CN106405072A - Activated fluorescent latex microspheres used for 25-hydroxyvitamin D fluorescence immunochromatography - Google Patents
Activated fluorescent latex microspheres used for 25-hydroxyvitamin D fluorescence immunochromatography Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract
The invention discloses activated fluorescent latex microspheres used for 25-hydroxyvitamin D fluorescence immunochromatography test strip, and test strip. A preparation method of the activated fluorescent latex microspheres used for 25-hydroxyvitamin D fluorescence immunochromatography test strip comprises following steps: 1, a surfactant is added into a buffer solution with a pH value ranging from 8 to 10, and dimethylformamide, N,N'-dicyclohexylcarbodiimide, and N-hydroxysuccinimide are added; 2, pH value of a dispersion liquid of fluorescent latex microspheres with amino radicals on the surfaces is adjusted to 8 to 10 with the buffer solution, and then is added into a mixture obtained in step 1, stirring reaction is carried out, after reaction, an obtained supernate is removed via centrifugalization so as to obtain the fluorescent latex microspheres modified via surface activation, wherein the surfactant is composed of poly(ethylene glycol) monolaurate and sodium stearate at a weight ratio of 0.5-5:1.
Description
Technical field
The invention belongs to fluorescence immune chromatography technical field is and in particular to a kind of 25-hydroxy-vitamin D fluorescence immune chromatography
The activation fluorescent latex microballoon of test strips and its preparation and application.
Background technology
Fluorescence immune chromatography test paper bar（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.Adopt fluorescence immune chromatography kit more therefore this area, add sample buffer containing test strips.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 fluorescent test paper strip also can be made poor, be not easy to preserve and transport.
Vitamin D is a kind of steroid hormone, produces after skin illumination, and two kinds of important form of vitamin D include tieing up
Raw element D3（Vitamin D3）And vitamin D2（Ergocalciferol）, in human body, vitamin-binding protein and vitamin D3With dimension life
Plain D2Combine, and they are transported in liver, there both forms, all by hydroxylating, generate vitamin D, i.e. 25-
Hydroxy-vitamine D.25-hydroxy-vitamin D is a kind of metabolism mixture, can detect in blood, because it is vitamin in human body
The major storage form of D, can promote enteron aisle to the absorption of calcium and to adjust calcium balance.Vitamin D is to maintain bone health
Essential element.Childhood vitamin D famine will lead to skeleton deformity, i.e. rickets.25-hydroxy-vitamin D concentration drops
Low relevant with bone density decline.In conjunction with other clinical datas, testing result contributes to assessing Bone m etabolism situation.
Content of the invention
The technical problem to be solved is：The fluorescent latex microballoon of fluorescence immune chromatography test paper bar after the drying,
Easily reunite, dispersion is uneven, thus leading to chromatograph unsuccessfully or make detection sensitivity to reduce.
In order to solve above-mentioned technical problem, the invention provides a kind of 25-hydroxy-vitamin D fluorescence immune chromatography test paper bar
Activation fluorescent latex microballoon and its preparation, apply the 25-hydroxy-vitamin D dry type of this activation fluorescent latex microballoon preparation glimmering
Light immuno-chromatographic test paper strip, this test strips can be used for Quantitative in vitro and measures 25-hydroxy-vitamin D in human serum, blood plasma or whole blood
Content.The test strips of the present invention can solve fluorescent latex microballoon, in spraying, drying glass fibre element film, reunion occur
Phenomenon, so that preparation labeling pad, thus obtaining dry type fluorescence immune chromatography test paper bar, configures detection reagent without dilution
Box.
The 25-hydroxy-vitamin D fluorescence immune chromatography test paper bar that the present invention the provides preparation of activation fluorescent latex microballoon
Method, comprises the 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 contains polyethylene glycol monolaurate and odium stearate, and the weight of the two is than for 0.5 ~ 5:
1.
Preferably, described surfactant also contains cocoyl glutamic acid triethanolamine salt, polyethylene glycol mono laurate
The weight of ester, odium stearate and cocoyl glutamic acid triethanolamine salt is than for 0.5 ~ 5:1:2~4.
It is highly preferred that described surfactant is polyethylene glycol monolaurate, odium stearate and cocoyl glutamic acid
Triethanolamine salt weight is than for 2:1:3 mixture.
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 25-hydroxy-vitamin D fluorescence immune chromatography test paper bar that above-mentioned preparation method obtains to live
Change fluorescent latex microballoon.
The present invention also provides above-mentioned 25-hydroxy-vitamin D fluorescence immune chromatography test paper bar to activate fluorescent latex microballoon
Label working solution preparation method：Take 25-hydroxy-vitamin D fluorescence immune chromatography test paper bar activation fluorescent latex microballoon
It is scattered in microballoon buffer solution, obtain label working solution；The fluorescent latex microballoon of surface active institute in label working solution
The ratio accounting for 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 25-hydroxy-vitamin D fluorescence immune chromatography test paper bar, including
Following steps:
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 mouse anti-human 25-hydroxy-vitamin D monoclonal antibody：Take
The label working solution that above-mentioned preparation method obtains, centrifugation, redissolved with mark buffer solution after abandoning supernatant, and be simultaneously introduced carbon
Diimine and mark mouse anti-human 25-hydroxy-vitamin D monoclonal antibody, stirring reaction, are then centrifuged for, abandon 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 25-hydroxy-vitamin D fluorescence immune chromatography test paper bar, including above-mentioned labeling pad, 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 25-
Hydroxy-vitamine D comlete antigen.
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
Chromatograph test strip（Reagent card）, it is user-friendly to, and testing result is stable, accurate, reliable.
2nd, the present invention selects the fluorescence being suitable for 25-hydroxy-vitamin D fluorescence immune chromatography test paper bar by testing sieve
The specific surfactant combination of latex beads.The fluorescent latex processing chemical modification using specific surfactant in advance is micro-
Ball, the latex beads mark 25-hydroxy-vitamin D antibody after process, it is used for preparing 25-hydroxy-vitamin D fluorescence immune chromatography
The labeling pad of test strips, realizes latex beads even application, soilless sticking phenomenon.And, after adding sample to be tested, mark
Fluorescent latex microballoon can quickly, smoothly be realized chromatographing, and processes test strips without special sample dilution or buffer solution.
3rd, the 25-hydroxy-vitamin D dry type fluorescence immune chromatography test paper bar prepared by the present invention, stability is strong, operation letter
Just, user friendly, testing result is accurate, reliable, and sensitivity is high.
Brief description
Fig. 1 is the section decomposition texture schematic diagram of the 25-hydroxy-vitamin D fluorescence immune chromatography reagent card of the present invention.
Fig. 2 is the overlooking the structure diagram of the 25-hydroxy-vitamin D fluorescence immune chromatography reagent card of the present invention.
The foundation of Fig. 3 25-hydroxy-vitamin D (25-OH-D) calibration curve.
25-hydroxy-vitamin D (25-OH-D) assay curve in Fig. 4 whole blood sample.
Fig. 5 is the fluorescence signal curve of the test strips of embodiment 5.
Fig. 6 is the fluorescence signal curve of the test strips of embodiment 6.
Fig. 7 is the fluorescence signal curve of the test strips of embodiment 7.
Fig. 8 is the fluorescence signal curve of the test strips of embodiment 8.
Fig. 9 is the fluorescence signal curve of the test strips 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 25-hydroxy-vitamin D fluorescence immune chromatography test paper bar activation fluorescent latex microballoon, bag
Include following steps：
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 contains polyethylene glycol monolaurate and odium stearate, and the weight of the two is than for 0.5 ~ 5:
1.
The present invention adopts polyethylene glycol monolaurate and odium stearate（May also include cocoyl glutamic acid triethanolamine
Salt）Latex beads outer surface is modified so as to have hydrophobicity by the method for covalent coupling.Using the activation fluorescence breast after processing
Glue microballoon marks 25-hydroxy-vitamin D antibody.The 25-hydroxy-vitamin D antibody of fluorescent latex microballoon mark is micro- with fluorescent latex
The rabbit igg of ball mark 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, for preparing dry type fluorescence
Immunochromatography reagent card, detection line is coated 25-hydroxy-vitamin D comlete antigen.After adding sample to be tested, the dimension life of 25- hydroxyl
The 25-hydroxy-vitamin D antibody haptoreaction that plain D is marked with fluorescent latex microballoon, 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（4mg polyethylene glycol monolaurate, 2mg stearic acid sodium and 6mg cocoyl glutamic acid three ethanol
Amine salt）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, 0.1ml's
In DMF, add N, N '-dicyclohexylcarbodiimide (DCC) 1mg and 0.6mg N-hydroxy-succinamide（NHS）Room temperature is stirred afterwards
Mix 3 hours.
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：The poly- second of 5mg
Glycol monolaurate, 1mg stearic acid sodium and 4mg cocoyl glutamic acid triethanolamine salt.
Embodiment 3
The method of operating of the present embodiment is similar to Example 1, and difference is, step 1）Middle surfactant is：The poly- second of 1mg
Glycol monolaurate, 2mg stearic acid sodium and 7mg cocoyl glutamic acid triethanolamine salt.
Embodiment 4
The method of operating of the present embodiment is similar to Example 1, and difference is, step 1）Middle surfactant is：The poly- second of 5mg
Glycol monolaurate and 5mg stearic acid sodium.
2nd, taking the 25-hydroxy-vitamin D fluorescence immune chromatography test paper bar as a example activation fluorescence breast to embodiment 1 ~ 4 preparation
The application of glue microballoon and effect illustrate.
Embodiment 5
The fluorescence immune chromatography test paper bar 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 mouse anti-human 25-hydroxy-vitamin D 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, add the mark mouse anti-human 25-hydroxy-vitamin D monoclonal of 2mg
Antibody mixes, another addition 10mg carbodiimides, reaction 1h is stirred at room temperature, is then centrifuged for 15min（Rotating speed is 10000r/min）,
Abandon supernatant, redissolved with the label diluted of 20mL 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 determinand antibody and the step 1 of the fluorescent latex microballoon mark obtaining）The fluorescent latex microballoon obtaining
The rabbit igg of mark mixes, and the two volume ratio is 2:1.Then will be equal by the amount of 1 microlitre/centimetre for the fluorescent latex microballoon mixing
The even drying baker being sprayed on 45 DEG C of transposition on glass fibre element paper dries the preparation completing labeling pad for 2 hours.
4）It is coated liquid preparation：Take 25-hydroxy-vitamin D comlete antigen to add in phosphate buffer, 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）Test strips assemble：
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 test strips of 4mm ± 0.1mm.
As illustrated in fig. 1 and 2, test strips include：It is coated pad 2（Nitrocellulose filter）, it is respectively arranged at the two ends with a linkage section（As
First linkage section 20, the second linkage section 22 shown in figure）, for detection section 21 in the middle of two linkage sections, detection section 21 surface is provided with detection
Line 5 and nature controlling line 7；Labeling pad 1（Glass fibre element film）, its one end is provided with linkage section 10, and the linkage section 10 of labeling pad covers
Cover and be fixed on the first linkage section 20 being coated pad 2；Label 8 is coated with labeling pad 1, specifically in an embodiment
In, it is near linkage section 10（Away from linkage section 2mm, label is 5mm along the length being coated pad length direction）Spraying mark
Note thing 8（The mark of the rabbit igg containing fluorescent latex microballoon mark and fluorescent latex microballoon mark ties up life with mouse anti-human 25- hydroxyl
Plain D monoclonal antibody）.
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 test strips adopts competition law reaction principle when measuring, by 25-hydroxy-vitamin D comlete antigen and goat-anti rabbit
LgG is coated on NC film, respectively as T line（Detection line）With C line（Nature controlling line）, by anti-human for mouse 25-hydroxy-vitamin D monoclonal
Antibody and rabbit lgG are marked on latex fluorescent microsphere respectively, and mixing is sprayed on glass fibre element film as label, in blood
Antibody in 25-OH-D antigen and envelope antigen competition binding label, the label being closed by T line and C knot shows necessarily
The ratio of intensity optical signal, T line and the optical signal of C line becomes negatively correlated with concentration, can be drawn to be measured by calibration curve calculating
Sample concentration.
（One）Test strips 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,
Test strips are 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 5-120 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 | |
| 4.83 | 23.56 | 50.24 | 85.35 | 107.02 | |
| 4.56 | 25.63 | 54.39 | 90.14 | 105.35 | |
| 4.89 | 26.39 | 52.63 | 86.35 | 117.38 | |
| Mean value | 4.76 | 25.19 | 52.42 | 87.28 | 109.92 |
| SD | 0.18 | 1.46 | 2.08 | 2.53 | 6.52 |
| CV | 3.69% | 5.81% | 3.97% | 2.90% | 5.93% |
| Theoretical value | Mean value | ||||
| x1 | 4.76 | 4.76 | |||
| x2 | 25.19 | 31.05 | |||
| x3 | 52.42 | 57.34 | |||
| x4 | 87.28 | 83.63 | |||
| x5 | 109.92 | 109.92 |
Collect clinical sample 200, with Siemens（SIEMENS）The total vitamin D of medical diagnostic prods measures kit（Chemistry 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>It was demonstrated that and Siemens when 0.975（SIEMENS）
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 test strips, 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 test strips, 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 test paper bar of the label working solution preparation being obtained using embodiment 2
The fluorescence immune chromatography test paper bar 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, with embodiment 5, result is shown in Fig. 6, this enforcement to method
The having the effect that of fluorescence immune chromatography test paper bar that example is obtained：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 test strips, 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 test paper bar of the label working solution preparation being obtained using embodiment 3
The fluorescence immune chromatography test paper bar 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
The having the effect that of fluorescence immune chromatography test paper bar that example is obtained：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 test strips, 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 test paper bar of the label working solution preparation being obtained using embodiment 3
The fluorescence immune chromatography test paper bar 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
The having the effect that of fluorescence immune chromatography test paper bar that example is obtained：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 test strips, 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 test paper bar 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 25-hydroxy-vitamin D fluorescence immune chromatography test paper bar preparation method of activation fluorescent latex microballoon, its feature
It is, comprise the 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 contains polyethylene glycol monolaurate and odium stearate, and the weight of the two is than for 0.5 ~ 5:
1.
2. preparation method according to claim 1 is it is characterised in that described surfactant also contains cocoyl paddy ammonia
Triethylenetetraminehexaacetic acid alcohol amine salt, the weight ratio of polyethylene glycol monolaurate, odium stearate and cocoyl glutamic acid triethanolamine salt is
0.5~5:1:2~4.
3. preparation method according to claim 1 is it is characterised in that described surfactant is polyethylene glycol mono laurate
Ester, odium stearate and cocoyl glutamic acid triethanolamine salt weight are than for 2:1:3 mixture.
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 25-hydroxy-vitamin D fluorescence immune chromatography test paper bar that the preparation method described in any one of claim 1 ~ 5 obtains is used
Activation fluorescent latex microballoon.
7. the mark of activation fluorescent latex microballoon of the 25-hydroxy-vitamin D fluorescence immune chromatography test paper bar described in claim 6
The preparation method of thing working solution is it is characterised in that take 25-hydroxy-vitamin D fluorescence immune chromatography test paper bar activation fluorescence breast
Glue microballoon is scattered in microballoon buffer solution, obtains label working solution；The fluorescent latex microballoon of surface active works in label
In liquid, shared ratio 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%.
The preparation method of the labeling pad of 9.25- hydroxy-vitamine D fluorescence immune chromatography test paper bar it is characterised in that include as
Lower step:
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 mouse anti-human 25-hydroxy-vitamin D monoclonal antibody：Take
The label working solution that preparation method described in claim 7 or 8 obtains, centrifugation, redissolved with mark buffer solution after abandoning supernatant, and
And it is simultaneously introduced carbodiimide and mark mouse anti-human 25-hydroxy-vitamin D monoclonal antibody, stirring reaction, stirring, Ran Houli
The heart, abandons supernatant, is 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 25-hydroxy-vitamin D fluorescence immune chromatography test paper bar is it is characterised in that include the mark described in claim 9
Remember thing pad, be coated pad and absorption pad, wherein said labeling pad is overlapped on the one end being coated pad, and absorption pad is overlapped in and is coated pad
The other end 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, in detection line
It is coated with 25-hydroxy-vitamin D comlete antigen.
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Address after: 101111 Four Floor F4E of Building No. 2, Kechuang East Five Street, Tongzhou District, Beijing Patentee after: Beijing Huaketai Biotechnology Co., Ltd. Address before: 100070 Room 501, 2 building, 8 Haiying Road, Science City, Fengtai District, Beijing (Park) Patentee before: Beijing Elcoteq Bio-Technology Co., Ltd. |
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Inventor after: Lin Si Inventor after: Hu Chenguang Inventor after: Xiao Jun Inventor after: Fu Qiao Inventor before: Lin Si |