CN103305441B - High-efficiency rapid vibrio parahemolyticus enrichment and separation method - Google Patents
High-efficiency rapid vibrio parahemolyticus enrichment and separation method Download PDFInfo
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Abstract
The invention discloses a high-efficiency rapid enrichment and separation method, mainly relates to a method for enriching and separating vibrio parahemolyticus from a matrix, aims at providing basis for follow-up study on target bacteria, and relates to the field of biotechnology. The method comprises the following steps of: carrying out covalent coupling on an arborescence hyper-branched polymer and an antibody, enveloping the antibody-modified arborescence hyper-branched polymer with long-chain biotin molecules, capturing the target bacteria in a sample liquid by using the arborescence hyper-branched polymer which is co-modified by the antibody and the long-chain biotin, recognizing and coupling the long-chain biotin arborescence hyper-branched polymer in the sample liquid by using streptavidin-modified nano magnetic beads, separating and heavy-suspending the captured bacteria, and the like. The captured bacteria can be subjected to follow-up analysis directly; and compared with the conventional bacterium separation method, the method disclosed by the invention is more applicable to magnetic separation of bacteria in a complex matrix, and the target bacterium separation efficiency in the sample is improved.
Description
Technical field
The present invention relates to biological technical field, specifically relate to the food-borne pathogens separation method based on nanometer magnetic bead.
Background technology
Vibrio parahemolyticus (
vibrio parahaemolyticus,vP), Vibrio is the polymorphic bacterium of a kind of Grain-negative addicted to salt.Be distributed widely in the sea-foods such as inshore seawater, seabed silt, planktonic organism and fish, shrimp, shellfish.Can cause acute gastroenteritis and primary septicemia, according to Ministry of Health's report, the food poisoning that in the second quarter in 2008 whole nation food poisoning situation, Vibrio parahemolyticus causes occupies the 2nd of microbes food poisoning, is only second to Bacillus cereus.The novel method setting up a kind of efficient rapid detection Vibrio parahemolyticus seems particularly urgent, and in view of needs are set up a kind of efficient, detection method fast, immune magnetic separation technique obtains and develops rapidly in Surveillance for foodborne pathogen.
Immunity magnetic separation technique is one of important component part of food-borne pathogens rapid screening technology, this technology can efficient capture, object bacteria in concentrated enrichment liquid, improve pathogenic microbes detect sensitivity and shorten detection time.In recent years, object bacteria antibody is connected on magnetic bead by the immunomagnetic separation (IMS) based on magnetic micro-beads, then the magnetic bead being connected with antibody is dropped in sample liquid object bacteria is caught, enrichment, Magneto separate (concrete principle is shown in Fig. 2 A).But, many limitation should be there is based on the isolation technique of micron order immunomagnetic beads at present: 1) specific surface area of micron magnetic bead is relatively little, reduces magnetic capture efficiency; 2) due to the particle properties of micron magnetic bead self, combined by heterogeneous reaction (multiphase reaction) between itself and bacterial cell, usually need the time more grown to go specificity to catch bacterial cell in food substrate; 3) micron magnetic bead monodispersity is poor, self assemble easily occurs in food substrate liquid or forms precipitation; 4) traditional immune magnetic separation technique, often antibody molecule is directly coupled on immunomagnetic beads, this process usually can cause the activity of antibody to reduce widely and cause the direction in space of antibody to change the space steric effect increased between antibody, thus reduce the capture rate 5 of antibody) food substrate character is complicated and wherein the miscellaneous bacteria concentration of non-object pathogenic bacterium is large, micron magnetic bead easily produces non-specific adsorption, is difficult to realize the specific isolation to object bacterium in food sample liquid; 6) excessive concentration of micron magnetic bead can cause the breakage of bacterial cell (magnetic field causes cell surface magnetic bead to be attracted each other, and cell is squeezed and even breaks), causes the failure be separated; 7), during magnetic bead coupled antibody, activated for tool antibody is connected in magnetic bead surfaces by general hydrophobic adsorbent or the chemical coupling mode of adopting.Too closely, the hydrophobic or strong hydrophilicity group of magnetic bead nature and remained on surface thereof easily causes antibody space conformation to change, and causes antibody bioactive to decline for antibody and magnetic bead surfaces distance.
Summary of the invention
For the defect of prior art, the object of this invention is to provide that a kind of capture rate is high, easy, disengaging time is short, the method for quick from the food substrate of complexity, the special separation object bacterium Vibrio parahemolyticus that (is less than 30 T/m) under low gradient magnetic.
A kind of efficient concentration and separation Vibrio parahemolyticus method fast, comprises the following steps:
(1) the tree-shaped over-expense polymer dissolution of 1 mg is often got in 2 mL 0.02 M, pH 6.5 phosphoric acid buffer PBS, adds 0.6 mg N-hydroxysuccinimide NHSS, 0.4 mg ethyl 3-(3-dimethylamino) carbodiimide hydrochloride EDC, room temperature is placed on blending instrument and stirs, and activates 15 min; Add 2.68 mg Vibrio parahemolyticus specific antibodies, room temperature is placed on blending instrument and stirs 30 min; Decompression is spin-dried for solvent, deionized water dissolving, and dialyse 1 d in PBS and deionized water; Dialysis terminates the solution lyophilize obtained to obtain tree-shaped over-expense polymer-antibody complex; (2) 15 mg long-chain biological elements are got, 3.6 mgN-hydroxysuccinimide NHSS, 2.4 mg ethyl 3-(3-dimethylaminos) carbodiimide hydrochloride EDC is dissolved in 2 mL 0.02 M pH 6.5 PBS damping fluids; Add the tree-shaped over-expense polymer-antibody complex of 2.24 mg, room temperature is placed on blending instrument and stirs 30 min; Decompression is spin-dried for solvent, deionized water dissolving, and dialyse 1 d in PBS and deionized water; Dialysis terminates the solution lyophilize obtained to obtain long-chain biological element-tree-shaped over-expense polymer-antibody complex; (3) 1 mL testing sample solution is got, add 0.1 mg Vibrio parahemolyticus antibody and long-chain biological element co-modified tree-shaped over-expense polymkeric substance and long-chain biological element-tree-shaped over-expense polymer-antibody complex, be placed on blending instrument, with rotating speed incubated at room 15 min of 30 rpm; Add the nanometer magnetic bead that 0.1 mg is modified with Streptavidin, be placed on blending instrument, with rotating speed incubated at room 15 min of 30 rpm, conventional magnetic frame is separated 3 min; (4), after magneticseparation, after 0.1% PBST washing, the nanometer magnetic bead-Streptavidin-long-chain biological element-tree-shaped over-expense polymer-antibody-Vibrio parahemolyticus antigenic compound of catching Vibrio parahemolyticus is namely obtained with PBS damping fluid is resuspended.
Described tree-shaped over-expense polymkeric substance is for being amidized dendroid polyamide-amide, and its molecular weight is 56000 Da.Structure is as Fig. 1.
Described nanometer magnetic bead particle diameter is 20-50nm, is preferably 30 nm.
Tree-shaped over-expense polymkeric substance realizes the covalent coupling of tree-shaped over-expense polymkeric substance and antibody by carboxyl that is amino and Vibrio parahemolyticus specific antibody.
Tree-shaped over-expense polymkeric substance, by carboxyl that is amino and long-chain biological element molecule, realizes the covalent coupling of tree-shaped over-expense polymkeric substance and long-chain biological element; Add excessive long-chain biological element to ensure amino sites exposed on closed tree-shaped over-expense polymkeric substance.
Concrete principle is shown in Fig. 2 B.
Present method is applicable to the separation of object bacterium in sample, particularly complex matrices, as food samples, whole blood sample etc.Food samples comprises the food material after all kinds of fresh or freeze cutting, as products such as fresh vegetables, meat, seafood and milks.Sample preparation according to national standard method process, as made solution to be measured by after sample sterile crushing.
Technical solution of the present invention is adopted to have following beneficial effect:
1, the present invention by means of the Cascaded amplification effect of tree-shaped over-expense polymkeric substance, magnetic bacterium signal exponentially level is expanded, the separation of magnetic bacterium just can be realized under lower magneticstrength, and within the identical time, comparatively routine immunization Beads enrichment method is compared, be separated to object bacterium ability stronger, be specially adapted to the separation of complex sample, as food samples, whole blood sample etc.For the defect that object bacterium speed is slow, magnetic field requirements is high in the simple 20-50 nm immuno magnetic cell separation complex matrices sample adopted after antibody modification, tree-shaped over-expense polymkeric substance is adopted to realize the amplification of nanometer magnetic bead magnetic signal, thus improve object bacterium separation efficiency in complex matrices sample, achieve object bacterium specificity sharp separation in the food substrate that (to be less than 30 T/m) under low gradient magnetic complicated.
2, this programme is for be coupled on tree-shaped over-expense polymkeric substance by antibody molecule, avoid in ordinary method antibody molecule is coupled to magnetic bead surfaces cause antibody activity reduce and sterically hindered large shortcoming.
3, the present invention adopts tree-shaped over-expense polymkeric substance, reaction soln can be made more stable, not easily precipitate, add the chance that antibody molecule contacts with object bacteria, is conducive to improving capture rate; Simultaneously, tree-shaped over-expense polymkeric substance is connected with a large amount of long-chain biological element molecules, the nanometer magnetic bead can modified in conjunction with Streptavidin, thus makes on tree-shaped over-expense polymkeric substance in conjunction with a large amount of nanometer magnetic beads, achieve the Cascaded amplification of magnetic bacterium signal, be conducive to the disengaging time shortening magnetic bacterium.
4, after replacing micron order magnetic particle with nanometer magnetic bead (20-50 nm), because nanometer magnetic bead particle diameter is little, specific surface area is large, the steric hindrance be combined with bacterial surface antigen is little, the covering efficiency of bacterium surface magnetic bead significantly improves, and the bacterium of magnetic nano particle subcovering can keep normal shape, nanometer magnetic bead also has dispersed and stability preferably in complex matrices, and therefore the use of nanometer magnetic bead can overcome above-mentioned all defects owing to using micron magnetic bead to cause.
5, the present invention is in sepn process, introduce tree-shaped over-expense polymkeric substance, tree-shaped over-expense polymkeric substance is connected with a large amount of long-chain biological element molecules, can special and high-affinity ground be dispersed in coupling in matrix solution and have the identification of Streptavidin nanometer magnetic bead, thus make on tree-shaped over-expense polymkeric substance in conjunction with a large amount of nanometer magnetic beads, considerably increase the magnetic bead quantity of target bacteria surface bonding, achieve the target bacteria that sharp separation is caught under magnetic field.Compared with traditional bacterial magnetic separation method, be nanometer magnetic bead more stable in matrix because of what add, the method is more suitable for carries out Magneto separate to bacterium in complex matrices, improves object bacterium separation efficiency in complex matrices sample.
6, during magnetic bead coupled antibody, activated for tool antibody is connected in magnetic bead surfaces by general hydrophobic adsorbent or the chemical coupling mode of adopting.Too closely, the hydrophobic or strong hydrophilicity group of magnetic bead nature and remained on surface thereof easily causes antibody space conformation to change, and causes antibody bioactive to decline for antibody and magnetic bead surfaces distance.But this experimental program introduces tree-shaped over-expense polymkeric substance in coupling process, it has certain space size, thus makes antibody molecule away from magnetic bead and magnetic bead surfaces, avoids the disadvantageous effect of magnetic bead nature and surperficial antagonist molecule.Meanwhile, the tree-shaped over-expense polymkeric substance of introducing but can not affect antibody space conformation, thus serves the bioactive effect of protection antibody molecule.
Accompanying drawing explanation
The structural representation of the tree-shaped over-expense polymkeric substance of Fig. 1.
The operational flowchart of the conventional magnetic separation technique (A) of Fig. 2 and magnetic separation technique involved in the present invention (B).
Embodiment
In order to make the present invention clearly understand, below in conjunction with embodiment, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
Long-chain biological element is for buying in the carboxylated long-chain biological element of Thermo Fisher Scientific company of the U.S. (EZ-Link Sulfo-NHS-LC-Biotin, molecular weight 556.59).
The nanometer magnetic bead (30 nm) being modified with Streptavidin is bought in Ocean NanoTech company of the U.S..
Amidized tree-shaped over-expense polymkeric substance is amidized dendroid polyamide-amide, and its molecular weight is 56000 Da, purchased from Weihai Chen Yuan new chemical materials company limited.
Conventional magnetic frame is separated magneticstrength and is less than 30T/m.
N-hydroxysuccinimide NHSS, ethyl 3-(3-dimethylamino) carbodiimide hydrochloride EDC etc. is conventional reagent, repeats no more.
0.1%PBST compound method: 8.0 g NaCl, 0.2 g KCl, 0.24 g KH
2pO
4, 1.44 g Na
2hPO
4be dissolved in 800 mL distilled water, adjust pH to 7.4 with 5 M NaOH, then be settled to 1000 mL and namely obtain 0.01 M PBS.Add Tween 20 with the volume ratio of 1/1000 (V/V) again, namely obtain 0.1%PBST.
Embodiment 1
1. tree-shaped over-expense polymer-antibody complex, prepare in accordance with the following steps:
(1) often get the tree-shaped over-expense polymer dissolution of 1 mg in 2 mL, 0.02 M, pH 6.5 PBS, add 0.6 mg NHSS, 0.4 mg EDC, room temperature is placed on blending instrument and stirs, and activates 15 min;
(2) getting 2.68 mg Vibrio parahemolyticus specific antibodies adds in above-mentioned reaction solution, and room temperature is placed on blending instrument and stirs 30 min;
(3) above-mentioned solution decompression is spin-dried for solvent, deionized water dissolving, dialyse 1 d in PBS and deionized water; Dialysis terminates the solution lyophilize that will obtain.
2. long-chain biological element-tree-shaped over-expense polymer-antibody complex is prepared in accordance with the following steps:
(1) often get 15 mg long-chain biological elements, 3.6 mg NHSS, 2.4 mg EDC are dissolved in 2 mL 0.02 M pH 6.5 PBS damping fluids;
(2) join in above-mentioned solution by 2.24 mg tree-shaped over-expense polymer-antibody complex, room temperature is placed on blending instrument and stirs 30 min;
(3) above-mentioned solution decompression is spin-dried for solvent, deionized water dissolving, dialyse 1 d in PBS and deionized water; Dialysis terminates the solution lyophilize that will obtain.
3. enrichment is caught: get testing sample solution 1mL, add 0.1 mg long-chain biological element-tree-shaped over-expense polymer-antibody complex, be placed on blending instrument, form long-chain biological element-tree-shaped over-expense polymer-antibody-Vibrio parahemolyticus antigenic compound with rotating speed incubated at room 15 min of 30 rpm; Add the nanometer magnetic bead that 0.1 mg is modified with Streptavidin, be placed on blending instrument, with the rotating speed of 30 rpm incubated at room 15 min again, centrifuge tube is inserted conventional magnetic frame and be separated 3 min;
4., after deionized water cleans gently, mix with PBS damping fluid and resuspendedly namely obtain the mixture nanometer magnetic bead-Streptavidin-vitamin H-tree-shaped over-expense polymer-antibody-Vibrio parahemolyticus antigen being enriched with Vibrio parahemolyticus.
Embodiment 2 concentration effect is tested
(1) getting 1 mL concentration is 10
4the Vibrio parahemolyticus of cfu/mL is in 1.5 mL sterile centrifugation tube, and centrifugal 5 min of 12000 rpm, abandon supernatant, resuspended by the aseptic PBS solution of equal-volume.
(2) enrichment is caught: arrange technical solution of the present invention group (Vibrio parahemolyticus antibody and the plain co-modified tree-shaped over-expense polymkeric substance group of long-chain biological), the nanometer magnetic bead group of Vibrio parahemolyticus specific antibody modification, the micron magnetic bead group enrichment object bacterium of Vibrio parahemolyticus specific antibody modification respectively.
(3), after Magneto separate, supernatant liquor is poured in sterile centrifugation tube, separate catch Vibrio parahemolyticus immunomagnetic beads then with PBST cleaning twice, to mix, and with the resuspended immunomagnetic beads mixture of the aseptic PBS solution of 1 mL.
(4) capture rate calculates: after the object bacterium re-suspension liquid of each group of enrichment is carried out gradient dilution, count each gradient with flat board, and by the capture rate of capture rate formulae discovery object bacteria, each experiment in triplicate.The calculation formula of each group of capture rate is as follows: (total number of bacterial colony of being adsorbed by enrichment/all total plate count) × 100%.
The scheme that object bacterium is caught in described each group of enrichment is as follows:
A. technical solution of the present invention group (Vibrio parahemolyticus antibody and the plain co-modified tree-shaped over-expense polymkeric substance group of long-chain biological) enrichment catches object bacterium scheme as embodiment 1, specific as follows:
0.1 mg Vibrio parahemolyticus antibody and the co-modified tree-shaped over-expense polymkeric substance of vitamin H and vitamin H-tree-shaped over-expense polymer-antibody complex are joined containing in object bacteria centrifuge tube, is placed on blending instrument, with rotating speed incubated at room 15 min of 30 rpm.Then add the nanometer magnetic bead that 0.1 mg is modified with Streptavidin, be placed on blending instrument, with the rotating speed of 30 rpm incubated at room 15 min again.Finally, centrifuge tube is inserted conventional magnetic frame and be separated 3 min.
B. it is specific as follows that object bacterium scheme is caught in the nanometer magnetic bead group enrichment that Vibrio parahemolyticus specific antibody is modified:
The nanometer magnetic bead that the Vibrio parahemolyticus specific antibody prepared by 0.1 mg is modified joins containing in object bacteria centrifuge tube, is placed on blending instrument, with rotating speed incubated at room 15 min of 30 rpm.Finally, centrifuge tube is inserted conventional magnetic frame and be separated 3 min.
The nanometer magnetic bead preparation that described Vibrio parahemolyticus specific antibody is modified: (1) is got 10 mg nanometer magnetic beads (30 nm do not have coupling Streptavidin) and used dehydrated alcohol successively, 1 M NaOH, 1 M HCl respectively washs once, PBS(0.02 M, pH 4.0) wash three times, aseptic PBS is resuspended.Add NHSS 0.4 mg, EDC 0.35 mg, be placed on blending instrument and keep magnetic bead to suspend, 37 DEG C of activation 2 h.(2) magnetic frame reclaim magnetic bead, PBS(0.02 M, pH 4.0) washing three times after, magnetic bead is resuspended in aseptic PBS, adds 80 μ g Vibrio parahemolyticus specific antibodies by every mg magnetic bead, is placed in 37 DEG C of coupling 2 h on blending instrument.(3) add thanomin room temperature and close 2 h.Magnet stand reclaims magnetic bead, and PBS washs three times, and 10 ml PBS(are containing 0.05% NaN
3, 0.5% BSA, pH 7.4) and resuspended immunomagnetic beads for subsequent use in 4 DEG C of Refrigerator stores.
C. it is specific as follows that object bacterium scheme is caught in the micron magnetic bead group enrichment that Vibrio parahemolyticus specific antibody is modified:
The micron magnetic bead that the Vibrio parahemolyticus specific antibody prepared by 0.1 mg is modified joins containing in object bacteria centrifuge tube, is placed on blending instrument, with rotating speed incubated at room 15 min of 30 rpm.Finally, centrifuge tube is inserted conventional magnetic frame and be separated 3 min.
The micron magnetic bead preparation that described Vibrio parahemolyticus specific antibody is modified: (1) is got 10 mg micron magnetic beads (1150 nm do not have coupling Streptavidin) and used dehydrated alcohol successively, 1 M NaOH, 1 M HCl respectively washs once, PBS(0.02 M, pH 4.0) wash three times, aseptic PBS is resuspended.Add NHSS 0.4 mg, EDC 0.35 mg, be placed on blending instrument and keep magnetic bead to suspend, 37 DEG C of activation 2 h.(2) magnetic frame reclaim magnetic bead, PBS(0.02 M, pH 4.0) washing three times after, magnetic bead is resuspended in aseptic PBS, adds 80 μ g Vibrio parahemolyticus specific antibodies by every mg magnetic bead, is placed in 37 DEG C of coupling 2 h on blending instrument.(3) add thanomin room temperature and close 2 h.Magnet stand reclaims magnetic bead, and PBS washs three times, and 10 ml PBS(are containing 0.05% NaN
3, 0.5% BSA, pH 7.4) and resuspended immunomagnetic beads for subsequent use in 4 DEG C of Refrigerator stores.
Each group of capture rate is as follows:
| The micron magnetic bead group capture rate that Vibrio parahemolyticus specific antibody is modified | The nanometer magnetic bead group capture rate that Vibrio parahemolyticus specific antibody is modified | Vibrio parahemolyticus antibody and the plain co-modified tree-shaped over-expense polymkeric substance group capture rate of long-chain biological |
| 56.1% | 25.2% | 90.3% |
Experimental result shows, the capture rate of the micron magnetic bead group that Vibrio parahemolyticus specific antibody is modified is apparently higher than the capture rate of nanometer magnetic bead group, this illustrates contrast nanometer magnetic bead group, because micron magnetic bead volume is large, magnetic strong, and the object bacteria that just energy separation and concentration is more at short notice.But, the capture rate of technical solution of the present invention group is far longer than again the micron magnetic bead group of Vibrio parahemolyticus specific antibody modification, this shows that technical solution of the present invention can increase object bacteria nano surface magnetic bead fraction of coverage by tree-shaped over-expense polymkeric substance, thus magnetic is improved greatly, and then achieve (3min) high efficiency separation enrichment Vibrio parahemolyticus at short notice.
Experiment is caught in embodiment 3 enrichment
Conventional magnetic frame disengaging time is 30min, and all the other are with embodiment 2.
Each group of capture rate is as follows:
| The micron magnetic bead group capture rate that Vibrio parahemolyticus specific antibody is modified | The nanometer magnetic bead group capture rate that Vibrio parahemolyticus specific antibody is modified | Vibrio parahemolyticus antibody and the plain co-modified tree-shaped over-expense polymkeric substance group capture rate of long-chain biological |
| 58.7% | 39.1% | 92.3% |
Experimental result shows, 3min is separated in comparative example 2, when reaching 30min when disengaged, the capture rate of three groups is obtained for raising, particularly the capture rate of the nanometer magnetic bead group of Vibrio parahemolyticus specific antibody modification improves the most obvious, this shows to improve the capture rate of nanometer magnetic bead group widely by time expand, but the capture rate of Vibrio parahemolyticus antibody and long-chain biological element co-modified tree-shaped over-expense polymkeric substance group when it is still separated (3min) lower than the short period of time.This shows that technical solution of the present invention can (3min) high efficiency separation enrichment Vibrio parahemolyticus at short notice.
Embodiment 4
Aseptic meat is pulverized, makes testing sample solution in the usual way, add Vibrio parahemolyticus and regulate bacterium colony concentration to 10
4cfu/mL is for subsequent use.
The Vibrio parahemolyticus antibody prepared and the co-modified tree-shaped over-expense polymkeric substance (0.1 mg) of long-chain biological element are joined in sample solution respectively, is placed on blending instrument, with rotating speed incubated at room 15 min of 30 rpm.Then add the nanometer magnetic bead (0.1 mg) being modified with Streptavidin, be placed on blending instrument, with the rotating speed of 30 rpm incubated at room 15 min again.Finally, conventional magnetic frame is separated 3 min.After Magneto separate, supernatant liquor is poured in sterile centrifugation tube, separate catch Vibrio parahemolyticus immunomagnetic beads then with PBST cleaning twice, to mix, and with the resuspended immunomagnetic beads of the aseptic PBS solution of 1 mL.Capture rate such as embodiment 2 method obtains, and all the other are with embodiment 2.The results are shown in Table 1, show the Vibrio parahemolyticus in this programme energy efficiently concentrating sample separation.
Embodiment 5
Germ-free milk is sample testing sample solution, adds Vibrio parahemolyticus and regulates bacterium colony concentration to 10
4cfu/mL.All the other are with embodiment 4.
Embodiment 6
Aseptic grates vegetables, makes testing sample solution in the usual way, adds Vibrio parahemolyticus and regulates bacterium colony concentration to 10
4cfu/mL.All the other are with embodiment 4.
Embodiment 7
Testing sample is aseptic whole blood, adds Vibrio parahemolyticus and regulates bacterium colony concentration to 10
4cfu/mL.All the other are with implementing 4.
The comparison of Vibrio parahemolyticus separating effect in the different actual sample of table 1
| Actual sample | Vibrio parahemolyticus antibody and the plain co-modified tree-shaped over-expense polymkeric substance group capture rate of long-chain biological |
| Embodiment 2 meat | 84.4% |
| Embodiment 3 milk | 87.2% |
| Embodiment 4 vegetables | 89.3% |
| Embodiment 5 whole blood | 83.6% |
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (3)
1. an efficient concentration and separation Vibrio parahemolyticus method fast, is characterized in that comprising the following steps:
(1) the tree-shaped over-expense polymer dissolution of 1 mg is often got in 2 mL 0.02 M, pH 6.5 phosphoric acid buffer PBS, adds 0.6 mg N-hydroxysuccinimide NHSS, 0.4 mg ethyl 3-(3-dimethylamino) carbodiimide hydrochloride EDC, room temperature is placed on blending instrument and stirs, and activates 15 min; Add 2.68 mg Vibrio parahemolyticus specific antibodies, room temperature is placed on blending instrument and stirs 30 min; Decompression is spin-dried for solvent, deionized water dissolving, and dialyse 1 d in PBS and deionized water; Dialysis terminates the solution lyophilize obtained to obtain tree-shaped over-expense polymer-antibody complex; (2) 15 mg long-chain biological elements are got, 3.6 mgN-hydroxysuccinimide NHSS, 2.4 mg ethyl 3-(3-dimethylaminos) carbodiimide hydrochloride EDC is dissolved in 2 mL 0.02 M pH 6.5 PBS damping fluids; Add the tree-shaped over-expense polymer-antibody complex of 2.24 mg, room temperature is placed on blending instrument and stirs 30 min; Decompression is spin-dried for solvent, deionized water dissolving, and dialyse 1 d in PBS and deionized water; Dialysis terminates the solution lyophilize obtained to obtain long-chain biological element-tree-shaped over-expense polymer-antibody complex; (3) 1 mL testing sample solution is got, add 0.1 mg Vibrio parahemolyticus antibody and long-chain biological element co-modified tree-shaped over-expense polymkeric substance and long-chain biological element-tree-shaped over-expense polymer-antibody complex, be placed on blending instrument, with rotating speed incubated at room 15 min of 30 rpm; Add the nanometer magnetic bead that 0.1 mg is modified with Streptavidin, be placed on blending instrument, with rotating speed incubated at room 15 min of 30 rpm, conventional magnetic frame is separated 3 min; (4), after magneticseparation, after 0.1% PBST washing, the nanometer magnetic bead-Streptavidin-long-chain biological element-tree-shaped over-expense polymer-antibody-Vibrio parahemolyticus antigenic compound of catching Vibrio parahemolyticus is namely obtained with PBS damping fluid is resuspended; Described nanometer magnetic bead particle diameter is 20-50nm.
2. method according to claim 1, it is characterized in that described tree-shaped over-expense polymkeric substance is for being amidized dendroid polyamide-amide, its molecular weight is 56000 Da.
3. method according to claim 1, is characterized in that described nanometer magnetic bead particle diameter is 30 nm.
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| 树枝状大分子复合磁性颗粒的制备与表征;司宝财;《中国优秀硕士毕业论文全文数据库 工程科技I辑》;20070610;第3页 * |
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