CN103513026A - Signal amplification type immunofluorescence probe, and preparation method and application thereof - Google Patents
Signal amplification type immunofluorescence probe, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a signal amplification type immunofluorescence probe, and a preparation method and application thereof. The preparation method for the probe comprises the following steps: under the existence of a condensing agent, performing condensation reaction on p-phenylenediamine and trimesic acid to obtain multi-carboxyl macromolecules; after activating the multi-carboxyl macromolecules, sequentially adding an antibody and polylysine to perform reaction; then marking with a fluorescence indicator to obtain the probe. Or, the preparation method for the signal amplification type immunofluorescence probe comprises the following steps: respectively adding the antibody and the polylysine into a biotin solution to perform reaction to generate corresponding compounds and then marking with the fluorescence indicator; mixing a fluorescence-marked antibody-biotin compound and a streptavidin solution to perform the reaction and then adding a fluorescence-marked polylysine-biotin compound to continuously perform the reaction to obtain the probe. The probe prepared by the method is marked with a great amount of fluorescence indicators, has a stable structure, is easy to store, is used for fluorescence immunodetection and has the characteristics of high detection sensitivity, short detection time, low cost and the like.
Description
The application is that application number is " 201110456124.7 ", and the applying date is " on Dec 30th, 2011 ", and what denomination of invention was " signal scale-up version immune fluorescent probe and its preparation method and application " application for a patent for invention divides an application
Technical field
The present invention relates to immune express-analysis technical field, relate in particular to a kind of signal scale-up version immune fluorescent probe and its preparation method and application.
Background technology
Immunoassay technology is that the fields such as current medical science, biology, food security are substantially the most also one of the most frequently used detection techniques.It is widely used in Medical Biology research, clinical medicine, environment measuring, and in the detection of food and microbial contamination.Immunoassay technology is that the principle based on antibody antigen reaction is carried out the detection method of quantitative and qualitative analysis to determinand, there is high specificity, the advantage such as highly sensitive, easy, be the important research means of modern life science, at bioanalysis detection field, have a wide range of applications.
Fluorescence immunoassay refers to immunological method (antigen-antibody specific bond) and fluorescence labeling technology (fluorescent marker antagonist or antigen carry out mark) is combined, and the method detecting by fluorescence signal.Final purpose is by antibody being combined with specific reaction and the fluorescence signal of antigen, reaching the object of quantitative detection.
Spot fluorescence immunoassay permeability test, on the basis of spot gold immunity infiltration test, grow up, after develop into spot fluorescence immunoassay permeability test, spot fluorescence immunoassay permeability test is to have replaced the collaurum thing that serves as a mark with fluorescent substance, a kind of detection method that adopts immunity infiltration technology, fluorescent marker and solid phase carrier to combine.
Fluorescence immune chromatography is to grow up on the basis of colloidal gold immunochromatographimethod, is to take NC film as carrier, utilizes the capillarity of microporous barrier, makes the liquid that drips Mo Tiao one end slowly to the other end, ooze and move, as chromatography.
At present, the fluorescent marker such as FITC, rhodamine, quantum dot, fluorescent microsphere has been widely used in fluoroimmunoassay technology.Fluorescence immunoassay detection method has high specific, high, the fireballing feature of susceptibility.But this detection method, when the very micro-material of reply, also exists signal low, and noise is large, classic method such as is difficult to detect at the problem.Therefore, develop new immunologic detection method, improve an urgent demand that the sensitivity detecting is the development of present analysis detection field.
Summary of the invention
The invention provides the preparation method of signal scale-up version immune fluorescent probe, by fluorescent marker be marked at be connected with the large molecule of many carboxyls and polylysine or be connected with biotin-Streptavidin and the antibody complex of polylysine on, immunoreactive fluorescence signal is amplified greatly, improved the sensitivity that fluorescence immunoassay detects.
A preparation method for signal scale-up version immune fluorescent probe, comprising:
(1) under the existence of condensation agent, p-phenylenediamine (PPD) and trimesic acid are added in organic solvent, in 80-100 ℃ of condensation reaction 3-5h, obtain the large molecule of many carboxyls;
(2) the large molecule of many carboxyls step (1) being obtained adds in the solution that contains 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxy-succinamide and activates, and then adds antibody, reaction 0.5-2h; Add again polylysine, continue reaction 0.5-2h, obtain the large molecule of many carboxyls that surface is connected with antibody and polylysine;
(3) with the large molecule of many carboxyls that the surface that fluorescent marker makes step (2) is connected with antibody and polylysine, carry out mark, make signal scale-up version immune fluorescent probe.
In step (1), described condensation agent can be the potpourri of triphenyl phosphite (TPP) with pyridine (Py); Described organic solvent can be METHYLPYRROLIDONE (NMP).
The mol ratio of described p-phenylenediamine (PPD) and trimesic acid is preferably 1:0.8-1:1.2; 1:1 more preferably.P-phenylenediamine (PPD) and trimesic acid, as reaction monomers, can condensation obtain the large molecule of many carboxyls under the existence of condensation agent.
Preferably, in every 80-100ml METHYLPYRROLIDONE, the addition of described p-phenylenediamine (PPD) is 10mmol, and the addition of trimesic acid is 10mmol, and the addition of triphenyl phosphite is 20-30mmol, and the addition of pyridine is 5-15ml.
In described condensation reaction, solution is stirred, guarantee that potpourri dissolves each other into homogeneous phase.
After described condensation reaction completes, reaction product is carried out to aftertreatment and obtain the large molecule of many carboxyls; Described aftertreatment can be: the reaction product after condensation reaction 3-5h is added cessation reaction in the methanol solution that contains hydrochloric acid, separation obtain condensation product precipitation, then condensation product precipitation is carried out to recrystallization, washing and dry processing successively.
Wherein, the concentration that contains hydrochloric acid in the methanol solution of hydrochloric acid described in is 10-15mol/L; Described recrystallization reagent used is METHYLPYRROLIDONE/methyl alcohol mixed liquor; Described washing reagent used is hot methyl alcohol; Described dry being treated to: condensation product precipitation is placed in to the dry 10-15h of 80-120 ℃ of baking oven.
In step (2), described in contain 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) and N-hydroxy-succinamide (NHS) the solvent of solution can be the mixed liquor of phosphate buffer (PBS) and dimethyl sulfoxide (DMSO) (DMSO).
Described antibody can be monoclonal antibody or how anti-.
The mol ratio of the large molecule of many carboxyls, antibody and polylysine that described step (1) obtains is preferably 1:2:20-1:3:50.
Described soak time is preferably 10-15min.
In step (3), described fluorescent marker can be FITC, rhodamine, quantum dot or fluorescent microsphere.
The present invention also provides the preparation method of another kind of signal scale-up version immune fluorescent probe, comprising:
(1) antibody is added in biotin solution, stirring reaction 1-2h, then adds fluorescent marker to carry out mark, makes fluorescently-labeled antibody-biotin composite;
(2) polylysine is added in biotin solution, stirring reaction 1-2h, then adds fluorescent marker to carry out mark, makes fluorescently-labeled polylysine-biotin composite;
(3) fluorescently-labeled antibody-biotin composite step (1) being made mixes with solution of streptavidin, reaction 0.5-1h; Then the fluorescently-labeled polylysine-biotin composite that adds step (2) to make, continues reaction 0.5-1h, makes signal scale-up version immune fluorescent probe.
In step (1), the mol ratio of described antibody and biotin is preferably 1:2-1:4.
Described antibody adds in biotin solution and reacts after 1-2h, the removal of impurities of can dialysing, and described dialysis removal of impurities is: reactant liquor is loaded in bag filter, is placed in concentration 10mmol/L, 48h dialyses in the phosphate buffer of pH7.4.
In step (2), the mol ratio of described polylysine and biotin is preferably 1:1-1:2.
Described polylysine adds in biotin solution and reacts after 1-2h, the removal of impurities of can dialysing, and dialysis removal step is the same.
In step (3), the mol ratio of described fluorescently-labeled antibody-biotin composite, Streptavidin and fluorescently-labeled polylysine-biotin composite is preferably 1:3:10-1:5:15.
The present invention also provides the signal scale-up version immune fluorescent probe that adopts above-mentioned two kinds of preparation methods to make, this signal scale-up version immune fluorescent probe is marked with more fluorescent marker, and intermolecular adhesion is strong, Stability Analysis of Structures can be stablized and preserve more than 1 year under 4 ℃ of gnotobasiss.
The application of signal scale-up version immune fluorescent probe described in the present invention also provides in fluorescence immunoassay detects.Test shows, signal scale-up version immune fluorescent probe of the present invention can detect for the fluorescence immunoassay based on antibody antigen specific reaction, be particularly useful for spot fluorescence immunoassay permeability test or fluorescence immune chromatography test, detection sensitivity is higher, than even tens times of the responsive several times of traditional fluorescence labeling method.
The present invention by fluorescent marker be marked at be connected with the large molecule of many carboxyls and polylysine or be connected with biotin-Streptavidin and the antibody complex of polylysine on, immunoreactive fluorescence signal is amplified greatly, improved the sensitivity that fluorescence immunoassay detects.
While being used for the signal amplification of immune fluorescent probe with the large molecule of many carboxyls and polylysine: p-phenylenediamine (PPD) and trimesic acid can condensation form surface with the large molecule of many carboxyls of a large amount of carboxyls, under suitable reaction sequence and dosage ratio, the large molecular energy of many carboxyls connects antibody and polylysine, a large amount of amino groups of polylysine can be used for connecting more fluorescence molecule, thereby form one and take the large molecule of many carboxyls as basis, surface is connected with certain antibody, the compound of a large amount of polylysines and a large amount of fluorescence molecules, with antibody, fluorescence molecule directly connects and compares, fluorescence signal amplifies greatly.
While amplifying for the signal of immune fluorescent probe with biotin-Streptavidin and polylysine: because biotin and Streptavidin have good pair relationhip, under suitable reaction sequence and dosage ratio, one of the formation that can be connected with polylysine with antibody be take antibody as starting point, Streptavidin, biotin and polylysine be compound from beginning to end mutually, and immunofluorescence signal amplification factor is improved greatly.
Adopt the present invention, there is following beneficial effect:
(1) quantitatively detect, highly sensitive: colloidal gold immunochromatographimethod also has easy to operate, advantage efficiently, but it is more suitable for qualitative detection; ELISA is applicable to a large amount of number of samples objects and detects, and required experimental facilities requires high; And fluorescence immune chromatography not only has quantitative measuring ability, and there is the advantages such as equipment needed thereby is simple, easy to operate; And the quantitative detection of the fluorescence immune chromatography amplifying through signal, its detection sensitivity is higher, can be used for the detection of denier material.
(2) detection time is short, cost is low: the fluorescence immunoassay detection sensitivity of amplifying due to signal is higher, can directly apply to fluorescence immune chromatography and spot fluorescence immunoassay permeability test, compared to ELISA, detect, greatly shortened detection time, be 10-15min the detection time of a general sample, can in 1 hour, detect 50-60 sample; Be particularly suitable for the quantitative detection of a small amount of number of samples, with respect to ELISA, detect, cost is lower.
(3) probe structure is stable, easily preservation: the intermolecular adhesion of signal scale-up version immune fluorescent probe of the present invention is strong, and Stability Analysis of Structures can be stablized and preserve more than 1 year under 4 ℃ of gnotobasiss.
Accompanying drawing explanation
Fig. 1 is the schematic diagram of the embodiment of the present invention 1 signal scale-up version immune fluorescent probe preparation process, and wherein, 1 is the large molecules of many carboxyls, and 2 is antibody, and 3 is polylysine, and 4 is fluorescent marker; NMP is METHYLPYRROLIDONE, and TPP is triphenyl phosphite, and Py is pyridine, and DMSO is dimethyl sulfoxide (DMSO), and PBS is phosphate buffer, and EDAC is 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide;
Fig. 2 is the schematic diagram of the embodiment of the present invention 4 signal scale-up version immune fluorescent probe preparation process, and wherein, 5 is antibody, and 6 is biotin, and 7 is fluorescent marker, and 8 is Streptavidin, and 9 is polylysine.
Embodiment
The preparation of embodiment 1 signal scale-up version immune fluorescent probe
(1) in three-necked bottle, monomer 1.08g (10mmol) p-phenylenediamine (PPD) and 2.10g (10mmol) trimesic acid are under agitation dissolved in 80ml METHYLPYRROLIDONE, add again condensation agent 7.5ml pyridine and 20mmol triphenyl phosphite, constantly be heated with stirring to 80 ℃, potpourri dissolves each other into after homogeneous phase, continues stirring reaction 3h; Stop after reaction, reaction mixture is dissolved in the methyl alcohol that 500ml contains hydrochloric acid (concentration of hydrochloric acid is 12mol/L), filter and be precipitated product; Thick product carries out recrystallization with METHYLPYRROLIDONE/methyl alcohol, and end product washs with hot methyl alcohol, and dries 12h in 100 ℃ of baking ovens; Obtain the large molecule of many carboxyls, the large molecule of called after PT.
(2) the large molecule 5mg of PT in step (1) is dissolved in the solution of 10ml PBS (10mM pH7-8) and 1ml DMSO; Get the large molecular solution of 30ul PT, add wherein 5ul EDAC(10mg/ml) and 3ul NHS(5mg/ml), after room temperature activation 10min, to be taken up in order of priority and to add 3ug antibody and 5ug polylysine, room temperature is respectively reacted 1h.By gel chromatographic columns Superdex on above-mentioned reaction solution separated unnecessary EDAC, NHS and the antibody not being connected, polylysine, and with 0.22 μ m filter opening film degerming, 4 ℃ of preservations.
(3) by the NaHCO that adds 10% in the solution 100ul after above-mentioned purifying
3, regulate pH value to being about 8-9, and add 5ul fluorescent marker, room temperature reaction 1h, and every 10-15min, mix once.Again purifying resin for above potpourri (Thermo Scientific Zeba) is removed to unnecessary fluorescent marker, the centrifugal 5min of 1100g, collect the solution after centrifugal, obtain antibody-many carboxyls large molecule-polylysine-fluorescence labeling compound, be signal scale-up version immune fluorescent probe.
(1) antibody of 10ug/ml concentration is coated on to nitrocellulose filter (NC film) above, and uses 10mM PBS(pH7-8) cleaning 3-5 time, 37 ℃ of dry for standby.
(2) plastics capsule is opened, be divided into Di Hegai.Order is installed to be followed successively by from top to bottom: the end, absorbent material, nitrocellulose filter, lid.
(1) first use the series standard product 0ppb that AFP standard items is configured to respectively to concentration, 1ppb, 5ppb, 20ppb, 50ppb, 100ppb, 200ppb, 400ppb.
(2) get the detection box that embodiment 2 makes, 40ul confining liquid (10mM PBS, pH7-8, containing 0.2%BSA and 0.05%Tween-20) is dropped on the NC film that detects box, sealing NC film, treats that it infiltrates in box.
(3) on NC film, drip the AFP solution of 10ul normal concentration, and drip 30ul cleansing solution (50mM PBS, pH7-8); After solution is slightly dry, then (dilution, for the 10mM PBS solution containing 1%BSA and 1% sucrose, pH7.2), and drips 30ul cleansing solution to drip the signal scale-up version immune fluorescent probe solution that the embodiment 1 of 1000 times of 10ul dilutions makes; After solution is slightly dry, utilize quantitative fluorescence analysis instrument to detect, read fluorescence signal (each sample detects respectively 3 times, averages); Take AFP concentration as horizontal ordinate, and fluorescence intensity is ordinate, drawing standard curve.The AFP lowest detection line of this fluorescence immunoassay test strips is 2ppb.
(4) on NC film, drip 10ul AFP solution to be measured, and drip 30ul cleansing solution; After solution is slightly dry, then (dilution, for the 10mM PBS solution containing 1%BSA and 1% sucrose, pH7.2), and drips 30ul cleansing solution to drip the signal scale-up version immune fluorescent probe solution that the embodiment 1 of 1000 times of 10ul dilutions makes; After solution is slightly dry, utilize quantitative fluorescence analysis instrument to detect, read fluorescence signal; The typical curve that fluorescence signal value substitution step (3) is made draws the AFP concentration of sample to be measured.
(5) repeated experiment: by 3 parts of positive samples at one time section measure 20 times, calculate variation within batch coefficient: by 3 parts of same positive sample METHOD FOR CONTINUOUS DETERMINATION 10 times, once a day, calculate interassay coefficient of variation.By result, known, variation within batch coefficient and interassay coefficient of variation are all less than 10%, show that this signal scale-up version immune fluorescent probe is for spot fluorescence immunoassay permeability test, and repeatability better.
(6) stability experiment: 1) reagent (the signal scale-up version immune fluorescent probe that embodiment 1 makes) is placed 12 days in 37 ℃ of incubators, 3 parts of positives of reagent Parallel testing and 1 part of ' negative ' specimens with being placed on 4 ℃ of refrigerators, carry out heat stabilization test.Result demonstration, this reagent at least can be stablized and deposit more than 1 year.2) the test strips sealing detecting in box is deposited in respectively to 37 ℃, 25 ℃ and 4 ℃, observe the impact of different temperatures on test strips.The test strips that is stored in 37 ℃ is taken out 70 examination criteria product respectively every month; Be stored in 70 difference examination criteria product of test strips taking-up in each month of 25 ℃.Result demonstration, test strips is preserved respectively and is preserved 6 months for 12 months and 37 ℃ at 25 ℃, and testing result is not all found to change.
The preparation of embodiment 4 signal scale-up version immune fluorescent probes
(1) biotin of 20mmol activation is reacted respectively to stirring at room 1h with 10mmol antibody and 10mmol polylysine; Then be loaded in bag filter and be placed in PBS buffer solution (10mM, pH7.4) dialysis 48h, make biotin-antibody, biotin-poly-ammonia compound that relies; Use again 0.22 μ m filter opening film degerming, 4 ℃ of preservations.
(2) get 20ul biotin-antibody and biotin-polylysine solution (protein concentration>=1mg/ml), use respectively 10% NaHCO
3, regulate pH value to being about 8-9, add fluorescent marker to carry out mark, and softly stir.Room temperature reaction 1h, and every 10-15min, mix once.After reaction finishes, with purifying resin (Thermo Scientific Zeba), remove unreacted fluorescent marker, the centrifugal 5min of 1100g, collects the solution after centrifugal, obtains fluorescently-labeled biotin-antibody, biotin-poly-bad ammonia compound.
(3) fluorescence labeling biotin-antibody complex step (2) being made mixes with solution of streptavidin, under room temperature, reacts 1h; Add the fluorescence labeling biotin that step (2) makes-poly-ammonia compound that relies again, continue reaction 1h under room temperature, wherein, it is 1:5:5 that fluorescence labeling biotin-antibody complex, Streptavidin, fluorescence labeling biotin-poly-rely the mol ratio of ammonia compound; Reaction finishes rear acquisition antibody-biotin-Streptavidin-polylysine-fluorescence labeling compound, is signal scale-up version immune fluorescent probe.
The preparation of embodiment 5 fluorescence immune chromatography test paper bars
(1) sample pad preparation: sample pad is cut into 1.5 * 30cm, puts it in treating fluid (3%BSA+1%Triton X-100), soak after 30min, 37 ℃ of oven dry 3h are standby.
(2) pad preparation: be cut into 1.0 * 30cm in connection with pad, the signal scale-up version that embodiment 4 is made dilution for immune fluorescent probe (containing the PBS of 1%BSA and 1% sucrose, 10mM, pH7-8) dilutes after 3000 times, be sprayed on pad, dry 30min for 37 ℃.
(3) NC film preparation: NC film is cut into 2.5 * 30cm, sprays sheep anti-mouse igg and antigen according to the amount of 1ul/cm with point sample instrument on NC film diverse location, respectively as nature controlling line and detectability, dry 30min for 37 ℃.
(4) fluorescence immunoassay test strips assembling: sample pad, pad, NC film and adsorptive pads are bonded on base plate respectively, and are cut into the wide test strips of 4cm.
Embodiment 6 fluorescence immune chromatography test paper bars detect clenbuterol hydrochloride (competition law)
(1) clenbuterol hydrochloride standard items are mixed with to the series standard product 0ppb of variable concentrations, 0.1ppb, 0.3ppb, 0.9ppb, 2.7ppb, 8.1ppb.
(2) above-mentioned series standard product are respectively got to 80ul, join in the test tube that contains the signal scale-up version immune fluorescent probe that embodiment 4 makes, mix rear 2min, get 70ul loading, chromatography continues 8min, uses fluorescence detector reading out data.Utilize quantitative fluorescence analysis instrument to detect respectively the fluorescence intensity (each sample detects respectively 3 times, averages) of T line and C line, make the typical curve of antigen concentration; The Clenbuterol lowest detection line of this fluorescence immunoassay test strips is 0.05ppb.
(3) determined antigen detects by the test strips that embodiment 5 makes, and utilizes the above-mentioned typical curve of ratio substitution of T/C to try to achieve determined antigen concentration.
(4) repeated experiment: by 3 parts of positive samples at one time section measure 20 times, calculate variation within batch coefficient: by 3 parts of same positive sample METHOD FOR CONTINUOUS DETERMINATION 10 times, once a day, calculate interassay coefficient of variation.By result, known, variation within batch coefficient and interassay coefficient of variation are all less than 10%, show that this signal scale-up version immune fluorescent probe is for fluorescence immune chromatography, and repeatability better.
(5) stability experiment: 1) reagent (the signal scale-up version immune fluorescent probe that embodiment 4 makes) is placed 12 days in 37 ℃ of incubators, 3 parts of positives of reagent Parallel testing and 1 part of ' negative ' specimens with being placed on 4 ℃ of refrigerators, carry out heat stabilization test.Result demonstration, this reagent at least can be stablized and deposit more than 18 months.2) test strips sealing is deposited in respectively to 37 ℃, 25 ℃ and 4 ℃, observe the impact of different temperatures on test strips.The test strips that is stored in 37 ℃ and 25 ℃ is taken out 70 examination criteria product 0ppb respectively, 0.05ppb, 0.1ppb, 0.3ppb, 0.9ppb, 2.7ppb, 8.1ppb every month.Result demonstration, test strips is preserved respectively and is preserved 6 months for 12 months and 37 ℃ at 25 ℃, and testing result is not all found to change.
Claims (6)
1. a preparation method for signal scale-up version immune fluorescent probe, comprising:
(1) antibody is added in biotin solution, stirring reaction 1-2h, then adds fluorescent marker to carry out mark, makes fluorescently-labeled antibody-biotin composite;
(2) polylysine is added in biotin solution, stirring reaction 1-2h, then adds fluorescent marker to carry out mark, makes fluorescently-labeled polylysine-biotin composite;
(3) fluorescently-labeled antibody-biotin composite step (1) being made mixes with solution of streptavidin, reaction 0.5-1h; Then the fluorescently-labeled polylysine-biotin composite that adds step (2) to make, continues reaction 0.5-1h, makes signal scale-up version immune fluorescent probe.
2. preparation method according to claim 1, is characterized in that, in step (1), the mol ratio of described antibody and biotin is 1:2-1:4.
3. preparation method according to claim 1, is characterized in that, in step (2), the mol ratio of described polylysine and biotin is 1:1-1:2.
4. preparation method according to claim 1, is characterized in that, in step (3), the mol ratio of described fluorescently-labeled antibody-biotin composite, Streptavidin and fluorescently-labeled polylysine-biotin composite is 1:3:10-1:5:15.
5. the signal scale-up version immune fluorescent probe that the preparation method of an employing as described in as arbitrary in claim 1-4 makes.
6. the application of signal scale-up version immune fluorescent probe as claimed in claim 5 in fluorescence immunoassay detects.
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Cited By (2)
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| CN106198962A (en) * | 2016-08-30 | 2016-12-07 | 四川迈克生物科技股份有限公司 | For the method closing biomagnetic beads |
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| CN105738616A (en) * | 2016-02-02 | 2016-07-06 | 基蛋生物科技股份有限公司 | Preparation method and application of dual-amplifying fluorescent immune labeling probe and method for preparing fluorescent immune chromatography reagent strip from probe |
| CN106198962A (en) * | 2016-08-30 | 2016-12-07 | 四川迈克生物科技股份有限公司 | For the method closing biomagnetic beads |
| CN106198962B (en) * | 2016-08-30 | 2018-01-30 | 迈克生物股份有限公司 | Method for closing biomagnetic beads |
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