It is that internal iron content is estimated that Ferritin Levels, which are measured, at treatment initial stage
Typical method.Determination of Serum Ferritin is clinically usually used in the diagnosis of hypoferric anemia.Low ferritin levels are simultaneously
With microcytic hypochromic anemia, then asiderosis is prompted.Such as ferritin levels rise simultaneously can exclude iron distribution disorders simultaneously
Possibility, then it represents that internal iron content is too high.
The content of the invention
The technical problems to be solved by the invention are：Ferritin（Fer）The fluorescent latex of fluorescence immune chromatography detection card is micro-
Ball 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 activation of ferritin fluorescence immune chromatography detection card
Fluorescent latex microballoon and its preparation, the ferritin dry type fluorescence immune chromatography prepared using the activation fluorescent latex microballoon detect
Card, content of this detection card available for ferritin in Quantitative in vitro measure human serum, blood plasma or whole blood.The detection card of the present invention can
To solve the phenomenon that fluorescent latex microballoon is reunited in spraying, drying glass fibre element film, to prepare labeling pad, from
And dry type fluorescence immune chromatography detection card is obtained, configure detection kit without dilution.
The preparation method of ferritin fluorescence immune chromatography detection card activation fluorescent latex microballoon provided by the invention, including
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 amino acid type surfactant, polyethylene glycol monolaurate and stearate
Mixture, the weight ratio of three is 1 ~ 10:1~5:1~10.
Preferably, the stearate is one kind in stearic potassium, sodium, ammonium salt and tri ethanol ammonium salt or its two kinds
Combination above；The amino acid type surfactant is dodecyl alanine salt.
Preferably, the surfactant is dodecyl alanine salt, polyethylene glycol monolaurate and stearic acid
The mixture of salt, the weight ratio of three is 2:3:5.
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 ferritin fluorescence immune chromatography detection card activation fluorescence breast that above-mentioned preparation method obtains
Glue microballoon.
The present invention also provides the label of above-mentioned ferritin fluorescence immune chromatography detection card activation fluorescent latex microballoon
The preparation method of working solution：Ferritin fluorescence immune chromatography detection card is taken to be scattered in microballoon buffer solution with activation fluorescent latex microballoon
In, obtain label working solution；The fluorescent latex microballoon of surface active ratio shared in label working solution for 0.5 ~
2wt%；
Wherein, the preparation method of microballoon buffer solution is：By BSA（Bovine serum albumin）, biological preservative, Lamepon A and sweet tea
Dish alkali soluble is in 0.01M, pH7.4 phosphate buffer.
Preferably, the concentration of BSA, biological preservative, Lamepon A and glycine betaine in the microballoon buffer solution is
0.1wt%。
The present invention also provides the preparation method of the labeling pad of ferritin 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 ferritin monoclonal antibody of fluorescent latex microballoon mark：Take above-mentioned preparation side
The label working solution that method obtains, centrifugation, redissolved after abandoning supernatant with mark buffer solution, while adding carbodiimide and mark
Note uses ferritin monoclonal antibody, stirring reaction, is then centrifuged for, 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.
A kind of ferritin fluorescence immune chromatography detection card of present invention offer, including above-mentioned labeling pad, coating pad and suction
Pad is received, 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
Nature controlling line and detection line are provided with, the antibody of goat anti-rabbit igg is coated with nature controlling line, coating ferritin is coated with detection line
Monoclonal antibody.
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 selects the fluorescent latex microballoon for being suitable for ferritin fluorescence immune chromatography detection card by testing sieve
Specific surfactant combination.Handle the fluorescent latex microballoon of chemical modification in advance using specific surfactant, handle
Latex beads mark ferritin antibody afterwards, for preparing the labeling pad of ferritin fluorescence immune chromatography detection card, realizes breast
Glue microballoon even application, soilless sticking phenomenon.Also, after sample to be tested is added, the fluorescent latex microballoon of mark can quickly,
Chromatography is smoothly realized, without special sample dilution or buffer solution processing detection card.
3rd, the ferritin dry type fluorescence immune chromatography detection card prepared by the present invention, stability is strong, easy to operate, Yong Huyou
Good, testing result is accurate, reliable, high sensitivity.
Brief description of the drawings
Fig. 1 is the section decomposition texture schematic diagram of the ferritin fluorescence immune chromatography reagent card of the present invention.
Fig. 2 is the overlooking the structure diagram of the ferritin fluorescence immune chromatography reagent card of the present invention.
Fig. 3 is the foundation of ferritin standard curve.
Ferritin Levels determine 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 ferritin fluorescence immune chromatography to detect card activation fluorescent latex microballoon, including following step
Suddenly：
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 amino acid type surfactant, polyethylene glycol monolaurate and stearate
Mixture, the weight ratio of three is 1 ~ 10:1~5:1~10.
The present invention uses the mixture of amino acid type surfactant, cithrol and stearate（It is cloudy from
Subtype, nonionic and amphoteric surfactant mixing）Latex beads outer surface is modified by the method for covalent coupling, makes its tool
There is hydrophobicity.Ferritin antibody is marked using the activation fluorescent latex microballoon after processing.The ferritin of fluorescent latex microballoon mark
The rabbit igg of antibody and fluorescent latex microballoon mark is sprayed on glass fibre element film after mixing and is prepared into labeling pad 1, coating pad
2（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 detection line, use
In preparing dry type fluorescence immune chromatography reagent card, coating ferritin antibody is coated with detection line.After adding sample to be tested, iron egg
Dispersity can be kept not reunite with the ferritin antibody haptoreaction of fluorescent latex microballoon mark, latex beads in vain, 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
1）Take surfactant（2mg sodium dodecyl aminopropionitriles, 3mg polyethylene glycol monolaurates and 5mg are stearic
Sour sodium）It is initially dissolved in 0.5M pH9.6 carbonic acid buffer（H values are to 9.6）, add in 0.1ml DMF, 0.1ml's
In DMF, N, N '-dicyclohexylcarbodiimide (DCC) 1mg and 0.6mg n-hydroxysuccinimides are added（NHS）Room temperature is stirred afterwards
Mix 3 hours.
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：
0.5mg sodium dodecyl aminopropionitriles, 2.5mg polyethylene glycol monolaurates and 5mg odium stearate.
Embodiment 3
The operating method of the present embodiment is similar to Example 1, and difference is, step 1）Middle surfactant is：5mg
Sodium dodecyl aminopropionitrile, 0.5mg polyethylene glycol monolaurates and 4.5mg odium stearate.
Embodiment 4
The operating method of the present embodiment is similar to Example 1, and difference is, step 1）Middle surfactant is：5mg
Sodium dodecyl aminopropionitrile, 2.5mg polyethylene glycol monolaurates and 0.5mg odium stearate.
2nd, the activation fluorescent latex microballoon prepared so that ferritin fluorescence immune chromatography detects card as an example to embodiment 1 ~ 4
Using 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：The label working solution that 5mL embodiments 1 obtain is taken, uses centrifuge
30min（Rotating speed is 10000r/min）, supernatant is abandoned after having centrifuged, is redissolved with 5mL mark buffer solutions, adds 1mg rabbit igg
Mix, it is another to add 5mg carbodiimides, reaction 1h is stirred at room temperature, is then centrifuged for 15min（Rotating speed is 10000r/min）, abandon
Clear liquid, redissolved with 10mL label diluted standby after mixing.
2）The mark ferritin 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 to be abandoned after having centrifuged, is redissolved with 10mL mark buffer solutions, the mark for adding 2mg is mixed with ferritin monoclonal antibody,
It is another to add 10mg carbodiimides, reaction 1h is stirred at room temperature, is then centrifuged for 15min（Rotating speed is 10000r/min）, supernatant is abandoned,
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 ferritin monoclonal antibody and step 1 of obtained fluorescent latex microballoon mark）What is obtained is glimmering
The rabbit igg of light latex beads mark mixes, and the two volume ratio is 2:1.Then by the fluorescent latex microballoon of mixing by 1 microlitre/
Centimetre amount even application 45 DEG C of transposition on glass fibre element paper drying baker dry 2 hours i.e. complete labeling pad system
It is standby.
4）It is prepared by coating buffer：Take coating ferritin monoclonal antibody to add in phosphate buffer, detection line bag is made
By liquid；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 ferritin monoclonal antibody of rabbit igg containing fluorescent latex microballoon mark and fluorescent latex microballoon mark）.
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 ferritin monoclonal antibody（Coating uses ferritin monoclonal antibody）In nitric acid
Rule and be coated with cellulose membrane, as p-wire, resist coating of being rule on nitrocellulose filter that nature controlling line is made with goat-anti rabbit more.
Another plant of ferritin monoclonal antibody that latex fluorescent microsphere is marked（Mark uses ferritin monoclonal antibody）With latex fluorescence
The rabbit igg of microballoon mark is sprayed on glass fibre element film after mixing and is prepared into labeling pad.Nitrocellulose filter is close to Quality Control
The upper adsorptive pads of one end covering of line, close to the other end overlay marks thing pad of p-wire.On toward labeling pad add standard items or
Blood sample to be measured, antigen will with label hybrid reaction and being chromatographed along nitrocellulose filter, respectively with p-wire and nature controlling line
Reaction.When test result is effective, nature controlling line shows certain luminous intensity.At this moment the light signal strength on p-wire is than Quality Control linear light
The ratio of signal intensity（T/C) with concentration of specimens into positive correlation, testing sample concentration can be drawn by being calculated by standard curve.
（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 10-500 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 | |
| 10.32 | 32.65 | 60.36 | 253.54 | 512.36 | |
| 11.23 | 34.25 | 56.67 | 250.24 | 520.24 | |
| 9.68 | 32.87 | 58.45 | 258.41 | 508.35 | |
| Average value | 10.41 | 33.26 | 58.49 | 254.06 | 513.65 |
| SD | 0.78 | 0.87 | 1.85 | 4.11 | 6.05 |
| CV | 7.48% | 2.61% | 3.15% | 1.62% | 1.18% |
| Theoretical value | Average value | ||||
| x1 | 10.41 | 10.41 | |||
| x2 | 32.45 | 33.26 | |||
| x3 | 54.49 | 58.49 | |||
| x4 | 262.03 | 254.06 | |||
| x5 | 513.65 | 513.65 |
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 ferritin detection kit（Electrification
Learn luminescence 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 ferritin fluorescence immune chromatography detection card activation fluorescent latex microballoon, it is characterised in that bag Include 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 the amino surface is aminopolystyrene fluorescent microsphere；Wherein, the surfactant is the mixed of dodecyl alanine salt, polyethylene glycol monolaurate and stearate Compound, the weight ratio of three is 1 ~ 10:1~5:1~10；The surfactant and the fluorescent latex microballoon of the amino surface Mass ratio is 500 ~ 2000:1.
- 2. preparation method according to claim 1, it is characterised in that the stearate is stearic potassium, sodium, ammonium salt And one kind in tri ethanol ammonium salt or its two or more combination.
- 3. preparation method according to claim 2, it is characterised in that the surfactant is dodecyl alanine The mixture of salt, polyethylene glycol monolaurate and stearate, the weight ratio of three is 2:3:5.
- 4. the ferritin fluorescence immune chromatography detection card activation fluorescence that the preparation method described in any one of claim 1 ~ 3 obtains Latex beads.
- 5. the label working solution of the ferritin fluorescence immune chromatography detection card activation fluorescent latex microballoon described in claim 4 Preparation method, it is characterised in that take ferritin fluorescence immune chromatography detection card with activate fluorescent latex microballoon be scattered in microballoon In buffer solution, 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：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 ferritin 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 ferritin monoclonal antibody of fluorescent latex microballoon mark：Take described in claim 5 The label working solution that preparation method obtains, centrifugation, redissolved after abandoning supernatant with mark buffer solution, while it is sub- to add carbon two Amine and mark use ferritin monoclonal antibody, stirring reaction, are then centrifuged for, abandon supernatant, are 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；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 ferritin fluorescence immune chromatography detection card, it is characterised in that be prepared including the method described in claim 6 Labeling pad, coating pad and absorption pad, wherein the labeling pad be overlapped in coating pad one end on, absorption pad is overlapped in bag On the other end padded, bag is covered with provided with nature controlling line and detection line, and the antibody of goat anti-rabbit igg, detection are coated with nature controlling line Coating ferritin monoclonal antibody is coated with line.
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| CN201302571Y (en) * | 2008-08-29 | 2009-09-02 | 天津中新科炬生物制药有限公司 | Kit for quickly testing human serum ferritin nano immune magnetic bead |
| WO2010082681A1 (en) * | 2009-01-16 | 2010-07-22 | 国立大学法人筑波大学 | Immuno-latex particles and process for producing same |
| CN104034893A (en) * | 2013-03-08 | 2014-09-10 | 北京普析通用仪器有限责任公司 | Latex-based melamine rapid-detection method and kit |
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| CN201302571Y (en) * | 2008-08-29 | 2009-09-02 | 天津中新科炬生物制药有限公司 | Kit for quickly testing human serum ferritin nano immune magnetic bead |
| WO2010082681A1 (en) * | 2009-01-16 | 2010-07-22 | 国立大学法人筑波大学 | Immuno-latex particles and process for producing same |
| CN104034893A (en) * | 2013-03-08 | 2014-09-10 | 北京普析通用仪器有限责任公司 | Latex-based melamine rapid-detection method and kit |
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