CN106405108B - Whole C reactive proteins fluorescence immune chromatography activation fluorescent latex microballoon - Google Patents
Whole C reactive proteins fluorescence immune chromatography activation fluorescent latex microballoon Download PDFInfo
- Publication number
- CN106405108B CN106405108B CN201610804731.0A CN201610804731A CN106405108B CN 106405108 B CN106405108 B CN 106405108B CN 201610804731 A CN201610804731 A CN 201610804731A CN 106405108 B CN106405108 B CN 106405108B
- Authority
- CN
- China
- Prior art keywords
- whole
- fluorescent latex
- microballoon
- reactive protein
- mark
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- 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
-
- 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
-
- 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
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- Pathology (AREA)
- Food Science & Technology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a kind of whole C reactive proteins fluorescence immune chromatography activation fluorescent latex microballoon and application.A kind of whole C reactive proteins fluorescence immune chromatography preparation method of activation fluorescent latex microballoon of the present invention, comprises the following steps：1）Take surfactant to add in the cushioning liquid that pH is 8 ~ 10, add dimethylformamide, N, N ' dicyclohexylcarbodiimides and N HOSu NHSs；2）The dispersion liquid of the fluorescent latex microballoon of amino surface is taken, after adjusting pH to 8 ~ 10 with cushioning liquid, is added to step 1）In the mixture of gained, stirring reaction, after completion of the reaction, centrifugation remove supernatant, obtain the fluorescent latex microballoon of surface active；Wherein, the surfactant is the mixture of amino acid type surfactant, cithrol and stearate, and the weight ratio of three is 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 C reactive protein 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, fast inspection field is widely used in.Determinand
Antibody labeling fluorescent latex microballoon, there is surface electronic repulsion and Van der Waals force in the liquid phase to cause in liquid phase for latex beads
Latex particle keeps being uniformly distributed.But as solution is evaporated dry, surface tension after being sprayed at glass fibre element film and drying
It is unlimited close to causing intermolecular repulsion to disappear between promotion latex particle, Van der Waals force increase, finally cause multiple latex micels
It is polymerized to big and small different particle.When sample redissolution latex beads is added when immunochromatography, latex beads no longer uniformly divides
Dissipating causes chromatography to fail.Therefore this area uses fluorescence immune chromatography kit more, and sample buffer is added containing test strips.Increase
Detection complexity.Or due to latex beads partial agglomeration so that detection sensitivity reduces, and false negative occurs in testing result.
In addition, latex beads is unstable, it can also make it that the stability of fluorescent test paper strip is 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 inflammatory cytokine such as IL-6 discharge
With the presence of inflammation.Because C reactive protein generally increases after bacterium infection, and do not increase during virus infection, so measure people's blood
Middle CRP contents are for having discriminating bacteria and virus infection significance.
The content of the invention
The technical problems to be solved by the invention are：The fluorescent latex microballoon of fluorescence immune chromatography test paper bar after the drying,
Easily reunite, it is scattered uneven, so as to cause chromatography failure or cause detection sensitivity to reduce.
In order to solve the above-mentioned technical problem, the invention provides a kind of whole C reactive protein fluorescence immune chromatography test paper bar
Activation fluorescent latex microballoon and its preparation, the whole C reactive protein dry type prepared using the activation fluorescent latex microballoon are glimmering
Light immuno-chromatographic test paper strip, this test strips is available for whole C reactive protein in Quantitative in vitro measure human serum, blood plasma or whole blood
Content.The test strips of the present invention can solve what fluorescent latex microballoon was reunited in spraying, drying glass fibre element film
Phenomenon, to prepare labeling pad, so as to obtain dry type fluorescence immune chromatography test paper bar, detection reagent is configured without dilution
Box.
The preparation method of whole C reactive protein fluorescence immune chromatography activation fluorescent latex microballoon provided by the invention, bag
Include following steps：
1）Take surfactant to add in the cushioning liquid that pH is 8 ~ 10, add dimethylformamide, N, the hexamethylenes of N '-two
Base carbodiimide and n-hydroxysuccinimide；
2）The dispersion liquid of the fluorescent latex microballoon of amino surface is taken, after adjusting pH to 8 ~ 10 with cushioning liquid, is added to step
1）In the mixture of gained, stirring reaction, after completion of the reaction, centrifugation remove supernatant, obtain activating fluorescent latex microballoon；
Wherein, the surfactant is amino acid type surfactant, cithrol and stearate
Mixture, the weight ratio of three is 1 ~ 5: 1~2:0.5~3.
Preferably, the amino acid type surfactant is lauroyl glutamate, N- acyl glutamates or dodecane
One kind or its two or more combination in base dimethylene amino diformate；The stearate be stearic potassium, sodium,
One kind or its two or more combination in ammonium salt and tri ethanol ammonium salt.
Preferably, the amino acid type surfactant is sodium lauroyl glutamate；The stearate is odium stearate；
The cithrol is polyethylene glycol monolaurate.
Preferably, the mass ratio of the surfactant and the fluorescent latex microballoon of the amino surface is 500 ~ 2000:
1。
Preferably, step 1）With step 2）Described in cushioning liquid be carbonic acid buffer；Step 2）In stirring reaction
Time is 2 ~ 4 hours, and temperature is 20 ~ 30 DEG C；The fluorescent latex microballoon of amino surface is aminopolystyrene fluorescent microsphere.
The present invention also provides the whole C reactive protein fluorescence immune chromatography activation fluorescence that above-mentioned preparation method obtains
Latex beads.
The present invention also provides the mark of above-mentioned whole C reactive protein fluorescence immune chromatography activation fluorescent latex microballoon
The preparation method of thing working solution：Take whole C reactive protein fluorescence immune chromatography to be scattered in microballoon with activation fluorescent latex microballoon to delay
In fliud flushing, label working solution is obtained；The fluorescent latex microballoon of surface active ratio shared in label working solution is 0.5
~2wt%；
Wherein, the preparation method of microballoon buffer solution is：BSA, biological preservative, Lamepon A and glycine betaine are dissolved in
In 0.01M, pH7.4 phosphate buffer.
Preferably, BSA（Bovine serum albumin）, biological preservative, Lamepon A and glycine betaine be in the microballoon buffer solution
Concentration be 0.1wt%.
The present invention also provides the preparation method of the labeling pad of whole C reactive protein fluorescence immune chromatography test paper bar, including
Following steps:
1）Activate the preparation of the rabbit igg of fluorescent latex microballoon mark：The label working solution that above-mentioned preparation method obtains is taken,
Centrifugation, after abandoning supernatant, redissolved with mark buffer solution, while adding carbodiimide and rabbit igg, stirring reaction, Ran Houli
The heart, supernatant is abandoned, finally redissolved with mark dilution；
2）Activate the preparation method of the whole C reactive protein monoclonal antibody of mark of fluorescent latex microballoon mark：Take
The label working solution that preparation method obtains is stated, is centrifuged, is redissolved after abandoning supernatant with mark buffer solution, while adding carbon two
Imines and the whole C reactive protein monoclonal antibody of mark, stirring reaction, are then centrifuged for, abandon supernatant, finally dilute with marking
Release liquid redissolution；
3）Take step 1）Gained dispersion liquid and step 2）Gained dispersion liquid, mixing, is sprayed on glass fibre, dries；
It is described mark buffer solution formula be：Sodium carbonate 4.33g, sodium acid carbonate 2.96g, are dissolved in 1000mL water；It is described
Mark dilution formula be：Trisodium citrate 7.33g, citric acid 4.44g, sodium hydroxide 1g, are dissolved in 1000mL water.
The present invention provides a kind of whole C reactive protein fluorescence immune chromatography test paper bar, including above-mentioned labeling pad, bag
Padded and absorption pad, wherein the labeling pad is overlapped on one end of coating pad, absorption pad is overlapped in the other end of coating pad
On, bag is covered with provided with nature controlling line and detection line, and the antibody of goat anti-rabbit igg is coated with nature controlling line, bag is coated with detection line
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 microballoon through the activation of specific surfactant keeps particle in drying
Between relative distance be not easy to reunite.During chromatography process, adding sample can at once redissolve and smoothly chromatograph, to prepare dry type fluorescence
Immuno-chromatographic test paper strip（Reagent card）, it is user-friendly, and testing result is stable, accurate, reliable.
2nd, the present invention selects the fluorescence for being suitable for whole C reactive protein fluorescence immune chromatography test paper bar by testing sieve
The specific surfactant combination of latex beads.The fluorescent latex for handling chemical modification in advance using specific surfactant is micro-
Ball, the latex beads after processing marks whole C reactive protein antibody, for preparing whole C reactive protein fluorescence immune chromatography
The labeling pad of test strips, realize latex beads even application, soilless sticking phenomenon.Also, after sample to be tested is added, mark
Fluorescent latex microballoon can quickly, smoothly realize chromatography, without special sample dilution or buffer solution processing test strips.
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, high sensitivity.
Brief description of the drawings
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 samples.
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.
Embodiment
The invention will be further described with specific embodiment below in conjunction with the accompanying drawings, so that those skilled in the art can be with
It is better understood from the present invention and can be practiced, but illustrated embodiment is not as a limitation of the invention.
Used the invention provides a kind of whole C reactive protein fluorescence immune chromatography and activate fluorescent latex microballoon, including it is as follows
Step：
1）Take surfactant to add in the cushioning liquid that pH is 8 ~ 10, add dimethylformamide, N, N '-dicyclohexyl
Carbodiimide and n-hydroxysuccinimide, stirring reaction；
2）The dispersion liquid of the fluorescent latex microballoon of amino surface is taken, after adjusting pH to 8 ~ 10 with cushioning liquid, is added to step
1）In the mixture of gained, stirring reaction, after completion of the reaction, centrifugation remove supernatant, obtain activating fluorescent latex microballoon；
Wherein, the surfactant is amino acid type surfactant, cithrol and stearate
Mixture, the weight ratio of three is 1 ~ 5:1~2:0.5~3.
The present invention uses the mixture of amino acid type surfactant, cithrol and stearate（It is cloudy from
Subtype, nonionic and amphoteric surfactant mixing）Latex beads outer surface is modified by the method for covalent coupling, makes its tool
There is hydrophobicity.Whole C reactive protein antibody is marked using the activation fluorescent latex microballoon after processing.Fluorescent latex microballoon marks
The rabbit igg that marks of whole C reactive protein antibody and fluorescent latex microballoon mix after be sprayed on glass fibre element film and be prepared into
Labeling pad 1, it is coated with pad 2（Nitrocellulose filter）Close to the upper adsorptive pads of one end of nature controlling line covering, close to the another of detection line
Overlay marks thing pad is held, be coated with coating for preparing dry type fluorescence immune chromatography reagent card, in detection line reacts egg with whole C-
Bai Kangti.After adding sample to be tested, whole C reactive protein connects with the whole C reactive protein antibody that fluorescent latex microballoon marks
Reaction is touched, latex beads can keep dispersity not reunite, and can be rapid, dispersed after contact measured sample, and edge
Nitrocellulose filter flash chromatography.
Fluorescent latex microballoon used refers to the polyphenyl second for the amino surface being modified by sulphation in following examples of the present invention
Alkene fluorescent microsphere, diameter 100-500nm.
First, the preparation of fluorescent latex microballoon is activated
Embodiment 1
1）Take surfactant（4 mg sodium lauroyl glutamates, 4mg polyethylene glycol monolaurates, 2mg odium stearate）
It is initially dissolved in 0.5M pH9.6 carbonic acid buffer（H values are to 9.6）, add in 0.1ml DMF, in 0.1ml DMF,
Add N, N '-dicyclohexylcarbodiimide (DCC) 1mg and 0.6mg n-hydroxysuccinimides（NHS）After to be stirred at room temperature 3 small
When.
2）By the fluorescent latex microspheres solution 0.5M pH9.6 of amino surfaces of the 1ml containing 1% solids carbonic acid buffer
PH value is adjusted to being added to step 1 after 9.6）In, continue stirring reaction 3 hours.Both the latex beads that must have been activated.
3）Fluorescent latex microballoon reaction solution is centrifuged at a high speed with supercentrifuge, removes reaction solution supernatant, is used
1mL microballoons buffer solution redissolves fluorescent latex microballoon, forms label working solution.
Microballoon phosphate buffer is prepared：0.01M pH7.4 phosphate buffer, BSA containing 0.1wt%, given birth to containing 0.1 wt %
Thing preservative, containing 0.1 wt % Lamepon As, containing 0.1 wt % glycine betaines.Microballoon phosphate buffer provides certain Ionic strength
And pH value, make microballoon dispersed.
Embodiment 2
The operating method of the present embodiment is similar to Example 1, and difference is, step 1）Middle surfactant is：5
Mg sodium lauroyl glutamates, 1mg polyethylene glycol monolaurates, 3mg odium stearate.
Embodiment 3
The operating method of the present embodiment is similar to Example 1, and difference is, step 1）Middle surfactant is：6
Mg N- acyl glutamic acid sodium, 4mg polyethylene glycol monolaurates, 0.5mg potassium stearates.
Embodiment 4
The operating method of the present embodiment is similar to Example 1, and difference is, step 1）Middle surfactant is：4
Mg dodecyl dimethylene amino sodium diformates, 4mg polyethylene glycol monolaurates, 2mg odium stearate.
2nd, the activation fluorescence breast prepared by taking whole C reactive protein fluorescence immune chromatography test paper bar as an example to embodiment 1 ~ 4
The application of glue microballoon and effect illustrate.
Embodiment 5
Fluorescence immune chromatography test paper bar prepared by the label working solution obtained using embodiment 1
1）The rabbit igg of fluorescent latex microballoon mark：Take the label working solution that 5mL embodiments 1 obtain, with centrifuge from
Heart 30min（Rotating speed is 10000r/min）, supernatant is abandoned after having centrifuged, is redissolved with 5mL mark buffer solutions, adds 1mg rabbit
IgG is mixed, another to add 5mg carbodiimides, and reaction 1h is stirred at room temperature, is then centrifuged for 15min（Rotating speed is 10000r/min）, abandon
Supernatant, redissolved with 10mL label diluted standby after mixing.
2）The whole C reactive protein monoclonal antibody of the mark of fluorescent latex microballoon mark
The label working solution that 10mL embodiments 1 obtain is taken, with centrifuge 30min（Rotating speed is 10000r/min）,
Supernatant is abandoned after having centrifuged, is redissolved with 10mL mark buffer solutions, the mark for adding 2mg is resisted with whole C reactive protein monoclonal
Body mixes, another to add 10mg carbodiimides, and reaction 1h is stirred at room temperature, is then centrifuged for 15min（Rotating speed is 10000r/min）, abandon
Supernatant, redissolved with 20mL label diluted standby after mixing.
Step 1）With 2）It is middle mark buffer solution formula be：Sodium carbonate 4.33g, sodium acid carbonate 2.96g, are dissolved in 1000mL water
In；Mark dilution formula be：Trisodium citrate 7.33g, citric acid 4.44g, sodium hydroxide 1g, are dissolved in 1000mL water.
3）It is prepared by labeling pad
Step 2）Mark the determinand antibody and step 1 of obtained fluorescent latex microballoon mark）Obtained fluorescent latex
The rabbit igg of microballoon mark mixes, and the two volume ratio is 2:1.Then by the fluorescent latex microballoon of mixing by 1 microlitre/centimetre
The drying baker for measuring even application 45 DEG C of transposition on glass fibre element paper dries the preparation for completing labeling pad in 2 hours.
4）It is prepared by coating buffer：Take coating to be added with whole C reactive protein monoclonal antibody in phosphate buffer, be made
Detection line coating buffer；Take goat anti-rabbit igg to add in phosphate buffer, nature controlling line coating buffer is made.The phosphate buffer
It is formulated and is：0.99g sodium dihydrogen phosphates, 5.16g disodium hydrogen phosphates are dissolved in 1000mL purified waters.
5）It is coated with the processing of pad：Detection line coating buffer is covered with line coating in bag and forms detection line, nature controlling line coating buffer
Nature controlling line is formed being covered with line coating in bag, is then dried.
6）Test strips assemble：
By dried labeling pad and the absorption pad cut out（Blotting paper）, padded by coating（Nitrocellulose filter）It is close
The upper absorption pad of one end covering of nature controlling line, the requirement close to the other end overlay marks thing pad of detection line carry out joint strip.By slitting
Machine standard practice instructions is operated, and is cut into the wide test strips of 4mm ± 0.1mm.
As illustrated in fig. 1 and 2, test strips include：It is coated with pad 2（Nitrocellulose filter）, its both ends is respectively provided with a linkage section（Such as
First linkage section 20 shown in figure, the second linkage section 22）, two linkage sections centre is detection section 21, and detection section 21 surface, which is provided with, to be detected
Line 5 and nature controlling line 7；Labeling pad 1（Glass fibre element film）, its one end is provided with linkage section 10, and the linkage section 10 of labeling pad covers
Cover and be fixed on the first linkage section 20 of coating pad 2；Label 8 is coated with labeling pad 1, specifically in an embodiment
In, it is near linkage section 10（Away from linkage section 2mm, length of the label along coating pad length direction is 5mm）Spraying mark
Remember thing 8（The whole C reactive protein Dan Ke of the mark of rabbit igg containing fluorescent latex microballoon mark and fluorescent latex microballoon mark
Grand 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 coating pad
On 2 the second linkage section 22；
Bottom plate 4, coating pad 2, labeling pad 1 and absorption pad 3 are both secured on bottom plate.
This test strips is when in use with one plant of whole C reactive protein monoclonal antibody（The whole C reactive protein list of coating
Clonal antibody）Rule and be coated with nitrocellulose filter, as p-wire, resisted with goat-anti rabbit more and rule on nitrocellulose filter
Nature controlling line is made in coating.Another plant of whole C reactive protein monoclonal antibody that latex fluorescent microsphere is marked（Mark is whole
C reactive protein monoclonal antibody）It is sprayed on glass fibre element film and prepares after being mixed with the rabbit igg of latex fluorescent microsphere mark
Into labeling pad.Nitrocellulose filter is close to the upper adsorptive pads of one end of nature controlling line covering, close to the other end covering mark of p-wire
Remember thing pad.Standard items or blood sample to be measured are added on toward labeling pad, antigen will be with label hybrid reaction and fine along nitric acid
Plain film layer analysis is tieed up, is reacted respectively with p-wire and nature controlling line.When test result is effective, nature controlling line shows certain luminous intensity.This
When p-wire on light signal strength than nature controlling line light signal strength ratio（T/C) mark is passed through into positive correlation with concentration of specimens
Directrix curve, which calculates, can draw testing sample concentration.
（One）The measure of test strips calibration curve and sample to be tested made from the present embodiment
50ul calibration objects or sample to be tested are slowly added dropwise in labeling pad.Stand 15 minutes instead at ambient temperature
Should, test strips are put into Savant-100 fluorescence immune chromatography analyzers and detected by reaction after terminating.Calibration object it is each dense
Degree point repeat does 3 times, take T/C areas than average value after with concentration generate a standard curve（Range of linearity 0.5-200 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 | |
| Average value | 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 | Average value | ||||
| 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 samples in the range of line taking repeat detection 15 times, its coefficient of variation（CV%）15.0% should be not higher than, to verify
The repeatability of test strips：
Clinical sample 200 is collected, with German Roche（ROCHE）Medical diagnostic prods c reactive protein detection kit（Exempt from
Epidemic disease turbidimetry）Contrasting detection is carried out, pattern detection value is as shown in Figure 4, it is seen that works as r>When 0.975, it was demonstrated that with German Roche
（ROCHE）The testing result of medical diagnostic prods is equal, and has same validity.
（Two）On the dispersiveness of fluorescent latex microballoon and springing up property
After the completion of the chromatography of test strips, the fluorescence signal in the detection section 21 of coating pad is gathered using instrument, it will detection
Section is bisected into 300 sections along its length, and every section is that unit exports electric signal（Fluorescence intensity）.300 electric signals are exported altogether, such as
Shown in Fig. 5.
In Fig. 5, ordinate：Fluonescence Intensity luminous intensities；Abscissa：Detect the position length of section 21
Degree（It is 0 that section 21, which is detected, close to a side of the second linkage section 22）；What abscissa 50-100 was surveyed is nature controlling line 7, and value is 50-
The peak area of 100 this sections.What abscissa 200-250 was surveyed is detection line 5, and value is the peak area of this sections of 200-250.
As can be seen from Figure 5, the fluorescent latex microballoon of surface active easily forms monodisperse status, improves springing up property of microballoon
Energy.When blood is moved to the detection line of antigen, the compound of determinand and reagent can be sufficiently to be specifically bound
（200-250 section peak values）.Peak is clear, and unstressed configuration microballoon remains in addition to detection line and nature controlling line in detection section 21.
Test strips made from the present embodiment, detection sensitivity height, high specificity, luminous efficiency are high and being capable of quantitative reaction blood
The concentration of detectable substance in liquid.
Embodiment 6
Fluorescence immune chromatography test paper bar prepared by the label working solution 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）It is middle to use in fact
Apply the label working solution that the label working solution substitution embodiment 1 that example 2 obtains obtains.
The dispersiveness of fluorescent latex microballoon and springing up property are detected, method is shown in Fig. 6, this implementation with the result of embodiment 5
Fluorescence immune chromatography test paper bar has the effect that made from example：The fluorescent latex microballoon of surface active easily forms single dispersing shape
State, improve microballoon and spring up performance.When blood is moved to the detection line of antigen, the compound of determinand and reagent can be with progress
Sufficiently specific binding（200-250 section peak values）.Peak is clear, detects in section 21 unstressed configuration in addition to detection line and nature controlling line
Microballoon remains.
Test strips made from the present embodiment, detection sensitivity height, high specificity, luminous efficiency are high and being capable of quantitative reaction blood
The concentration of detectable substance in liquid.
Embodiment 7
Fluorescence immune chromatography test paper bar prepared by the label working solution 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）It is middle to use in fact
Apply the label working solution that the label working solution substitution embodiment 1 that example 3 obtains obtains.
The dispersiveness of fluorescent latex microballoon and springing up property are detected, as a result method is shown in Fig. 7, this implementation with embodiment 5
Fluorescence immune chromatography test paper bar has the effect that made from example：The fluorescent latex microballoon of surface active easily forms single dispersing shape
State, improve microballoon and spring up performance.When blood is moved to the detection line of antigen, the compound of determinand and reagent can be with progress
Sufficiently specific binding（200-250 section peak values）.Peak is clear, detects in section 21 unstressed configuration in addition to detection line and nature controlling line
Microballoon remains.
Test strips made from the present embodiment, detection sensitivity height, high specificity, luminous efficiency are high and being capable of quantitative reaction blood
The concentration of detectable substance in liquid.
Embodiment 8
Fluorescence immune chromatography test paper bar prepared by the label working solution 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）It is middle to use in fact
Apply the label working solution that the label working solution substitution embodiment 1 that example 4 obtains obtains.
The dispersiveness of fluorescent latex microballoon and springing up property are detected, as a result method is shown in Fig. 8, this implementation with embodiment 5
Fluorescence immune chromatography test paper bar has the effect that made from example：The fluorescent latex microballoon of surface active easily forms single dispersing shape
State, improve microballoon and spring up performance.When blood is moved to the detection line of antigen, the compound of determinand and reagent can be with progress
Sufficiently specific binding（200-250 section peak values）.Peak is clear, detects in section 21 unstressed configuration in addition to detection line and nature controlling line
Microballoon remains.
Test strips made from the present embodiment, detection sensitivity height, high specificity, luminous efficiency are high and being capable of quantitative reaction blood
The concentration of detectable substance in liquid.
Comparative example
The fluorescence immune chromatography test paper bar of this comparative example is similar to Example 5, and difference is step 1）With 2）It is middle to use city
The label working solution that the aminopolystyrene fluorescent microsphere dispersion liquid substitution embodiment 1 sold obtains.
Dispersiveness and springing up property detection method are with embodiment 1, and testing result is shown in Fig. 9, as can be seen from Figure 9：It is 1. unactivated micro-
Ball, it is poor to climb film effect.Latex beads easily condenses, while because of its hydrophobicity, non-specific adsorption easily occurs, causes particle coalescence.
2. in the chromatography process in detection, the fluorescent microsphere of powerful adsorptivity conglomerate is difficult to re-form mono-dispersion microballoon layer
Analysis.3. because coating pads（NC films）It is porous network structure film, it is thus possible to be unfavorable for the non-of chromatography detection with larger particles
Specific adsorption and cause bulky grain fluorescent microsphere release property poor.4. before causing NC films（Between the linkage section of detection line 5 to the first 20
Detection section 21）There are obvious a large amount of fluorescent microsphere residuals at end.200-300 sections in Fig. 9, test peak are difficult to differentiate.
Embodiment described above is only to absolutely prove preferred embodiment that is of the invention and being lifted, protection model of the invention
Enclose not limited to this.The equivalent substitute or conversion that those skilled in the art are made on the basis of the present invention, in the present invention
Protection domain within.Protection scope of the present invention is defined by claims.
Claims (7)
- A kind of 1. preparation method of whole C reactive protein fluorescence immune chromatography activation fluorescent latex microballoon, it is characterised in that Comprise the following steps：1）Take surfactant to add in the carbonic acid buffer that pH is 8 ~ 10, add dimethylformamide, N, N '-dicyclohexyl Carbodiimide and n-hydroxysuccinimide；2）The dispersion liquid of the fluorescent latex microballoon of amino surface is taken, after adjusting pH to 8 ~ 10 with carbonic acid buffer, is added to step 1） In the mixture of gained, stirring reaction 2 ~ 4 hours at 20 ~ 30 DEG C, after completion of the reaction, centrifugation remove supernatant, are activated Fluorescent latex microballoon, the fluorescent latex microballoon of amino surface is aminopolystyrene fluorescent microsphere；Wherein, the surfactant is the mixing of amino acid type surfactant, cithrol and stearate Thing, the weight ratio of three is 1 ~ 5:1~2:0.5~3；The matter of the surfactant and the fluorescent latex microballoon of the amino surface Amount is than being 500 ~ 2000:1；The amino acid type surfactant is lauroyl salt, N- acyl glutamates or dodecyl two One kind or its two or more combination in methene amido diformate.
- 2. preparation method according to claim 1, it is characterised in that the stearate is stearic potassium, sodium, ammonium salt And one kind in tri ethanol ammonium salt or its two or more combination.
- 3. preparation method according to claim 2, it is characterised in that the amino acid type surfactant is lauroyl paddy Propylhomoserin sodium；The stearate is odium stearate；The cithrol is polyethylene glycol monolaurate.
- 4. the whole C reactive protein fluorescence immune chromatography activation that the preparation method described in any one of claim 1 ~ 3 obtains is glimmering Light latex beads.
- 5. the whole C reactive protein fluorescence immune chromatography described in claim 4 is worked with the label of activation fluorescent latex microballoon The preparation method of liquid, it is characterised in that take whole C reactive protein fluorescence immune chromatography to be scattered in activation fluorescent latex microballoon In microballoon buffer solution, label working solution is obtained；The fluorescent latex microballoon of surface active ratio shared in label working solution Example is 0.5 ~ 2wt%；Wherein, the preparation method of microballoon buffer solution is：By BSA, biological preservative, Lamepon A and glycine betaine be dissolved in 0.01M, In pH7.4 phosphate buffer；The BSA, biological preservative, Lamepon A and glycine betaine are dense in the microballoon buffer solution Degree is 0.1wt%.
- 6. the preparation method of the labeling pad of whole C reactive protein fluorescence immune chromatography test paper bar, it is characterised in that including such as Lower step:1）Activate the preparation of the rabbit igg of fluorescent latex microballoon mark：Take the label work that preparation method described in claim 5 obtains Make liquid, centrifuge, after abandoning supernatant, redissolved with mark buffer solution, while carbodiimide and rabbit igg are added, stirring reaction, so After centrifuge, abandon supernatant, finally with mark dilution redissolve；2）Activate the preparation method of the whole C reactive protein monoclonal antibody of mark of fluorescent latex microballoon mark：Weighting profit will The label working solution for asking 5 preparation methods to obtain, centrifugation, redissolved after abandoning supernatant with mark buffer solution, while plus Enter carbodiimide and the whole C reactive protein monoclonal antibody of mark, stirring reaction, be then centrifuged for, abandon supernatant, finally use Dilution is marked to redissolve；3）Take step 1）Gained dispersion liquid and step 2）Gained dispersion liquid, mixing, is sprayed on glass fibre, dries；It is described mark buffer solution formula be：Sodium carbonate 4.33g, sodium acid carbonate 2.96g, are dissolved in 1000mL water；The mark The formula of dilution is：Trisodium citrate 7.33g, citric acid 4.44g, sodium hydroxide 1g, are dissolved in 1000mL water.
- 7. a kind of whole C reactive protein fluorescence immune chromatography test paper bar, it is characterised in that including the method described in claim 6 Labeling pad, coating pad and the absorption pad being prepared, wherein the labeling pad is overlapped on one end of coating pad, absorption pad It is overlapped on the other end of coating pad, bag is covered with provided with nature controlling line and detection line, and the anti-of goat anti-rabbit igg is coated with nature controlling line Body, whole C reactive protein monoclonal antibody is coated with detection line.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610804731.0A CN106405108B (en) | 2016-09-06 | 2016-09-06 | Whole C reactive proteins fluorescence immune chromatography activation fluorescent latex microballoon |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610804731.0A CN106405108B (en) | 2016-09-06 | 2016-09-06 | Whole C reactive proteins fluorescence immune chromatography activation fluorescent latex microballoon |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106405108A CN106405108A (en) | 2017-02-15 |
| CN106405108B true CN106405108B (en) | 2018-01-16 |
Family
ID=57999820
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610804731.0A Active CN106405108B (en) | 2016-09-06 | 2016-09-06 | Whole C reactive proteins fluorescence immune chromatography activation fluorescent latex microballoon |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106405108B (en) |
Citations (3)
| 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 |
| 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 |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2322061T3 (en) * | 2003-07-28 | 2009-06-16 | Jsr Corporation | SURFACE OF BASE MATERIAL THAT IS INHIBITED IN NON-SPECIFIC ADSORTION. |
-
2016
- 2016-09-06 CN CN201610804731.0A patent/CN106405108B/en active Active
Patent Citations (3)
| 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 |
| 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 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106405108A (en) | 2017-02-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106380617B (en) | The fluorescent latex microballoon of surface active and its preparation and application | |
| CN106405071B (en) | 25 hydroxy-vitamine Ds3Fluorescence immune chromatography activation fluorescent latex microballoon | |
| CN106405110B (en) | Myoglobins fluorescence immune chromatography test paper bar activation fluorescent latex microballoon and application | |
| CN106501502B (en) | G17 fluorescence immune chromatography activation fluorescent latex microballoon | |
| CN103097895B (en) | Reagent composition for nucleic acid chromatography or immunochromatography, method for measurement by nucleic acid chromatography or immunochromatography, and kit for measurement by nucleic acid chromatography or immunochromatography | |
| FI102921B (en) | Method for Preparation of Biologically Active Reagents in Socks from iimide-Containing Polymers, Analytical Element and Methods for Using Them | |
| CN105467122A (en) | Kit and method for detection of thyroid peroxidase antibody | |
| CN103558381A (en) | Immunochromatographic test paper for detecting human immunodeficiency virus antibodies and preparation method thereof | |
| CN110873791A (en) | Indirect background fluorescent colloidal gold immunochromatographic test strip based on double-labeled signal amplification and application thereof | |
| CN108318691A (en) | Influenza A and Type B influenza virus lateral chromatography detection kit and method | |
| CN115993451B (en) | Quantitative detection kit for influenza A virus and adenovirus antigens, preparation method and quantitative detection method | |
| CN103063845A (en) | Nanometer magnetic particle chemiluminiscence kit, preparation method and detection method of hepatitis B virus surface-antibody | |
| CN103823064B (en) | A kind of vomitoxin immue quantitative detection reagent box and using method thereof | |
| CN104949948B (en) | A kind of magnetic flourescent nano material for magnetic and preparation method and application | |
| CN109459570A (en) | The colloid gold immune test paper preparation method of lead ion in a kind of quick detection blood of human body | |
| CN108548924A (en) | AngiotensinⅡ Magnetism particulate immuno chemistry luminescence method detection kit and detection method | |
| CN103048476A (en) | Thyroxine nanometer magnetic particle chemiluminiscence determinstion kit as well as preparation method and detection method thereof | |
| CN106405072B (en) | 25 hydroxy-vitamine D fluorescence immune chromatographies activation fluorescent latex microballoon | |
| CN103048477B (en) | Nanometer magnetic particle chemiluminescence detection kit for triiodothyronine as well as preparation method and detecting method of same | |
| CN106405108B (en) | Whole C reactive proteins fluorescence immune chromatography activation fluorescent latex microballoon | |
| CN106405109B (en) | Ferritin fluorescence immune chromatography detection card activation fluorescent latex microballoon | |
| CN109061198A (en) | inhibin A detection kit and preparation method thereof | |
| CN106405070B (en) | Procalcitonin fluorescence immune chromatography detection card activation fluorescent latex microballoon | |
| CN106370838B (en) | D dimers fluorescence immune chromatography detection card activation fluorescent latex microballoon | |
| CN112763703A (en) | Immunomagnetic bead and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CP03 | Change of name, title or address |
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. |
|
| CP03 | Change of name, title or address | ||
| CB03 | Change of inventor or designer information |
Inventor after: Lin Si Inventor after: Hu Chenguang Inventor after: Xiao Jun Inventor after: Chai Lijun Inventor after: Jiang Lin Inventor before: Lin Si |
|
| CB03 | Change of inventor or designer information |