CN103308680B - Magnetic enrichment and separation method of Shigellaspp in complex matrix - Google Patents
Magnetic enrichment and separation method of Shigellaspp in complex matrix Download PDFInfo
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- CN103308680B CN103308680B CN201310219457.7A CN201310219457A CN103308680B CN 103308680 B CN103308680 B CN 103308680B CN 201310219457 A CN201310219457 A CN 201310219457A CN 103308680 B CN103308680 B CN 103308680B
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Abstract
本发明公开了志贺氏菌(Shigellaspp.)在复杂基质中的磁富集分离的方法，该方法为目的菌更好地进行后续研究提供基础，涉及生物技术领域。方法包括超支化聚合物与抗体共价偶联、抗体修饰的超支化聚合物再包被长链生物素分子、抗体和长链生物素共修饰的超支化聚合物捕获样品液中的目的菌、链霉亲和素修饰的纳米磁珠识别并偶联样品液中的长链生物素化超支化聚合物、被捕获细菌的分离及重悬等步骤。捕获到的目的菌可以直接进行后续分析，与传统的细菌磁分离方法相比，该方法更适用于在复杂基质中对细菌进行磁分离，提高了样品中目的菌分离效率。
The invention discloses a method for magnetic enrichment and separation of Shigella spp. in a complex matrix. The method provides a basis for better follow-up research on target bacteria and relates to the field of biotechnology. The method includes covalent coupling of hyperbranched polymers and antibodies, antibody-modified hyperbranched polymers are coated with long-chain biotin molecules, antibody and long-chain biotin co-modified hyperbranched polymers capture target bacteria in the sample liquid, Streptavidin-modified nano-magnetic beads recognize and couple the long-chain biotinylated hyperbranched polymers in the sample solution, the separation and resuspension of captured bacteria, and other steps. The captured target bacteria can be directly analyzed. Compared with the traditional bacterial magnetic separation method, this method is more suitable for magnetic separation of bacteria in complex matrices, and improves the separation efficiency of target bacteria in samples.
Description
Technical field
The present invention relates to biological technical field, specifically relate to the Shigella separation method based on nanometer magnetic bead.
Background technology
Shigella common name shigella dysenteriae, belongs to pathogenic bacteria in gram-negative cells, ranks first in China infectious diarrhea pathogen.Shigella is propagated mainly through alimentary canal approach, and invasion and attack enterocyte, causes to generate heat, suffers from abdominal pain, suffers from diarrhoea as the typical dysentery symptom of feature.The annual whole world has 1.6 hundred million people ill, and the group of people at high risk of shigella infection main be the children in l-4 year.In adult patients, 10 ~ 100 cfu Shigellas are just by infecting enteropathic, set up a kind of efficient new method detecting salmonella typhimurium fast and seem particularly urgent, 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 technics 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 bacteria 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 Shigella that (is less than 30 T/m) under low gradient magnetic.
Shigella is magnetic enrichment and separation method in complex matrices, comprises the following steps:
(1) often get 1 mg dissaving polymer and be dissolved in 2 mL 0.02 M, pH 6.5 phosphate 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 18.75 mg Shigella 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 freeze drying obtained to obtain dissaving polymer-antibody complex;
(2) 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; Adding 0.33 mg dissaving polymer-antibody complex joins in above-mentioned solution, and 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 freeze drying obtained to obtain long-chain biological element-dissaving polymer-antibody complex;
(3) 1mL testing sample solution is got, add 0.1 mg Shigella antibody and long-chain biological element co-modified dissaving polymer and step (2) long-chain biological element-dissaving 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 the rotating speed of 30 rpm incubated at room 15 min again, conventional magnetic frame is separated 3 min;
(4), after magnetic separation, after 0.1% PBST washing, the nanometer magnetic bead-Streptavidin-long-chain biological element-dissaving polymer-antibody-Shigella antigenic compound of catching Shigella is namely obtained with PBS damping fluid is resuspended.
Described dissaving polymer is amidized ultrabranching polyamide-amine, and its molecular weight is 8000 Da.Structure is as Fig. 1.
Described nanometer magnetic bead particle diameter is 20-50nm, is preferably 30 nm.
Dissaving polymer realizes the covalent coupling of dissaving polymer and antibody by carboxyl that is amino and Shigella specific antibody.
Dissaving polymer, by carboxyl that is amino and long-chain biological element molecule, realizes the covalent coupling of dissaving polymer and long-chain biological element; Add excessive long-chain biological element to ensure amino sites exposed on closed dissaving polymer.
Concrete principle is shown in Fig. 2 B.
This 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 dissaving polymer, magnetic bacterium signal exponentially level is expanded, the separation of magnetic bacterium just can be realized under lower magnetic field intensity, 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, dissaving polymer 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 quick separating 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 dissaving polymer by antibody molecule, avoid in conventional method antibody molecule is coupled to magnetic bead surfaces cause antibody activity reduce and sterically hindered large shortcoming.
3, the present invention adopts dissaving polymer, reaction solution 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, dissaving polymer 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 dissaving polymer 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 sub-covering 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 detachment process, introduce dissaving polymer, dissaving polymer 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 dissaving polymer in conjunction with a large amount of nanometer magnetic beads, considerably increase the magnetic bead quantity of target bacteria surface conjunction, achieve the target bacteria that quick separating 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 dissaving polymer in coupling process, it has certain space size, thus makes antibody molecule away from magnetic bead and magnetic bead surfaces, avoids the adverse effect of magnetic bead nature and surperficial antagonist molecule.Meanwhile, the dissaving polymer 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 Fig. 1 dissaving polymer.
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..
Over-expense polymkeric substance is amidized ultrabranching polyamide-amine, and its molecular weight is 8000 Da, purchased from Weihai Chen Yuan new chemical materials company limited.
Conventional magnetic frame is separated magnetic field intensity 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, dissaving polymer-antibody complex, prepare in accordance with the following steps:
(1) often get the amidized ultrabranching polyamide-amine solvent of 1 mg dissaving polymer 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 18.75 mg Shigella specific antibodies adds in above-mentioned reactant liquor, 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 freeze drying that will obtain.
2, long-chain biological element-dissaving 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 18.75 mg dissaving polymer-antibody complexes, 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 freeze drying that will obtain.
3. enrichment is caught: get testing sample solution 1 mL, add 0.1 mg long-chain biological element-over-expense polymer-antibody complex, be placed on blending instrument, form long-chain biological element-over-expense polymer-antibody-Shigella 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 compound nanometer magnetic bead-Streptavidin-biotin-over-expense polymer-antibody-Shigella antigen being enriched with Shigella.
Embodiment 2 concentration effect is tested
(1) getting 1 mL concentration is 10
4the Shigella 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 (Shigella antibody and the plain co-modified over-expense polymkeric substance group of long-chain biological), the nanometer magnetic bead group of Shigella specific antibody modification, the micron magnetic bead group enrichment object bacterium of Shigella specific antibody modification respectively.
(3), after Magneto separate, supernatant is poured in sterile centrifugation tube, separate catch Shigella immunomagnetic beads then with PBST cleaning twice, to mix, and with the resuspended immunomagnetic beads compound 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 computing formula of each group of capture rate is as follows: (total plate count of being adsorbed by enrichment/all total number of bacteria) × 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 (Shigella antibody and the plain co-modified over-expense polymkeric substance group of long-chain biological) enrichment catches object bacterium scheme as embodiment 1, specific as follows:
0.1 mg Shigella antibody and the co-modified over-expense polymkeric substance of biotin and biotin-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 Shigella specific antibody is modified:
The nanometer magnetic bead that the Shigella 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 Shigella specific antibody is modified: (1) is got 10 mg nanometer magnetic beads (30 nm do not have coupling Streptavidin) and used absolute ethyl 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 Shigella specific antibodies by every mg magnetic bead, is placed in 37 DEG C of coupling 2 h on blending instrument.(3) add monoethanolamine 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 Shigella specific antibody is modified:
The micron magnetic bead that the Shigella 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 Shigella specific antibody is modified: (1) is got 10 mg micron magnetic beads (1150 nm do not have coupling Streptavidin) and used absolute ethyl 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 Shigella specific antibodies by every mg magnetic bead, is placed in 37 DEG C of coupling 2 h on blending instrument.(3) add monoethanolamine 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 Shigella specific antibody is modified | The nanometer magnetic bead group capture rate that Shigella specific antibody is modified | Shigella antibody and the plain co-modified over-expense polymkeric substance group capture rate of long-chain biological |
| 57.2% | 24.1% | 93.3% |
Experimental result shows, the capture rate of the micron magnetic bead group that Shigella 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 Shigella specific antibody modification, this shows that technical solution of the present invention can increase object bacteria nano surface magnetic bead coverage rate by over-expense polymkeric substance, thus magnetic is improved greatly, and then achieve (3min) at short notice efficient separation and concentration Shigella.
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 Shigella specific antibody is modified | The nanometer magnetic bead group capture rate that Shigella specific antibody is modified | Shigella antibody and the plain co-modified over-expense polymkeric substance group capture rate of long-chain biological |
| 58.2% | 36.3% | 93.9% |
Embodiment 4
Aseptic meat is pulverized, makes testing sample solution in the usual way, add Shigella and regulate bacterium colony concentration to 10
4cfu/mL is for subsequent use.
The Shigella antibody prepared and the co-modified dissaving polymer (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 is poured in sterile centrifugation tube, separate catch Shigella 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 Shigella in this programme energy efficiently concentrating sample separation.
Embodiment 5
Germ-free milk is sample testing sample solution, adds Shigella 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 Shigella 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 Shigella and regulates bacterium colony concentration to 10
4cfu/mL.All the other are with implementing 4.
The comparison of Shigella separating effect in the different actual sample of table 1
| Actual sample | Shigella antibody and the plain co-modified dissaving polymer group capture rate of long-chain biological |
| Embodiment 2 meat | 83.2% |
| Embodiment 3 milk | 87.5% |
| Embodiment 4 vegetables | 89.3% |
| Embodiment 5 whole blood | 80.2% |
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.
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