CN106405109A - Activated fluorescent latex microsphere used for ferritin fluorescence immunoassay chromatography detection card - Google Patents
Activated fluorescent latex microsphere used for ferritin fluorescence immunoassay chromatography detection card Download PDFInfo
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Abstract
The invention discloses an activated fluorescent latex microsphere used for a ferritin fluorescence immunoassay chromatography detection card, and application of the activated fluorescent latex microsphere. A preparation method of the activated fluorescent latex microsphere used for the ferritin fluorescence immunoassay chromatography detection card comprises the following steps: 1) adding a surfactant into buffered solution of which the pH is 8-10, and adding dimethylformamide, N,N'-dicyclohexylcarbodiimide and N-hydroxy succinimide; 2) taking dispersion liquid of the fluorescent latex microsphere on an amino surface, regulating the pH to 8-10 by the buffered solution, adding into a mixture obtained in 1), carrying out stirring reaction, and removing supernate by centrifugation after reaction is finished, so as to obtain a surface-activated fluorescent latex microsphere, wherein the surfactant is a mixture of an amino acid-based surfactant, polyethylene glycol monolaurate and stearate at the mass ratio of (1 to 10):(1 to 5): (1 to 10).
Description
Technical field
The invention belongs to fluorescence immune chromatography technical field is 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, it is widely used in fast inspection field.Determinand
Antibody labeling fluorescent latex microballoon, latex beads has 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 particle.When adding sample to redissolve latex beads when immunochromatography, latex beads no longer uniformly divides
Dissipate and lead to chromatograph unsuccessfully.Adopting fluorescence immune chromatography kit therefore this area, card containing detection adds sample buffer more.Increase
Detection complexity.Or due to latex beads partial agglomeration so that detection sensitivity reduces, false negative in testing result.
In addition, latex beads is unstable, the stability of fluoroscopic examination card also can be made 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 iron content in it is how many）.
Serum ferritin is the iron of bonding state in blood.It is to check sideropenic the most sensitive index in vivo.The mensure of ferritin is
Specify the usability methods of iron metabolism situation.Ferritin Levels are measured be that internal iron content is estimated at the 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 point out asiderosis.As ferritin levels raise and can exclude iron distribution disorders simultaneously
Possibility is then it represents that internal iron content is too high.
Content of the invention
The technical problem to be solved is：Ferritin（Fer）The fluorescent latex of fluorescence immune chromatography detection card is micro-
Ball 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 activation of ferritin fluorescence immune chromatography detection card
Fluorescent latex microballoon and its preparation, apply the ferritin dry type fluorescence immune chromatography detection of this activation fluorescent latex microballoon preparation
Card, this detection card can be used for the content that Quantitative in vitro measures ferritin in human serum, blood plasma or whole blood.The detection card of the present invention can
The phenomenon reunited with solving fluorescent latex microballoon to occur in spraying, drying glass fibre element film, so that preparation labeling pad, from
And obtain dry type fluorescence immune chromatography detection card, configure detection kit without dilution.
The ferritin fluorescence immune chromatography detection card that the present invention the provides preparation method of activation fluorescent latex microballoon, including
Following steps：
1）Take surfactant to add in the cushioning liquid 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 microballoon of amino surface, adjusted after pH to 8 ~ 10 with cushioning liquid, be added to step 1）Institute
In the mixture obtaining, stirring reaction, after completion of the reaction, centrifugation removes supernatant, obtains activating fluorescent latex microballoon；
Wherein, described surfactant is the mixed of amino acid type surfactant, polyethylene glycol monolaurate and stearate
Compound, the weight of three is than for 1 ~ 10:1~5:1~10.
Preferably, described stearate is one of stearic potassium, sodium, ammonium salt and tri ethanol ammonium salt or its two kinds
Above combination；Described amino acid type surfactant is dodecyl alanine salt.
Preferably, described surfactant is dodecyl alanine salt, polyethylene glycol monolaurate and stearic acid
The mixture of salt, the weight of three is than for 2:3:5.
Preferably, described surfactant and the mass ratio of the fluorescent latex microballoon of described amino surface are 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 above-mentioned ferritin fluorescence immune chromatography to detect that card activates the label of fluorescent latex microballoon
The preparation method of working solution：Ferritin fluorescence immune chromatography detection card activation fluorescent latex microballoon is taken to be scattered in microballoon buffer solution
In, obtain label working solution；The shared ratio in label working solution of the fluorescent latex microballoon of surface active is 0.5 ~
2wt%；
Wherein, the preparation method of microballoon buffer solution is：By BSA（Bovine serum albumin）, biological preservative, Lamepon A and glycine betaine
It is dissolved in the phosphate buffer of 0.01M, pH7.4.
Preferably, BSA, biological preservative, the Lamepon A and glycine betaine concentration in described 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, walks including following
Suddenly:
1）The preparation of the rabbit igg of activation fluorescent latex microballoon mark：Take the label working solution that above-mentioned preparation method obtains, from
The heart, after abandoning supernatant, is redissolved with mark buffer solution, and is simultaneously introduced carbodiimide and rabbit igg, stirring reaction, be then centrifuged for,
Abandon supernatant, finally redissolved with mark dilution；
2）The mark of the activation fluorescent latex microballoon mark preparation method of ferritin monoclonal antibody：Above-mentioned preparation method is taken to obtain
The label working solution arriving, centrifugation, redissolved with mark buffer solution after abandoning supernatant, and be simultaneously introduced carbodiimide and use with mark
Ferritin monoclonal antibody, stirring reaction, it is then centrifuged for, abandons supernatant, finally redissolved with mark dilution；
3）Take step 1）Gained dispersion liquid and step 2）Gained dispersion liquid, mixing, it is sprayed on glass fibre, dry；
Described mark buffer solution formula be：Sodium carbonate 4.33g, sodium acid carbonate 2.96g, are dissolved in 1000mL water；Described mark
The formula of dilution is：Trisodium citrate 7.33g, citric acid 4.44g, NaOH 1g, are dissolved in 1000mL water.
The present invention provides a kind of ferritin fluorescence immune chromatography detection card, including above-mentioned labeling pad, is coated pad and suction
Receive 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
It 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 be coated and uses ferritin
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 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 selects the fluorescent latex microballoon being suitable for ferritin fluorescence immune chromatography detection card by testing sieve
Specific surfactant combination.Process the fluorescent latex microballoon of chemical modification using specific surfactant in advance, process
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.And, after adding sample to be tested, the fluorescent latex microballoon of mark can quickly,
Smoothly realize chromatography, 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 and simple to handle, Yong Huyou
Good, testing result is accurate, reliable, and sensitivity is high.
Brief description
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 calibration curve.
In Fig. 4 whole blood sample, Ferritin Levels measure curve.
Fig. 5 is the fluorescence signal curve of the detection card of embodiment 5.
Fig. 6 is the fluorescence signal curve of the detection card of embodiment 6.
Fig. 7 is the fluorescence signal curve of the detection card of embodiment 7.
Fig. 8 is the fluorescence signal curve of the detection card of embodiment 8.
Fig. 9 is the fluorescence signal curve of the detection card of comparative example.
Specific embodiment
The invention will be further described with specific embodiment below in conjunction with the accompanying drawings, so that those skilled in the art is permissible
It is better understood from the present invention and can be practiced, but illustrated embodiment is not as a limitation of the invention.
The invention provides a kind of ferritin fluorescence immune chromatography detection card activation fluorescent latex microballoon, walk including following
Suddenly：
1）Take surfactant to add in the cushioning liquid 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 microballoon of amino surface, adjusted after pH to 8 ~ 10 with cushioning liquid, be added to step 1）Institute
In the mixture obtaining, stirring reaction, after completion of the reaction, centrifugation removes supernatant, obtains activating fluorescent latex microballoon；
Wherein, described surfactant is the mixed of amino acid type surfactant, polyethylene glycol monolaurate and stearate
Compound, the weight of three is than for 1 ~ 10:1~5:1~10.
The present invention adopts the mixture of amino acid type surfactant, cithrol and stearate（Cloudy from
Subtype, nonionic and amphoteric surfactant mixing）Latex beads outer surface is modified by the method for covalent coupling so as to have
There is hydrophobicity.Using the activation fluorescent latex microballoon mark ferritin antibody after processing.The ferritin of fluorescent latex microballoon mark
The rabbit igg that antibody is marked with fluorescent latex microballoon is sprayed on after mixing and is prepared into labeling pad 1 on glass fibre element film, is coated pad
2（Nitrocellulose filter）Cover upper adsorptive pads near one end of nature controlling line, near the other end overlay marks thing pad of detection line, use
In preparation dry type fluorescence immune chromatography reagent card, detection line is coated and uses ferritin antibody.After adding sample to be tested, iron egg
The ferritin antibody haptoreaction that white and fluorescent latex microballoon marks, latex beads can keep dispersity not reunite, and
Can be rapid, dispersed after contact measured sample, and along nitrocellulose filter flash chromatography.
Fluorescent latex microballoon 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 microballoon
Embodiment 1
1）Take surfactant（2mg sodium dodecyl aminopropionitrile, 3mg polyethylene glycol monolaurate and 5mg odium stearate）
It is initially dissolved in the carbonic acid buffer of 0.5M pH9.6（H value 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% solids
PH value is adjusted to be added to step 1 to after 9.6）In, continue stirring reaction 3 hours.Both the latex beads that must activate.
3）With supercentrifuge, fluorescent latex microballoon reactant liquor is centrifuged at a high speed, removes reaction solution supernatant, use
1mL microballoon buffer solution redissolves fluorescent latex microballoon, forms label working solution.
Microballoon 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.Microballoon phosphate buffer provides certain Ionic strength
And pH value, make microballoon dispersed.
Embodiment 2
The method of operating of the present embodiment is similar to Example 1, and difference is, step 1）Middle surfactant is：0.5mg ten
Dialkyl amido sodium propionate, 2.5mg polyethylene glycol monolaurate and 5mg odium stearate.
Embodiment 3
The method of operating of the present embodiment is similar to Example 1, and difference is, step 1）Middle surfactant is：5mg 12
Sodium alkyl amino-propionate, 0.5mg polyethylene glycol monolaurate and 4.5mg odium stearate.
Embodiment 4
The method of operating of the present embodiment is similar to Example 1, and difference is, step 1）Middle surfactant is：5mg 12
Sodium alkyl amino-propionate, 2.5mg polyethylene glycol monolaurate and 0.5mg odium stearate.
2nd, the activation fluorescent latex microballoon to embodiment 1 ~ 4 preparation as a example card is detected by ferritin fluorescence immune chromatography
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 microballoon mark：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 mark buffer solution, 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 mark ferritin monoclonal antibody of fluorescent latex microballoon mark
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 mark buffer solution, the mark adding 2mg is mixed with ferritin monoclonal antibody, separately adds
Enter 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）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, NaOH 1g, are dissolved in 1000mL water.
3）Prepared by labeling pad
Step 2）Mark ferritin monoclonal antibody and the step 1 of the fluorescent latex microballoon mark obtaining）The fluorescence breast obtaining
The rabbit igg of glue microballoon mark mixes, and the two volume ratio is 2:1.Then the fluorescent latex microballoon mixing is pressed 1 microlitre/centimetre
Amount even application on glass fibre element paper the drying baker of 45 DEG C of transposition dry and complete within 2 hours the preparation of labeling pad.
4）It is coated liquid preparation：Take to be coated and added in phosphate buffer with ferritin monoclonal antibody, make detection line bag
By 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（Blotting 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- operational procedure 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（Rabbit igg containing fluorescent latex microballoon mark and the mark ferritin monoclonal antibody of 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 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 ferritin monoclonal antibody（It is coated and use ferritin monoclonal antibody）In nitric acid
On cellulose membrane, line is coated, and as p-wire, makes nature controlling line with resisting line on nitrocellulose filter to be coated goat-anti rabbit more.
Another plant of ferritin monoclonal antibody by latex fluorescent microsphere mark（Mark uses ferritin monoclonal antibody）With latex fluorescence
The rabbit igg of microballoon mark is sprayed on after mixing and is prepared into labeling pad on glass fibre element film.Nitrocellulose filter is near Quality Control
One end of line covers upper adsorptive pads, near the other end overlay marks thing pad of p-wire.Toward in labeling pad add standard items or
Blood sample to be measured, antigen will be with label hybrid reaction and along nitrocellulose filter chromatography, 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 strength signal intensity（T/C) become positive correlation with concentration of specimens, calculated by calibration curve and can draw testing sample 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 analyzer after terminating by reaction.Each concentration point of calibration object
Repeat to do 3 times, after taking the mean value of T/C area ratio, generate a calibration curve with concentration（Range of linearity 10-500 ng/mL）.
Standard curve determination result is as shown in the table, and calibration 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 | |
| Mean 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 | Mean 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 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 ferritin detection kit（Electrochemistry is sent out
Light method）Carry out contrasting 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 validity.
（Two）Dispersiveness with regard to fluorescent latex microballoon 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 electric signal for unit（Fluorescence intensity）.Amount to and export 300 electric signals, such as Fig. 5
Shown.
In Fig. 5, ordinate：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 microballoon of surface active easily forms monodisperse status, improves springing up property of microballoon
Energy.When the detection line of blood movement to antigen, the compound 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 microballoon remains.
The present embodiment be obtained detection card, detection sensitivity height, high specificity, luminous efficiency height and being capable of quantitative reaction 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 dispersiveness and springing up property of fluorescent latex microballoon 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 microballoon of surface active easily forms single dispersing shape
State, improves microballoon and springs up performance.When the detection line of blood movement to antigen, the compound 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
Microballoon remains.
The present embodiment be obtained detection card, detection sensitivity height, high specificity, luminous efficiency height and being capable of quantitative reaction 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 dispersiveness and springing up property of fluorescent latex microballoon 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 microballoon of surface active easily forms single dispersing shape
State, improves microballoon and springs up performance.When the detection line of blood movement to antigen, the compound 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
Microballoon remains.
The present embodiment be obtained detection card, detection sensitivity height, high specificity, luminous efficiency height and being capable of quantitative reaction 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 dispersiveness and springing up property of fluorescent latex microballoon 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 microballoon of surface active easily forms single dispersing shape
State, improves microballoon and springs up performance.When the detection line of blood movement to antigen, the compound 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
Microballoon remains.
The present embodiment be obtained detection card, detection sensitivity height, high specificity, luminous efficiency height 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）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 dispersiveness and springing up property detection method：1. unactivated micro-
Ball, climbs film effect poor.Latex beads easily condenses, and simultaneously because of its hydrophobicity, easily non-specific adsorption 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 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 ferritin fluorescence immune chromatography detection card preparation method of activation fluorescent latex microballoon is it is characterised in that wrap
Include following steps：
1）Take surfactant to add in the cushioning liquid 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 microballoon of amino surface, adjusted after pH to 8 ~ 10 with cushioning liquid, be added to step 1）Institute
In the mixture obtaining, stirring reaction, after completion of the reaction, centrifugation removes supernatant, obtains activating fluorescent latex microballoon；
Wherein, described surfactant is the mixed of amino acid type surfactant, polyethylene glycol monolaurate and stearate
Compound, the weight of three is than for 1 ~ 10:1~5:1~10.
2. preparation method according to claim 1 is it is characterised in that described stearate is stearic potassium, sodium, ammonium salt
And one of tri ethanol ammonium salt or its two or more combination；Described amino acid type surfactant is dodecylamino
Propionate.
3. preparation method according to claim 2 is it is characterised in that described surfactant is dodecyl alanine
The mixture of salt, polyethylene glycol monolaurate and stearate, the weight of three is than for 2:3:5.
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 beads is 500 ~ 2000:1.
5. preparation method according to claim 1 is it is characterised in that step 1）With step 2）Described in cushioning liquid 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 ferritin fluorescence immune chromatography detection card activation fluorescence that the preparation method described in any one of claim 1 ~ 5 obtains
Latex beads.
7. the label working solution of activation fluorescent latex microballoon of the ferritin fluorescence immune chromatography detection card described in claim 6
Preparation method it is characterised in that take ferritin fluorescence immune chromatography detection card activate fluorescent latex microballoon be scattered in microballoon
In buffer solution, obtain label working solution；The ratio that the fluorescent latex microballoon of surface active is 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 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 microballoon buffer solution is 0.1wt%.
9. the preparation method of the labeling pad of ferritin 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 microballoon mark：Take the mark that preparation method described in claim 7 or 8 obtains
Thing working solution, centrifugation, after abandoning supernatant, redissolved with mark buffer solution, and be simultaneously introduced carbodiimide and rabbit igg, stirring is anti-
Should, it is then centrifuged for, abandons supernatant, finally redissolved with mark dilution；
2）The mark of the activation fluorescent latex microballoon mark preparation method of ferritin monoclonal antibody：Take claim 7 or 8 institute
State the label working solution that preparation method obtains, centrifugation, redissolved with mark buffer solution after abandoning supernatant, and be simultaneously introduced carbon two
Imines 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, it is sprayed on glass fibre, dry；
Described mark buffer solution formula be：Sodium carbonate 4.33g, sodium acid carbonate 2.96g, are dissolved in 1000mL water；Described mark
The formula of dilution is：Trisodium citrate 7.33g, citric acid 4.44g, NaOH 1g, are dissolved in 1000mL water.
10. a kind of ferritin fluorescence immune chromatography detection card is it is characterised in that include the labeling pad described in claim 9, bag
Padded and absorption pad, wherein said labeling pad is overlapped on the one end being coated pad, and absorption pad is overlapped in the other end being coated pad
On, 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 bag
Used ferritin monoclonal antibody.
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| CN112379102A (en) * | 2020-10-29 | 2021-02-19 | 广东工业大学 | Rhodium nanoparticle-ferritin polyclonal antibody compound and ferritin rapid detection test strip |
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