CN108267574A - A kind of preparation and application based on dendrimer dual amplification label small molecule competition probe - Google Patents
A kind of preparation and application based on dendrimer dual amplification label small molecule competition probe Download PDFInfo
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- CN108267574A CN108267574A CN201711271867.0A CN201711271867A CN108267574A CN 108267574 A CN108267574 A CN 108267574A CN 201711271867 A CN201711271867 A CN 201711271867A CN 108267574 A CN108267574 A CN 108267574A
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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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- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
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Abstract
The present invention relates to a kind of preparations and application based on dendrimer dual amplification label small molecule competition probe, belong to the chemiluminescent labeling field in chemiluminescence diagnosis in vitro.This pair enhancing amplification small molecule tags method includes the following steps：Step 1：The preparation of dendrimer Streptavidin chemistry luminous signal agent complex；Step 2：Compete the biotinylation label of small molecule；Step 3：Dual amplification chemiluminescence immunoassay marks the preparation of detection probe complex.Dual amplification chemiluminescence immunoassay detection probe in the present invention, the molecular detection binding capacity two ways for enhancing luminous intensity and increase magnetic microsphere is effectively combined, primary amplification is realized using combination between biotin avidin, another amplification is realized using the dendrimer that semiochemicals are marked, dual amplification pattern is greatly improved chemiluminescence immune assay signal strength simultaneously.
Description
Technical field
A kind of chemiluminescent labeling field in being diagnosed in vitro the invention belongs to chemiluminescence, and in particular to dual amplification
The method learned the preparation method and application of electrochemiluminescent immunoassay label probe, chemiluminescence detection kit is prepared with it.
Background technology
Chemiluminescence immune assay (Chemiluminescence Immunoassay, CLIA) is will be with high sensitivity
Chemical luminescent detecting technology be combined with the immune response of high specific, for various antigens, haptens, antibody, hormone,
The detection and analysis technology of enzyme, aliphatic acid, vitamin and drug etc..Be after radioimmunology analysis, enzyme exempt from analysis, fluoroimmunoassay and when
Between a newest immunoassay growing up after resolved fluorometric immunoassay.Exempt from analytic approach compared with traditional enzyme,
CLIA has the characteristics that sensitivity higher, detection time are shorter, labeling method is simpler and cost of material is lower.Although such as
This, with the gradually universal of analytical control technology and clinically for detecting further carrying for testing molecule sensitivity requirement
Height there is an urgent need to systematicness can further promote sensitivity on the basis of CLIA, reduces the new technology of detection time.At present, exist
Immunoassay field, common amplification means are biotin-avidin amplification systems, and 1981, Hsu etc. demonstrated an affinity
Element can be discussed in detail in combination with four biotin molecules, and to the method that the two is applied in immunology, pass through chain
The bridge joint effect of mould Avidin avidin-biotin, respectively in connection with antigen-antibody, marking signal substance etc., can greatly enhance
Antibody binding capacity or semiochemicals binding capacity, play the role of amplification.
General biotin, avidin amplification system using a kind of amplification mode, be utilized only to biotin and
Amplification effect between avidin or the increase and amplification only for antibody binding capacity, it is impossible to effectively put biotin and avidin
Large model fully utilizes, and often can seem helpless when in face of some trace detections.
To solve the above problems, using the amplification system of biotin-streptavidin plus dendrimer label hair
Light probe carrys out dual amplification chemiluminescence signal, and developing one kind can sensitiveer, the broader detection trace materials of the range of linearity
Chemical luminescence reagent kit.
Invention content
A kind of dual amplification chemiluminescence immunoassay detection is provided the purpose of the present invention is overcome the deficiencies in the prior art to visit
The preparation method of needle and the method for chemiluminescence detection kit is prepared with it, the dual amplification chemiluminescence immunoassay label probe
Using enhanced chemiluminescence intensity and increase molecular detection two kinds of amplification modes of binding capacity so that chemiluminescence detection kit is competing
Contention signal intensity had the amplification factor of bigger more in the past, realized trace detection.
The preparation method of dual amplification chemiluminescence competition immunological probe provided by the present invention, specifically may include walking as follows
Suddenly：
Step 1：The dendrimer of certain molal quantity is taken, EDC is added in and is activated, delayed in 0.05mol/L carbonate
In fliud flushing (pH 9.5), Streptavidin is added in, 37 DEG C are stirred to react 1h, after reaction according to the ratio of molar ratio 1: 10
The activation signals substance purchased is added in, 37 DEG C are stirred to react 1h, are eventually adding 0.05mol/LTris-HCl and carry out capping
30min, entirely after reaction, the unreacted small-molecule chemical luminous signal substance of dialysis removal in PBS, after collecting dialysis
Solution obtains the dendritic macromole of modification chemiluminescence signal substance and Streptavidin；
Step 2：The activated biotin purchased is taken, it is anti-with competitive small molecule mixing according to the ratio of molar ratio 1: 3
Should, it is spare after 37 DEG C of reaction 1h；
Step 3：The dendritic macromole that semiochemicals and Streptavidin are modified made from step 1 is added to step
Biotinylation competitiveness small molecule made from two continues 37 DEG C of reaction 30min and exempts to get dual amplification chemiluminescence competition
Epidemic disease probe.
Further, the preparation method of the dual amplification chemiluminescence immunoassay detection probe, wherein in step 1
The competitiveness small molecule is antigen such as T3, T4, T, E2, E3, P etc., and used carrier is dendrimer, the semiochemicals
For chemiluminescent substances such as acridinium ester, tris (bipyridine) ruthenium, luminols.
Further, the preparation method of dual amplification chemiluminescence competition immunological probe, wherein in step 1
The Streptavidin, carrier, semiochemicals molar ratio be 1: 3: 10.
Further, dual amplification chemiluminescence competition immunological probe, wherein biotin described in step 2
Change competitive small molecule, the molar ratio of biotin is 1: 3.
Further, dual amplification chemiluminescence competition immunological probe, wherein the label carbonate solution
Mass concentration be 50mmol/L.
Further, above-mentioned dual amplification chemiluminescence competition immunological probe, wherein modifies letter described in step 3
The dendritic macromole of number substance and Streptavidin, streptavidin, biotinylation competition small molecule molar ratio be 3: 1: 1.
The method for chemiluminescence detection kit is prepared with the dual amplification chemiluminescence competition immunological probe,
Including following preparation process：
Step 1, the preparation of Streptavidin MagneSphere：Purchase carboxyl magnetic microsphere is taken, EDC is added in and is activated, use MES
Buffer solution Streptavidin, adds in the magnetic microsphere after activation, and 1: 2,37 DEG C of shaking table reaction 3h of mass ratio are placed in magnetic force
Separator frame, Tris-HCl buffer solution for cleaning 1 time add in 10%BSA and are closed, and after shaking table reaction 30min, are placed in Magnetic Isolation
Frame Tris-HCl buffer solution for cleaning 3 times adds in 2 DEG C~8 DEG C preservations of Tris-HCl buffer solutions containing 2%BSA.
Step 2, the preparation of capture probe：The Streptavidin MagneSphere that step 1 is taken to obtain adds in biotinylated to be measured
37 DEG C of shaking table reaction 30min of substance capture probe molecule, Tris-HCl buffer solution for cleaning three times, add in the Tris- containing 2%BSA
2 DEG C~8 DEG C preservations of HCl buffer solutions, both obtain the separating trap probe complex containing magnetic particle.
The capture of preparation is detached probe and competition immunological probe by step 3, be respectively charged into kit both it is chemical
Method luminescence detection kit.
Further, prepared by the use dual amplification chemiluminescence immunoassay capture probe tries for chemiluminescence detection
The method of agent box, wherein buffer solution described in step 1 are 0.05mol/LTris-HCl (pH7.4), with the buffer solution
BSA a concentration of 2%.
The dendrimer, which carries body, arbitrarily to pass through acyl with the small molecule containing amino and carboxyl or albumen
Amine key is coupled.
Advantageous effect：Dual amplification chemiluminescence immunoassay detection probe of the present invention, diagnosing in vitro in immune detection will increase
Effective combination of extensive chemical luminous intensity and the molecular detection binding capacity two ways of increase magnetic microsphere, utilizes biotin affinity
Combination realizes primary amplification between element, while realizes another amplification using dendrimer and semiochemicals coupling, double
It resets large model and is greatly improved chemiluminescence immune assay luminous signal intensity, detection sensitivity and detection range etc. are immune
Analytical reagent performance is been significantly enhanced.
Description of the drawings
The dual amplification principle schematic of Fig. 1 dual amplification chemiluminescence competition immunological probes of the present invention, wherein, 1：It is magnetic
Microballoon； 2：Streptavidin；3：Biotin；4：Specific antibody；5：Competitive antigen small molecule；6：Semiochemicals (acridine
Ester, luminol etc.)；7：Dendrimer
Specific embodiment
Further the present invention is explained by the following examples, but is not limited thereto system, it is any to be familiar with this profession
Technical staff possibly also with the disclosure above technology contents make modifications or to change or modify the equivalent equivalent Example.But
Everything without departing from technical solution of the present invention content, the technique according to the invention any modification substantially made to the above embodiment,
Equivalent variations and remodeling, still fall within the protection domain of technical solution of the present invention.
Embodiment 1
Dual amplification chemiluminescence competes the preparation of immunological probe：
1) dendrimer of certain 10nmol/L is taken, the 10mg/mL EDC for adding in 100 μ L are activated, and are added in
3nmol/L Streptavidins, 0.05mol/L carbonate buffer solutions (pH 9.5) supplement 500 μ L of total volume, 37 DEG C are stirred to react
1h；
2) the 100nmol/L activation signals substances purchased after reaction according to the addition of the ratio of molar ratio 1: 10,37 DEG C
1 h is stirred to react, the 0.05mol/L Tris-HCl for being eventually adding 50 μ L carry out 37 DEG C of shaking table reaction 30min of capping；
3) the 2.7 μ L of 2mg/mL activated biotins purchased are taken, are detected according to the ratio of molar ratio 1: 10 with test substance
150 μ g of molecule carry out hybrid reaction, 37 DEG C of reaction 1h, and the 0.05mol/L Tris-HCl for being eventually adding 20 μ L close instead
Should, 37 DEG C of shaking tables react 30min；
4) modification semiochemicals-Streptavidin-dendritic macromole obtained is taken to be added to biotinylation obtained competing
Strive molar ratio 1: 1 in small molecule, the reaction was continued 37 DEG C reaction 30min
5) entire to be transferred in the bag filter of molecular cut off 8KD after reaction, dialyzate is the PBS (pH of 0.1mol/L
7.2), dialysis removes unreacted small-molecule chemical luminous signal substance, and solution is to get the dual amplification after collecting dialysis
Learn the competition immunological probe that shines.
Embodiment 2
The preparation of magnetic microsphere capture separation probe：
1) purchase carboxyl magnetic microsphere 10mg is taken to stand 1min on magnetic separation rack in centrifuge tube, magnetic bead is absorbed and protects
Liquid storage adds in the 0.1mol/LMES buffer solutions of 1.5mL, and it is spare that magnetic bead is resuspended.
2) EDC weighed up is dissolved to 10mg/mL with ice reaction buffer, the magnetic bead that 34 μ L is taken to be quickly adding into after being resuspended
In, mixing (or being shaken when being added dropwise) is shaken at once.
3) streptavidin weighed up is dissolved to 10mg/mL with reaction buffer, 60 μ L is taken to be added in magnetic bead, room temperature
(25 DEG C)/37 DEG C of shaking table concussion 3h.
4) ultrasound 3min~4min, is placed on magnetic separation rack and stands 1min, reaction solution is absorbed, with Tris-HCl buffer solutions
Cleaning 3 times adds in the Tris-HCl buffer solutions containing 2%BSA and (8mL, 1.25mg/mL) is resuspended, and 2 DEG C~8 DEG C save backup.
5) 1.5mL is taken to be added to what is prepared after biotinylated 200 times of test substance capture probe molecular dilution
In 1.25mg/L Streptavidin MagneSpheres 1mL, 37 DEG C of shaking tables react 30min
6) after reaction, Tris-HCl buffer solution for cleaning three times, adds in the Tris-HCl buffer solutions containing 2%BSA, 2 DEG C
~8 DEG C of preservations, both obtain the separating trap probe complex containing magnetic particle.
7) by the capture of preparation detach probe and competition immunological probe, be respectively charged into kit seal both chemical method
Luminescence detection kit.
Claims (9)
1. a kind of preparation and application based on dendrimer dual amplification label small molecule competition probe, which is characterized in that
Include the following steps：
Step 1：The dendrimer of certain molal quantity is taken, EDC is added in and is activated, at carbonate buffer solution (pH 9.5)
In, it adds in 37 DEG C of small molecule of competition and is stirred to react 1h, the activation purchased after reaction according to the addition of the ratio of molar ratio 1: 10
37 DEG C of semiochemicals are stirred to react 1h, are eventually adding 0.05mol/L Tris-HCl and carry out capping 30min, entire reaction knot
Shu Hou, the unreacted small-molecule chemical luminous signal substance of dialysis removal, collects solution after dialysis in PBS, obtains modificationization
Learn luminous signal substance-Streptavidin-dendritic macromole compound；
Step 2：The activated biotin purchased is taken, hybrid reaction is carried out with small molecule is competed according to the ratio of molar ratio 1: 3,
37 DEG C of reaction 1h, it is spare to obtain biotinylation test substance molecular detection；
Step 3：Semiochemicals-Streptavidin-dendritic macromole will be modified made from step 1 and meet object according to centainly rubbing
You are than being added to made from step 2 in biotinylation competition small molecule, and the reaction was continued 37 DEG C of reaction 30min are transferred to and stalk in bag
Dialysis removes unreacted small molecule and semiochemicals and competes probe to get the dual amplification.
2. the preparation method of dual amplification label small molecule competition probe according to claim 1, which is characterized in that described
Competitive probe is antigen such as T3, T4, T, E2, E3, P etc., and used carrier is dendrimer, and the semiochemicals are acridine
The chemiluminescent substances such as ester, tris (bipyridine) ruthenium, luminol.
3. the preparation method of dual amplification label small molecule competition probe according to claim 1, which is characterized in that step
Streptavidin described in one, carrier, semiochemicals molar ratio be 1: 3: 10.
4. the preparation method of dual amplification label small molecule competition probe according to claim 1, which is characterized in that step
The competition of biotinylation described in two small molecule, the molar ratio of biotin are 1: 3.
5. the preparation method of dual amplification label small molecule competition probe according to claim 1, which is characterized in that step
Described in label carbonate solution mass concentration be 0.05mol/L.
6. the preparation method of dual amplification label small molecule competition probe according to any one of claims 1 to 5, feature
It is, dendritic macromole, streptavidin, the biotinylation competition of semiochemicals and Streptavidin is modified described in step 3
The molar ratio of immunological probe molecule is 3: 1: 1.
7. dual amplification label small molecule competition probe obtained according to claim 1 is prepared for chemiluminescence detection
The method of kit, which is characterized in that including following preparation process：
Step 1, the preparation of Streptavidin MagneSphere：It takes purchase carboxyl magnetic microsphere appropriate, adds in EDC and activated, use MES
Buffer solution Streptavidin, adds in the magnetic microsphere after activation, and 1: 2,37 DEG C of shaking table reaction 3h of mass ratio are placed in magnetic force
Separator frame, Tris-HCl buffer solution for cleaning 1 time add in 10%BSA and are closed, and after shaking table reaction 30min, are placed in Magnetic Isolation
Frame Tris-HCl buffer solution for cleaning 3 times adds in 2 DEG C~8 DEG C preservations of Tris-HCl buffer solutions containing 2%BSA；
Step 2, the preparation of capture probe：The Streptavidin MagneSphere that step 1 is taken to obtain, adds in biotinylated test substance
37 DEG C of shaking table reaction 30min of capture probe molecule, Tris-HCl buffer solution for cleaning three times, add in the Tris-HCl containing 2%BSA and delay
Fliud flushing, 2 DEG C~8 DEG C preservations, both obtains the separating trap probe complex containing magnetic particle.
8. dual amplification label small molecule competition probe according to claim 7 is prepared for chemiluminescence detection kit
Method, which is characterized in that buffer solution described in step 1 be 0.05mol/L Tris-HCl (pH7.4), with the buffer solution
The BSA a concentration of 2% of dissolving.
9. application of the dual amplification label small molecule competition probe described in claim 1 in chemiluminescence immunoassay detection.
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| CN104698184A (en) * | 2015-02-10 | 2015-06-10 | 深圳市新产业生物医学工程股份有限公司 | Kit for detecting carbohydrate antigen as well as detection method and application thereof |
| CN106198982A (en) * | 2016-07-04 | 2016-12-07 | 复旦大学 | The preparation of the hydrophilic biomolecular that a kind of dendrimer is modified and the application in rapidly and efficiently cell capture thereof |
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2017
- 2017-11-27 CN CN201711271867.0A patent/CN108267574A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102565383A (en) * | 2011-12-30 | 2012-07-11 | 吴坚 | Signal amplification type immunofluorescence probe as well as preparation method and application thereof |
| CN103275902A (en) * | 2013-06-05 | 2013-09-04 | 南昌大学 | Method for enriching and separating helicobacter pylori |
| CN104698186A (en) * | 2015-02-10 | 2015-06-10 | 深圳市新产业生物医学工程股份有限公司 | Kit for detecting hyaluronic acid and detection method and application of kit |
| CN104698172A (en) * | 2015-02-10 | 2015-06-10 | 深圳市新产业生物医学工程股份有限公司 | Kit for detecting hepatitis B surface antigen and detection method and application of kit |
| CN104698184A (en) * | 2015-02-10 | 2015-06-10 | 深圳市新产业生物医学工程股份有限公司 | Kit for detecting carbohydrate antigen as well as detection method and application thereof |
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