CN106405108A - Activated fluorescent latex microsphere for fluorescent immunochromatography of whole C-reactive protein - Google Patents
Activated fluorescent latex microsphere for fluorescent immunochromatography of whole C-reactive protein Download PDFInfo
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- CN106405108A CN106405108A CN201610804731.0A CN201610804731A CN106405108A CN 106405108 A CN106405108 A CN 106405108A CN 201610804731 A CN201610804731 A CN 201610804731A CN 106405108 A CN106405108 A CN 106405108A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- 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
Abstract
The invention discloses an activated fluorescent latex microsphere for fluorescent immunochromatography of a whole C-reactive protein and a use thereof. The preparation method comprises 1) adding a surfactant into a buffer solution with the pH value of 8-10 and adding dimethylformamide, N, N'-dicyclohexylcarbodiimide and N-hydroxysuccinimide into the mixed solution, and 2) taking a dispersion liquid of fluorescent latex microspheres with amido groups on the surfaces, adjusting pH of the dispersion liquid to 8-10 through a buffer solution, adding the dispersion liquid into the mixture obtained in the step 1), carrying out stirring for a reaction, then carrying out centrifugation after the reaction, and removing the supernatant so that the surface-activated fluorescent latex microspheres are obtained. The surfactant is a mixture of an amino acid-type surfactant, polyethylene glycol fatty acid ester and a stearate according to a weight ratio of 1-5: 1-2: 0.5-3.
Description
Technical field
The invention belongs to fluorescence immune chromatography technical field and in particular to a kind of whole process C reactive protein fluorescence immune chromatography
The activation fluorescent latex microsphere 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 microsphere, latex beadses have surface electronic repulsion in the liquid phase and Van der Waals force makes in liquid phase
Latex particle keeps being uniformly distributed.But it is sprayed at glass fibre element film and evaporated dry, surface tension with solution after drying
Promote infinitely near leading to intermolecular repulsion to disappear between latex particle, Van der Waals force increases, and finally makes multiple latex micels
It is polymerized to big and small different granule.When adding sample to redissolve latex beadses when immunochromatography, latex beadses no longer uniformly divide
Dissipate and lead to chromatograph unsuccessfully.Adopt fluorescence immune chromatography test kit more therefore this area, add sample buffer containing test strips.Increase
Detection complexity.Or due to latex beadses partial agglomeration so that detection sensitivity reduces, false negative in testing result.
In addition, latex beadses is unstable, the stability of fluorescent test paper strip also can be made poor, be not easy to preserve and transport.
C reactive protein（CRP be) a kind of Acute phase protein, and be the Acute phase protein that is realized first in history it
One.The increase of its serum or plasma concentration is that it is nearly constant unchangeably to show caused by the release of inflammatory cytokine such as IL-6
With the presence of inflammation.Because C reactive protein generally increases after bacterium infection, and do not increase during virus infection, so measuring human blood
Middle CRP content for have discriminating bacteria and virus infection significance.
Content of the invention
The technical problem to be solved is：The fluorescent latex microsphere 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 whole process C reactive protein fluorescence immune chromatography test paper bar
Activation fluorescent latex microsphere and its preparation, apply the whole C reactive protein dry type of this activation fluorescent latex microsphere preparation glimmering
Light immuno-chromatographic test paper strip, this test strips can be used for Quantitative in vitro and measures whole C reactive protein in human serum, blood plasma or whole blood
Content.The test strips of the present invention can solve fluorescent latex microsphere, 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 detectable without diluent
Box.
The whole C reactive protein fluorescence immune chromatography that the present invention the provides preparation method of activation fluorescent latex microsphere, bag
Include following steps：
1）Take surfactant to add in the buffer solution that pH is 8 ~ 10, add dimethylformamide, N, N '-dicyclohexyl carbon
Diimine and N-hydroxy-succinamide；
2）Take the dispersion liquid of the fluorescent latex microsphere of amino surface, adjusted after pH to 8 ~ 10 with buffer solution, be added to step 1）Institute
In the mixture obtaining, stirring reaction, after completion of the reaction, centrifugation removes supernatant, obtains activating fluorescent latex microsphere；
Wherein, described surfactant is the mixing of amino acid type surfactant, cithrol and stearate
Thing, the weight of three is than for 1 ~ 5: 1~2:0.5~3.
Preferably, described amino acid type surfactant is lauroyl glutamate, N- acyl glutamate or dodecane
One of base dimethylene amino diformate or its two or more combination；Described stearate be stearic potassium, sodium,
One of ammonium salt and tri ethanol ammonium salt or its two or more combination.
Preferably, described amino acid type surfactant is sodium lauroyl glutamate；Described stearate is sodium stearate；
Described cithrol is polyethylene glycol monolaurate.
Preferably, described surfactant and the mass ratio of the fluorescent latex microsphere of described amino surface are 500 ~ 2000:
1.
Preferably, step 1）With step 2）Described in buffer solution be carbonic acid buffer；Step 2）In stirring reaction
Time is 2 ~ 4 hours, and temperature is 20 ~ 30 DEG C；The fluorescent latex microsphere of amino surface is aminopolystyrene fluorescent microsphere.
The present invention also provides the whole C reactive protein fluorescence immune chromatography activation fluorescence that above-mentioned preparation method obtains
Latex beadses.
The present invention also provides above-mentioned whole C reactive protein fluorescence immune chromatography to activate the labelling of fluorescent latex microsphere
The preparation method of thing working solution：Take whole C reactive protein fluorescence immune chromatography activation fluorescent latex microsphere to be scattered in microsphere to delay
Rush in liquid, obtain label working solution；The ratio that the fluorescent latex microsphere of surface active is shared in label working solution is 0.5
~2wt%；
Wherein, the preparation method of microsphere buffer is：By BSA, biological preservative, Lamepon A and glycine betaine be dissolved in 0.01M,
In the phosphate buffer of pH7.4.
Preferably, BSA（Bovine serum albumin）, biological preservative, Lamepon A and glycine betaine be in described microsphere buffer
Concentration be 0.1wt%.
The present invention also provides the preparation method of the labeling pad of whole C reactive protein fluorescence immune chromatography test paper bar, including
Following steps:
1）The preparation of the rabbit igg of activation fluorescent latex microsphere labelling：Take the label working solution that above-mentioned preparation method obtains, from
The heart, after abandoning supernatant, is redissolved with labelling buffer, and is simultaneously introduced carbodiimide and rabbit igg, stirring reaction, be then centrifuged for,
Abandon supernatant, finally use labelling diluent to redissolve；
2）The labelling of the activation fluorescent latex microsphere labelling preparation method of whole C reactive protein monoclonal antibody：Take above-mentioned system
The label working solution that Preparation Method obtains, centrifugation, redissolved with labelling buffer after abandoning supernatant, and be simultaneously introduced carbodiimide
With labelling whole C reactive protein monoclonal antibody, stirring reaction, it is then centrifuged for, abandons supernatant, finally use labelling diluent
Redissolve；
3）Take step 1）Gained dispersion liquid and step 2）Gained dispersion liquid, mixing, it is sprayed on glass fibre, dry；
The formula of described labelling buffer is：Sodium carbonate 4.33g, sodium bicarbonate 2.96g, are dissolved in 1000mL water；Described labelling
The formula of diluent is：Trisodium citrate 7.33g, citric acid 4.44g, sodium hydroxide 1g, are dissolved in 1000mL water.
The present invention provides a kind of whole process C reactive protein 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 bag
By with whole C reactive protein monoclonal antibody.
The present invention can reach following technique effect：
1st, present invention research finds that the fluorescent latex microsphere through the activation of specific surfactant keeps intergranular when drying
Relative distance is difficult to reunite.During chromatography process, sample is added can at once to redissolve and smoothly chromatograph, so that preparation dry type fluorescence immunoassay
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 whole C reactive protein fluorescence immune chromatography test paper bar by testing sieve
The specific surfactant combination of latex beadses.The fluorescent latex processing chemical modification using specific surfactant in advance is micro-
Ball, the latex beadses labelling whole process C reactive protein antibody after process, for preparing whole C reactive protein fluorescence immune chromatography
The labeling pad of test strips, realizes latex beadses even application, soilless sticking phenomenon.And, after adding sample to be tested, labelling
Fluorescent latex microsphere can quickly, smoothly be realized chromatographing, and processes test strips without special sample diluent or buffer.
3rd, the whole C reactive protein 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 whole C reactive protein fluorescence immune chromatography reagent card of the present invention.
Fig. 2 is the overlooking the structure diagram of the whole C reactive protein fluorescence immune chromatography reagent card of the present invention.
Fig. 3 is the foundation of whole C reactive protein standard curve.
Whole C reactive protein 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 whole process C reactive protein fluorescence immune chromatography activation fluorescent latex microsphere, including as follows
Step：
1）Take surfactant to add in the buffer solution that pH is 8 ~ 10, add dimethylformamide, N, N '-dicyclohexyl carbon two
Imines and N-hydroxy-succinamide, stirring reaction；
2）Take the dispersion liquid of the fluorescent latex microsphere of amino surface, adjusted after pH to 8 ~ 10 with buffer solution, be added to step 1）Institute
In the mixture obtaining, stirring reaction, after completion of the reaction, centrifugation removes supernatant, obtains activating fluorescent latex microsphere；
Wherein, described surfactant is the mixing of amino acid type surfactant, cithrol and stearate
Thing, the weight of three is than for 1 ~ 5:1~2:0.5~3.
The present invention adopts the mixture of amino acid type surfactant, cithrol and stearate（Cloudy from
Subtype, nonionic and amphoteric surfactant mixing）Latex beadses outer surface is modified by the method for covalent coupling so as to have
There is hydrophobicity.Using the activation fluorescent latex microsphere labelling whole process C reactive protein antibody after processing.Fluorescent latex microsphere labelling
Whole C reactive protein antibody and fluorescent latex microsphere labelling rabbit igg mix after be sprayed on and be prepared on glass fibre element film
Labeling pad 1, is coated pad 2（Nitrocellulose filter）Cover upper adsorptive pads near one end of nature controlling line, another near detection line
End overlay marks thing pad, for preparing dry type fluorescence immune chromatography reagent card, detection line is coated with whole C- reaction egg
Bai Kangti.After adding sample to be tested, whole C reactive protein is connect with the whole C reactive protein antibody of fluorescent latex microsphere labelling
Touch reaction, latex beadses can keep dispersity not reunite, and can be rapid, dispersed after contact measured sample, and edge
Nitrocellulose filter flash chromatography.
Fluorescent latex microsphere used in following examples of the present invention refers to the polyphenyl second of amino surface being modified by sulphation
Alkene fluorescent microsphere, diameter 100-500nm.
First, activate the preparation of fluorescent latex microsphere
Embodiment 1
1）Take surfactant（4 mg sodium lauroyl glutamates, 4mg polyethylene glycol monolaurate, 2mg sodium stearate）First
It is dissolved in the carbonic acid buffer of 0.5M pH9.6（H-number is to 9.6）, add in the DMF of 0.1ml, in the DMF of 0.1ml, add
N, N '-dicyclohexylcarbodiimide (DCC) 1mg and 0.6mg N-hydroxy-succinamide（NHS）After be stirred at room temperature 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% solidss
PH value is adjusted to be added to step 1 to after 9.6）In, continue stirring reaction 3 hours.Both the latex beadses that must activate.
3）With high speed centrifuge, fluorescent latex microsphere reactant liquor is centrifuged at a high speed, removes reaction solution supernatant, use
1mL microsphere buffer redissolves fluorescent latex microsphere, forms label working solution.
Microsphere phosphate buffer is prepared：The phosphate buffer of 0.01M pH7.4, BSA containing 0.1wt%, gives birth to containing 0.1 wt %
Thing preservative, containing 0.1 wt % Lamepon A, containing 0.1 wt % glycine betaine.Microsphere phosphate buffer provides certain Ionic strength
And pH value, make microsphere dispersed.
Embodiment 2
The operational approach of the present embodiment is similar to Example 1, and difference is, step 1）Middle surfactant is：The 5 mg months
Osmanthus acyl sodium glutamate, 1mg polyethylene glycol monolaurate, 3mg sodium stearate.
Embodiment 3
The operational approach of the present embodiment is similar to Example 1, and difference is, step 1）Middle surfactant is：6 mg N-
Acyl glutamic acid sodium, 4mg polyethylene glycol monolaurate, 0.5mg potassium stearate.
Embodiment 4
The operational approach of the present embodiment is similar to Example 1, and difference is, step 1）Middle surfactant is：4 mg ten
Dialkyl group dimethylene amino sodium diformate, 4mg polyethylene glycol monolaurate, 2mg sodium stearate.
2nd, taking the whole C reactive protein fluorescence immune chromatography test paper bar as a example activation fluorescence breast to embodiment 1 ~ 4 preparation
The application of glue microsphere 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 microsphere labelling：Take the label working solution that 5mL embodiment 1 obtains, use centrifuge
30min（Rotating speed is 10000r/min）, abandon supernatant after being centrifuged, redissolved with 5mL labelling buffer, add the rabbit igg of 1mg
Mix, another addition 5mg carbodiimides, reaction 1h is stirred at room temperature, is then centrifuged for 15min（Rotating speed is 10000r/min）, abandon
Clear liquid, is redissolved standby after mixing with the label diluted of 10mL.
2）The labelling of fluorescent latex microsphere labelling whole process C reactive protein monoclonal antibody
Take the label working solution that 10mL embodiment 1 obtains, use centrifuge 30min（Rotating speed is 10000r/min）, centrifugation
Abandon supernatant after complete, redissolved with 10mL labelling buffer, the labelling adding 2mg is mixed with whole C reactive protein monoclonal antibody
Even, another addition 10mg carbodiimides, reaction 1h is stirred at room temperature, is then centrifuged for 15min（Rotating speed is 10000r/min）, abandon supernatant
Liquid, is redissolved standby after mixing with the label diluted of 20mL.
Step 1）With 2）The formula of middle labelling buffer is：Sodium carbonate 4.33g, sodium bicarbonate 2.96g, are dissolved in 1000mL water
In；The formula of labelling diluent is：Trisodium citrate 7.33g, citric acid 4.44g, sodium hydroxide 1g, are dissolved in 1000mL water.
3）Prepared by labeling pad
Step 2）The labelling of the fluorescent latex microsphere labelling obtaining determinand antibody and step 1）The fluorescent latex microsphere obtaining
The rabbit igg of labelling mixes, and the two volume ratio is 2:1.Then will be equal by the amount of 1 microlitre/centimetre for the fluorescent latex microsphere mixing
The even drying baker being sprayed on 45 DEG C of transposition on glass fibre element paper dries the preparation completing labeling pad for 2 hours.
4）It is coated liquid preparation：Take to be coated and added in phosphate buffer with whole C reactive protein monoclonal antibody, make
Detection line is coated liquid；Take goat anti-rabbit igg to add in phosphate buffer, make nature controlling line and be coated liquid.This phosphate buffer
Formula 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（Absorbent paper）, by being coated pad（Nitrocellulose filter）Near Quality Control
One end of line covers upper absorption pad, and the requirement near the other end overlay marks thing pad of detection line carries out joint strip.By cutting cutter mark
Quasi- rule of operation is operated, and is cut into the wide 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 labelling of the rabbit igg containing fluorescent latex microsphere labelling and fluorescent latex microsphere labelling whole process C reactive protein Dan Ke
Grand antibody）.
Absorption pad 3（Absorbent paper）, its one end is provided with linkage section 30, and the linkage section 30 of absorption pad covers and is fixed on and is coated pad
On 2 the second linkage section 22；
Base plate 4, is coated pad 2, labeling pad 1 and absorption pad 3 and is both secured on base plate.
This test strips is when using with one plant of whole C reactive protein monoclonal antibody（It is coated with whole C reactive protein list
Clonal antibody）Nitrocellulose filter is rule and is coated, as p-wire, rule on nitrocellulose filter with goat-anti rabbit multi-resistance
It is coated and make nature controlling line.By another plant of latex fluorescent microsphere labelling whole C reactive protein monoclonal antibody（Labelling is whole
C reactive protein monoclonal antibody）It is sprayed on after mixing with the rabbit igg of latex fluorescent microsphere labelling and prepare on glass fibre element film
Become labeling pad.Nitrocellulose filter covers upper adsorptive pads near one end of nature controlling line, and the other end near p-wire covers mark
Note thing pad.Standard substance or blood sample to be measured are added toward on labeling pad, antigen will be with label hybrid reaction and fine along nitric acid
Dimension plain film layer analysis, is reacted with p-wire and nature controlling line respectively.When test result is effective, nature controlling line shows certain light intensity.This
When p-wire on light signal strength than nature controlling line light signal strength ratio（T/C) become positive correlation with concentration of specimens, by mark
Directrix curve calculates and can draw testing 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 analyser after terminating by reaction.Each concentration point of calibration object
Repeat to do 3 times, after taking the meansigma methodss of T/C area ratio, generate a standard curve with concentration（Range of linearity 0.5-200 μ g/mL）.
Standard curve determination result is as shown in the table, and standard curve is as shown in Figure 3.
Linearly
| x1 | x2 | x3 | x4 | x5 | |
| 0.62 | 4.54 | 51.14 | 87.12 | 187.44 | |
| 0.49 | 4.77 | 50.96 | 81.71 | 198.49 | |
| 0.55 | 5.82 | 46.57 | 80.51 | 192.35 | |
| Meansigma methodss | 0.55 | 5.04 | 49.56 | 83.11 | 192.76 |
| SD | 0.06 | 0.68 | 2.59 | 3.52 | 5.54 |
| CV | 11.76% | 13.53% | 5.22% | 4.24% | 2.87% |
| Theoretical value | Meansigma methodss | ||||
| x1 | 0.55 | 0.55 | |||
| x2 | 5.04 | 4.92 | |||
| x3 | 49.56 | 48.60 | |||
| x4 | 83.11 | 96.66 | |||
| x5 | 192.76 | 192.76 |
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 reagent paper
The repeatability of bar：
Collect clinical sample 200, with German Roche（ROCHE）Medical diagnostic prods c reactive protein detection kit（Immunity ratio
Turbid method）Carry out comparison and detection, pattern detection value is as shown in Figure 4 it is seen that work as r>When 0.975 it was demonstrated that with German Roche（ROCHE）
The testing result of medical diagnostic prods is equal to, and has same effectiveness.
（Two）Dispersibility with regard to fluorescent latex microsphere and springing up property
After the completion of the chromatography of 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 the signal of telecommunication for unit（Fluorescence intensity）.Amount to and export 300 signals of telecommunication, such as Fig. 5
Shown.
In Fig. 5, vertical coordinate：Fluonescence Intensity luminous intensity；Abscissa：The position of detection section 21 is long
Degree（Detection section 21 is 0 near a side of the second linkage section 22）；What abscissa 50-100 surveyed is nature controlling line 7, and value is 50-
The peak area of 100 this sections.What abscissa 200-250 surveyed is detection line 5, and value is the peak area of this section of 200-250.
As can be seen from Figure 5, the fluorescent latex microsphere of surface active easily forms monodisperse status, improves springing up property of microsphere
Energy.When the detection line of blood movement to antigen, the complex of determinand and reagent just can sufficiently be specifically bound
（200-250 section peak value）.Peak is clear, and in detection section 21, in addition to detection line with nature controlling line, unstressed configuration microsphere remains.
The present embodiment be obtained test strips, detection sensitivity height, high specificity, luminous efficiency height and being capable of quantitative response blood
The concentration of detectable substance in liquid.
Embodiment 6
The fluorescence immune chromatography 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 dispersibility and springing up property of fluorescent latex microsphere is detected, method is shown in Fig. 6, this enforcement with embodiment 5 result
The having the effect that of fluorescence immune chromatography test paper bar that example is obtained：The fluorescent latex microsphere of surface active easily forms single dispersing shape
State, improves microsphere and springs up performance.When the detection line of blood movement to antigen, the complex of determinand and reagent just can be carried out
Sufficiently specifically bind（200-250 section peak value）.Peak is clear, in detection section 21 in addition to detection line with nature controlling line unstressed configuration
Microsphere remains.
The present embodiment be obtained test strips, detection sensitivity height, high specificity, luminous efficiency height and being capable of quantitative response blood
The concentration of detectable substance in liquid.
Embodiment 7
The fluorescence immune chromatography 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 dispersibility and springing up property of fluorescent latex microsphere 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 microsphere of surface active easily forms single dispersing shape
State, improves microsphere and springs up performance.When the detection line of blood movement to antigen, the complex of determinand and reagent just can be carried out
Sufficiently specifically bind（200-250 section peak value）.Peak is clear, in detection section 21 in addition to detection line with nature controlling line unstressed configuration
Microsphere remains.
The present embodiment be obtained test strips, detection sensitivity height, high specificity, luminous efficiency height and being capable of quantitative response blood
The concentration of detectable substance in liquid.
Embodiment 8
The fluorescence immune chromatography test paper bar of the label working solution preparation being obtained using embodiment 4
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 dispersibility and springing up property of fluorescent latex microsphere 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 microsphere of surface active easily forms single dispersing shape
State, improves microsphere and springs up performance.When the detection line of blood movement to antigen, the complex of determinand and reagent just can be carried out
Sufficiently specifically bind（200-250 section peak value）.Peak is clear, in detection section 21 in addition to detection line with nature controlling line unstressed configuration
Microsphere remains.
The present embodiment be obtained test strips, detection sensitivity height, high specificity, luminous efficiency height and being capable of quantitative response blood
The concentration of detectable substance in liquid.
Comparative example
The fluorescence immune chromatography 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 dispersibility and springing up property detection method：1. unactivated micro-
Ball, climbs film effect poor.Latex beadses easily condense, and simultaneously because of its hydrophobicity, easily non-specific adsorption occur, cause granule coalescence.
2., in the chromatography process in detection, the fluorescent microsphere of powerful adsorptivity conglomerate is difficult to re-form mono-dispersion microballoon layer
Analysis.3. because being coated pad（NC film）It is porous network structure film it is thus possible to be unfavorable for chromatographing the non-of detection with larger particles
Specific adsorption and lead to bulky grain fluorescent microsphere release property poor.4. before causing NC film（Between detection line the 5 to the first linkage section 20
Detection section 21）There are substantially a large amount of fluorescent microsphere residuals at end.200-300 section in Fig. 9, test peak is difficult to differentiate.
Embodiment described above is only the preferred embodiment lifted for absolutely proving the present invention, the protection model of the present invention
Enclose not limited to this.Equivalent substitute or conversion that those skilled in the art are made on the basis of the present invention, all in the present invention
Protection domain within.Protection scope of the present invention is defined by claims.
Claims (10)
1. a kind of whole process C reactive protein fluorescence immune chromatography with activation fluorescent latex microsphere preparation method it is characterised in that
Comprise the steps：
1）Take surfactant to add in the buffer solution that pH is 8 ~ 10, add dimethylformamide, N, N '-dicyclohexyl carbon
Diimine and N-hydroxy-succinamide；
2）Take the dispersion liquid of the fluorescent latex microsphere of amino surface, adjusted after pH to 8 ~ 10 with buffer solution, be added to step 1）Institute
In the mixture obtaining, stirring reaction, after completion of the reaction, centrifugation removes supernatant, obtains activating fluorescent latex microsphere；
Wherein, described surfactant is the mixing of amino acid type surfactant, cithrol and stearate
Thing, the weight of three is than for 1 ~ 5:1~2:0.5~3.
2. preparation method according to claim 1 is it is characterised in that described amino acid type surfactant is lauroyl paddy
One of propylhomoserin salt, N- acyl glutamate or dodecyl dimethylene amino diformate or its two or more group
Close；Described stearate is one of stearic potassium, sodium, ammonium salt and tri ethanol ammonium salt or its two or more combination.
3. preparation method according to claim 2 is it is characterised in that described amino acid type surfactant is lauroyl paddy
Propylhomoserin sodium；Described stearate is sodium stearate；Described cithrol is polyethylene glycol monolaurate.
4. preparation method according to claim 1 it is characterised in that described surfactant and described amino surface glimmering
The mass ratio of light latex beadses is 500 ~ 2000:1.
5. preparation method according to claim 1 is it is characterised in that step 1）With step 2）Described in buffer solution be
Carbonic acid buffer；Step 2）In the stirring reaction time be 2 ~ 4 hours, temperature be 20 ~ 30 DEG C；The fluorescent latex of amino surface is micro-
Ball is aminopolystyrene fluorescent microsphere.
6. the whole C reactive protein fluorescence immune chromatography activation that the preparation method described in any one of claim 1 ~ 5 obtains is glimmering
Light latex beadses.
7. the label work of activation fluorescent latex microsphere of the whole C reactive protein fluorescence immune chromatography described in claim 6
The preparation method of liquid is it is characterised in that take whole C reactive protein fluorescence immune chromatography activation fluorescent latex microsphere to be scattered in
In microsphere buffer, obtain label working solution；The shared ratio in label working solution of the fluorescent latex microsphere of surface active
Example is 0.5 ~ 2wt%；
Wherein, the preparation method of microsphere buffer is：By BSA, biological preservative, Lamepon A and glycine betaine be dissolved in 0.01M,
In the phosphate buffer of pH7.4.
8. preparation method according to claim 7 is it is characterised in that BSA, biological preservative, Lamepon A and glycine betaine
Concentration in described microsphere buffer is 0.1wt%.
9. the labeling pad of whole C reactive protein fluorescence immune chromatography test paper bar preparation method it is characterised in that include as
Lower step:
1）The preparation of the rabbit igg of activation fluorescent latex microsphere labelling：Take the labelling that preparation method described in claim 7 or 8 obtains
Thing working solution, centrifugation, after abandoning supernatant, redissolved with labelling buffer, and be simultaneously introduced carbodiimide and rabbit igg, stirring is anti-
Should, it is then centrifuged for, abandons supernatant, finally use labelling diluent to redissolve；
2）The labelling of the activation fluorescent latex microsphere labelling preparation method of whole C reactive protein monoclonal antibody：Weighting profit will
Seek the label working solution that preparation method described in 7 or 8 obtains, centrifugation, redissolved with labelling buffer after abandoning supernatant, and simultaneously
Add carbodiimide and labelling whole C reactive protein monoclonal antibody, stirring reaction, be then centrifuged for, abandon supernatant, finally
Redissolved with labelling diluent；
3）Take step 1）Gained dispersion liquid and step 2）Gained dispersion liquid, mixing, it is sprayed on glass fibre, dry；
The formula of described labelling buffer is：Sodium carbonate 4.33g, sodium bicarbonate 2.96g, are dissolved in 1000mL water；Described labelling
The formula of diluent is：Trisodium citrate 7.33g, citric acid 4.44g, sodium hydroxide 1g, are dissolved in 1000mL water.
10. a kind of whole process C reactive protein 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 and be coated with whole C reactive protein monoclonal antibody.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1576842A (en) * | 2003-06-30 | 2005-02-09 | 希森美康株式会社 | Sample pretreatment solution for immunological test and method for using the same |
| US20060240438A1 (en) * | 2003-07-28 | 2006-10-26 | Yukio Nagasaki | Surface of base material being inhibited in non-specific adsorption |
| CN102023211A (en) * | 2010-11-19 | 2011-04-20 | 广州万孚生物技术有限公司 | Immunochromatographic test strip for full quantitative detection of C-reactive protein and preparation method thereof |
| CN104193873A (en) * | 2014-08-22 | 2014-12-10 | 深圳市汇松科技发展有限公司 | Polystyrene microspheres of fluorescence groups, preparation method and application of polystyrene microsphere, latex conjugate as well as preparation method and application of latex conjugate |
-
2016
- 2016-09-06 CN CN201610804731.0A patent/CN106405108B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1576842A (en) * | 2003-06-30 | 2005-02-09 | 希森美康株式会社 | Sample pretreatment solution for immunological test and method for using the same |
| US20060240438A1 (en) * | 2003-07-28 | 2006-10-26 | Yukio Nagasaki | Surface of base material being inhibited in non-specific adsorption |
| CN102023211A (en) * | 2010-11-19 | 2011-04-20 | 广州万孚生物技术有限公司 | Immunochromatographic test strip for full quantitative detection of C-reactive protein and preparation method thereof |
| CN104193873A (en) * | 2014-08-22 | 2014-12-10 | 深圳市汇松科技发展有限公司 | Polystyrene microspheres of fluorescence groups, preparation method and application of polystyrene microsphere, latex conjugate as well as preparation method and application of latex conjugate |
Non-Patent Citations (1)
| Title |
|---|
| 张改平: "《免疫层析试纸快速检测技术》", 31 August 2015, 河南科学技术出版社 * |
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