CN103289929A - Novel quick separation method of bacilluscereus - Google Patents
Novel quick separation method of bacilluscereus Download PDFInfo
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- CN103289929A CN103289929A CN2013102193979A CN201310219397A CN103289929A CN 103289929 A CN103289929 A CN 103289929A CN 2013102193979 A CN2013102193979 A CN 2013102193979A CN 201310219397 A CN201310219397 A CN 201310219397A CN 103289929 A CN103289929 A CN 103289929A
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Abstract
The invention discloses a novel quick separation method of bacilluscereus, which provides the basis for the subsequent research for a target bacterium and relates to the technical field of biology. The novel quick separation method comprises the steps of: carrying out covalent coupling on a multi-arm star-shaped polymer and an antibody, coating the antibody-modified multi-arc star-shaped polymer on a long-chain biotin molecule, capturing the target bacterium in a sample solution by the antibody and long-chain biotin co-modified multi-arc star-shaped polymer, recognizing and coupling the long-chain biotin multi-arm star-shaped polymer in the sample solution by a streptavidin-modified nano magnetic bead, separating and heavily-suspending the captured bacterium; a heavily-suspended liquid can be directly analyzed in subsequent. Compared with a conventional bacterium magnetic separation method, the novel quick separation method is more suitable for carrying out magnetic separation on the bacterium in a complex matrix; the separation efficiency of the target bacterium in a sample is increased.
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
The food-borne pathogenic fungi pollution is one of significant problem of China's food safety.According to the WHO statistics, developed country has 1/3rd people to infect food origin disease every year approximately, and the whole world has 2,200,000 people to die because suffering from food origin disease every year.In China, the routine number of annual food poisoning is 20～400,000 people, and except mishap, major part causes by food-borne pathogens.Bacillus cereus (
Bacillus cereus) as one of common food-borne pathogens, main meat, milk, vegetables, fish, rice and the wheaten food etc. of polluting, regarded as the easiest pathogeny microorganism that detects in the food by Norway and Holland at present, happened occasionally by its poisoning that causes, harm people's health.Its main toxicity symptom that causes shows as vomiting, diarrhoea, and is similar with some other microbial symptom of causing a disease, thereby usually is left in the basket.Therefore, development fast, the technology utmost point of bacillus cereus is necessary in the efficiently concentrating sample separation.
The immunity magnetic separation technique is one of important component part of food-borne pathogens rapid screening technology, but this technology efficient capture, concentrates object bacteria in the enrichment liquid, improves the pathogenic bacterium detection sensitivity.In recent years, based on the immunomagnetic separation (IMS) of magnetic micro-beads object bacteria antibody is connected on the magnetic bead, the magnetic bead that will be connected with antibody then drop in the sample liquid to object bacteria catch, enrichment, magnetic separates (concrete principle is seen Fig. 2 A).Yet should have many limitation based on the isolation technique of micron order immunomagnetic beads at present: 1) specific surface area of micron magnetic bead is less relatively, has reduced magnetic capture efficient; 2) because the particle properties of micron magnetic bead self, by heterogeneous reaction (multiphase reaction) combination, need the longer time go specificity to catch bacterial cell in the food substrate usually between itself and the bacterial cell; 3) micron magnetic bead monodispersity is relatively poor, and precipitation takes place self to assemble or form in food substrate liquid easily; 4) traditional immune magnetic separation technique, directly be coupled to antibody molecule on the immunomagnetic beads often, this process usually can cause the activity of antibody to reduce widely and cause the direction in space of antibody to change having increased space steric effect between antibody, thereby having reduced the capture rate 5 of antibody) food substrate character is complicated and assorted bacteria concentrations wherein non-purpose pathogenic bacterium are big, the micron magnetic bead is easy to generate non-specific adsorption, is difficult to realize the specific isolation to purpose bacterium in the food sample liquid; 6) excessive concentration of micron magnetic bead can cause the breakage (magnetic field causes the cell surface magnetic bead to be attracted each other, cell is squeezed even break) of bacterial cell, causes the failure that separates; 7) during magnetic bead coupling antibody, generally adopt hydrophobic absorption or chemical coupling mode will have active antibody and be connected in magnetic bead surfaces.Antibody and magnetic bead surfaces distance are too near, and the hydrophobic or strong hydrophilicity group of the character of magnetic bead own and remained on surface thereof causes that easily the antibody space conformation changes, and cause the antibody biological activity to descend.
Summary of the invention
At the defective of prior art, the purpose of this invention is to provide a kind of easy, capture rate is high, disengaging time is short, under the low gradient magnetic (less than 30 T/m) can be from the food substrate of complexity fast, the method for specific isolation bacillus cereus.
The sharp separation novel method of bacillus cereus, may further comprise the steps: (1) gets 2.8 mg multi-arm well star polymer dissolution in 2 mL, 0.02 M, pH 6.5 phosphoric acid buffer PBS, add 0.6 mg N-maloyl imines NHSS, 0.4 carbodiimide hydrochloride EDC mg ethyl 3-(3-dimethylamino), room temperature places on the mixing instrument and stirs, and activates 15 min; Get 6 mg bacillus cereus specific antibodies and add in the above-mentioned reaction solution, room temperature places and stirs 30 min on the mixing instrument; Decompression is spin-dried for solvent, deionized water dissolving, dialysis 1 d in PBS and deionized water; Dialysis finishes the solution lyophilize that obtains is got multi-arm well star polymkeric substance-antibody complex; (2) get 15 mg long-chain vitamin Hs, 3.6 mg NHSS, 2.4 mg EDC are dissolved in 2 mL PBS(0.02 M, and pH 6.5) in the damping fluid; Add 0.55 mg multi-arm well star polymkeric substance-antibody complex, room temperature places and stirs 30 min on the mixing instrument; Decompression is spin-dried for solvent, deionized water dissolving, dialysis 1 d in PBS and deionized water; Dialysis finishes the solution lyophilize that obtains is got long-chain vitamin H-multi-arm well star polymkeric substance-antibody complex; (3) get 1 mL testing sample solution, adding the co-modified multi-arm well star polymkeric substance of 0.1 mg bacillus cereus antibody and long-chain vitamin H is step (2) long-chain vitamin H-multi-arm well star polymkeric substance-antibody complex, place on the mixing instrument, with rotating speed incubated at room 15 min of 30 rpm; Add 0.1 mg and be modified with the nanometer magnetic bead of Streptavidin, place on the mixing instrument, with the rotating speed of 30 rpm incubated at room 15 min again, conventional magnetic force frame separates 3 min; (4) after the magneticseparation, after the 0.1%PBST washing, with the resuspended nanometer magnetic bead-Streptavidin-long-chain vitamin H-multi-arm well star polymkeric substance-antibody-bacillus cereus antigenic compound of catching bacillus cereus that namely gets of PBS damping fluid.
Described multi-arm well star polymkeric substance is multi-arm well star polyamide-amide, and its molecular weight is 70000 Da.Structure such as Fig. 1.
The described nanometer magnetic bead particle diameter of having modified Streptavidin is 20-50 nm, is preferably 30 nm.
Multi-arm well star polymkeric substance is realized the covalent coupling of multi-arm well star polymkeric substance and antibody by the carboxyl of amino and bacillus cereus specific antibody.
Multi-arm well star polymkeric substance is realized the covalent coupling of multi-arm well star polymkeric substance and long-chain vitamin H by the carboxyl of amino and long-chain biotin molecule; Add excessive long-chain vitamin H to guarantee exposed amino sites on the sealing multi-arm well star polymkeric substance.
Concrete principle is seen Fig. 2 B.
Present method is specially adapted to the separation of complex sample, as food samples, whole blood sample etc.Food samples comprises the food material after all kinds of fresh or freezing processing, as products such as fresh vegetables, meat, seafood and milks.Sample preparation gets final product according to conventional treatment method.
Adopt technical solution of the present invention to have following beneficial effect:
1, the present invention is by the cascade scale effect of multi-arm well star polymkeric substance, magnetic bacterium signal exponentially level is enlarged, under lower magneticstrength, just can realize the separation of magnetic bacterium, and in the identical time, compare with routine immunization magnetic bead separation method, it is stronger to be separated to purpose bacterium ability, is specially adapted to the separation of complex sample, as food samples, whole blood sample etc.At slow, the demanding defective in magnetic field of purpose bacterium speed in the 20-50 nm immunomagnetic beads separate complex matrix sample behind the simple employing antibody modification, adopt multi-arm well star polymkeric substance to realize the amplification of nanometer magnetic bead magnetic signal, thereby improved purpose bacterium separation efficiency in the complex matrices sample, realized purpose bacterium specificity sharp separation in (less than 30 T/m) are complicated under low gradient magnetic the food substrate.
2, this programme has been avoided in the ordinary method antibody molecule being coupled to magnetic bead surfaces and has been caused antibody activity to reduce and sterically hindered big shortcoming for antibody molecule is coupled on the multi-arm well star polymkeric substance.
3, the present invention adopts multi-arm well star polymkeric substance, can make reaction soln more stable, and difficult the precipitation increased the chance that antibody molecule contacts with object bacteria, is conducive to improve capture rate; Simultaneously, be connected with a large amount of long-chain biotin molecules on the multi-arm well star polymkeric substance, the nanometer magnetic bead that can modify in conjunction with Streptavidin, thus make on the multi-arm well star polymkeric substance in conjunction with a large amount of nanometer magnetic beads, realize the cascade amplification of magnetic bacterium signal, be conducive to shorten the disengaging time of magnetic bacterium.
4, with behind nanometer magnetic bead (20-50 nm) the replacement micron order magnetic particle, because the nanometer magnetic bead particle diameter is little, specific surface area is big, the steric hindrance of being combined with bacterium surface antigen is little, the covering efficient of bacterium surface magnetic bead significantly improves, and the bacterium of magnetic nano particle subcovering can keep normal shape, and nanometer magnetic bead also has dispersed and stable preferably in complex matrices, so the use of nanometer magnetic bead can overcome above-mentioned all because the defective of using the micron magnetic bead to cause.
5, the present invention is in sepn process, introduced multi-arm well star high-polymer molecular, be connected with a large amount of long-chain biotin molecules on the multi-arm well star polymkeric substance, can be special and high-affinity ground be dispersed in that coupling has the identification of Streptavidin nanometer magnetic bead in the matrix solution, thereby make on the multi-arm well star polymkeric substance in conjunction with a large amount of nanometer magnetic beads, increase the magnetic bead quantity of target bacteria surface bonding greatly, realized the target bacteria that sharp separation is caught under magnetic field.Comparing with traditional bacterial magnetic separation method, is stabilized nano magnetic bead more in matrix because of what add, and this method is applicable to that more in complex matrices bacterium being carried out magnetic separates, and has improved purpose bacterium separation efficiency in the complex matrices sample.
6, during magnetic bead coupling antibody, generally adopt hydrophobic absorption or chemical coupling mode will have active antibody and be connected in magnetic bead surfaces.Antibody and magnetic bead surfaces distance are too near, and the hydrophobic or strong hydrophilicity group of the character of magnetic bead own and remained on surface thereof causes that easily the antibody space conformation changes, and cause the antibody biological activity to descend.Yet this experimental program is introduced multi-arm well star polymkeric substance in coupling process, it has certain space size, thereby makes antibody molecule away from magnetic bead and magnetic bead surfaces, has avoided the disadvantageous effect of the character of magnetic bead own and surperficial antagonist molecule.Simultaneously, the multi-arm well star polymkeric substance of introducing but can not influence the antibody space conformation, thereby has played the bioactive effect of protection antibody molecule.
Description of drawings
The structural representation of the amidized multi-arm well of Fig. 1 star polymkeric substance.
The operational flowchart of the conventional magnetic separation technique (A) of Fig. 2 and magnetic separation technique (B) involved in the present invention.
Embodiment
In order to make the present invention clearer, below in conjunction with embodiment, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explaining the present invention, and be not used in restriction the present invention.
The long-chain vitamin H is for buying in the carboxylated long-chain vitamin H of U.S. Thermo Fisher Scientific company (EZ-Link Sulfo-NHS-LC-Biotin, molecular weight 556.59).
The nanometer magnetic bead (30 nm) that is modified with Streptavidin is bought the Ocean NanoTech company in the U.S..
Amidized multi-arm well star polymkeric substance is multi-arm well star polyamide-amide, and its molecular weight is 70000 Da, available from Weihai Chen Yuan new chemical materials company limited.
Conventional magnetic force frame separates magneticstrength less than 30T/m.
N-maloyl imines 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 the 800 mL distilled water, adjust pH to 7.4 with 5 M NaOH, be settled to 1000 mL again and namely get 0.01 M PBS.Volume ratio with 1/1000 (V/V) adds Tween 20 again, namely obtains 0.1%PBST.
Embodiment 1
1. multi-arm well star polymkeric substance-antibody complex prepares according to following steps:
(1) get 2.8 mg multi-arm well star polymkeric substance multi-arm well star polyamide-amides and be dissolved in 2 mL PBS(0.02 M, pH 6.5), add 0.6 mg NHSS, 0.4 mg EDC, room temperature places on the mixing instrument and stirs, and activates 15 min;
(2) get 6 mg bacillus cereus specific antibodies and add in the above-mentioned reaction solution, room temperature places and stirs 30 min on the mixing instrument;
(3) above-mentioned solution decompression is spin-dried for solvent, deionized water dissolving, dialysis 1 d in PBS and deionized water; Dialysis finishes the solution lyophilize that obtains.
2. long-chain vitamin H-multi-arm well star polymkeric substance-antibody complex prepares according to following steps:
(1) get 15 mg long-chain vitamin Hs, 3.6 mg NHSS, 2.4 mg EDC are dissolved in 2 mL PBS(0.02 M, and pH 6.5) in the damping fluid;
(2) 0.55 mg multi-arm well star polymkeric substance-antibody complex is joined in the above-mentioned solution, room temperature places and stirs 30 min on the mixing instrument;
(3) above-mentioned solution decompression is spin-dried for solvent, deionized water dissolving, dialysis 1 d in PBS and deionized water; Dialysis finishes the solution lyophilize that obtains.
3. enrichment is caught: get testing sample solution 1mL, add 0.1 mg long-chain vitamin H-multi-arm well star polymkeric substance-antibody complex, place on the mixing instrument, with rotating speed incubated at room 15 min formation long-chain vitamin H-multi-arm well star polymkeric substance-antibody-bacillus cereus antigenic compound of 30 rpm; Add 0.1 mg and be modified with the nanometer magnetic bead of Streptavidin, place on the mixing instrument, with the rotating speed of 30 rpm incubated at room 15 min again, centrifuge tube is inserted conventional magnetic force frame separate 3 min;
4. after deionized water cleans gently, mix the resuspended mixture nanometer magnetic bead-Streptavidin-vitamin H-multi-arm well star polymkeric substance-antibody-bacillus cereus antigen that is enriched with bacillus cereus that namely gets with the PBS damping fluid.
The experiment of embodiment 2 concentration effects
(1) getting 1 mL concentration is 10
4The bacillus cereus of cfu/mL is in the aseptic centrifuge tube of 1.5 mL, and centrifugal 5 min of 12000 rpm abandon supernatant, and is resuspended with the aseptic PBS solution of equal-volume.
(2) enrichment is caught: technical solution of the present invention group (the multi-arm well star polymkeric substance group that bacillus cereus antibody and long-chain vitamin H are co-modified), the nanometer magnetic bead group that the bacillus cereus specific antibody is modified, the micron magnetic bead group enrichment purpose bacterium that the bacillus cereus specific antibody is modified are set respectively.
(3) after magnetic separates, supernatant liquor is poured in the aseptic centrifuge tube, separated the immunomagnetic beads of catching bacillus cereus and then clean twice with PBST, mix, and with the resuspended immunomagnetic beads mixture of the aseptic PBS solution of 1 mL.
(4) capture rate calculates: after the resuspended liquid of purpose bacterium of each group enrichment is carried out gradient dilution, to each gradient counting, calculate the capture rate of object bacteria by the capture rate formula with dull and stereotyped, test triplicate at every turn.Each calculation formula of organizing capture rate is as follows: (total number of bacterial colony/all total plate count of being adsorbed by enrichment) * 100%.
Describedly respectively organize enrichment to catch the scheme of purpose bacterium as follows:
A. purpose bacterium scheme such as embodiment 1 are caught in technical solution of the present invention group (the multi-arm well star polymkeric substance group that bacillus cereus antibody and long-chain vitamin H are co-modified) enrichment, and be specific as follows:
Be that vitamin H-multi-arm well star polymkeric substance-antibody complex joins and contains in the object bacteria centrifuge tube with 0.1 mg bacillus cereus antibody and the co-modified multi-arm well star polymkeric substance of vitamin H, place on the mixing instrument, with rotating speed incubated at room 15 min of 30 rpm.Add 0.1 mg then and be modified with the nanometer magnetic bead of Streptavidin, place on the mixing instrument, with the rotating speed of 30 rpm incubated at room 15 min again.At last, centrifuge tube is inserted conventional magnetic force frame and separate 3 min.
B. it is specific as follows that purpose bacterium scheme is caught in the nanometer magnetic bead group enrichment of bacillus cereus specific antibody modification:
The nanometer magnetic bead that the bacillus cereus specific antibody that 0.1 mg is prepared is modified joins and contains in the object bacteria centrifuge tube, places on the mixing instrument, with rotating speed incubated at room 15 min of 30 rpm.At last, centrifuge tube is inserted conventional magnetic force frame and separate 3 min.
The nanometer magnetic bead preparation that described bacillus cereus specific antibody is modified: (1) is got 10 mg nanometer magnetic beads (30 nm do not have the coupling Streptavidin) and is used dehydrated alcohol, 1 M NaOH successively, each washing of 1 M HCl once, PBS(0.02 M, pH 4.0) give a baby a bath on the third day after its birth time, aseptic PBS is resuspended.Add NHSS 0.4 mg, EDC 0.35 mg places to keep magnetic bead to suspend on the mixing instrument 37 ℃ of activation 2 h.(2) the magnetic force frame reclaims magnetic bead, and PBS(0.02 M, pH 4.0) after the washing three times, magnetic bead is resuspended among the aseptic PBS, adds 80 μ g bacillus cereus specific antibodies by every mg magnetic bead, places 37 ℃ of coupling 2 h on the mixing instrument.(3) add the thanomin room temperature and seal 2 h.Magnet stand reclaims magnetic bead, PBS washing three times, and 10 ml PBS(contain 0.05% NaN
3, 0.5% BSA, pH 7.4) resuspended immunomagnetic beads and standby in 4 ℃ of refrigerators preservations.
C. it is specific as follows that purpose bacterium scheme is caught in the micron magnetic bead group enrichment of bacillus cereus specific antibody modification:
The micron magnetic bead that the bacillus cereus specific antibody that 0.1 mg is prepared is modified joins and contains in the object bacteria centrifuge tube, places on the mixing instrument, with rotating speed incubated at room 15 min of 30 rpm.At last, centrifuge tube is inserted conventional magnetic force frame and separate 3 min.
The micron magnetic bead preparation that described bacillus cereus specific antibody is modified: (1) is got 10 mg micron magnetic beads (1150 nm do not have the coupling Streptavidin) and is used dehydrated alcohol, 1 M NaOH successively, each washing of 1 M HCl once, PBS(0.02 M, pH 4.0) give a baby a bath on the third day after its birth time, aseptic PBS is resuspended.Add NHSS 0.4 mg, EDC 0.35 mg places to keep magnetic bead to suspend on the mixing instrument 37 ℃ of activation 2 h.(2) the magnetic force frame reclaims magnetic bead, and PBS(0.02 M, pH 4.0) after the washing three times, magnetic bead is resuspended among the aseptic PBS, adds 80 μ g bacillus cereus specific antibodies by every mg magnetic bead, places 37 ℃ of coupling 2 h on the mixing instrument.(3) add the thanomin room temperature and seal 2 h.Magnet stand reclaims magnetic bead, PBS washing three times, and 10 ml PBS(contain 0.05% NaN
3, 0.5% BSA, pH 7.4) resuspended immunomagnetic beads and standby in 4 ℃ of refrigerators preservations.
It is as follows that each organizes capture rate:
| The micron magnetic bead group capture rate that the bacillus cereus specific antibody is modified | The nanometer magnetic bead group capture rate that the bacillus cereus specific antibody is modified | The multi-arm well star polymkeric substance group capture rate that bacillus cereus antibody and long-chain vitamin H are co-modified |
| 57.0% | 25.6% | 92.4% |
Experimental result shows, the capture rate of the micron magnetic bead group that the bacillus cereus specific antibody is modified is apparently higher than the capture rate of nanometer magnetic bead group, this explanation contrast nanometer magnetic bead group, because micron magnetic bead volume is big, magnetic is strong, at short notice just can the more object bacteria of separation and concentration.But, the capture rate of technical solution of the present invention group is far longer than the micron magnetic bead group that the bacillus cereus specific antibody is modified again, this shows that technical solution of the present invention can increase object bacteria nano surface magnetic bead fraction of coverage by multi-arm well star polymkeric substance, thereby magnetic is improved greatly, and then realized (3min) high efficiency separation enrichment bacillus cereus at short notice.
Experiment is caught in embodiment 3 enrichments
Conventional magnetic force frame disengaging time is 30min, and all the other are with embodiment 2.
It is as follows that each organizes capture rate:
| The micron magnetic bead group capture rate that the bacillus cereus specific antibody is modified | The nanometer magnetic bead group capture rate that the bacillus cereus specific antibody is modified | The multi-arm well star polymkeric substance group capture rate that bacillus cereus antibody and long-chain vitamin H are co-modified |
| 60.1% | 45.1% | 93.2% |
Experimental result shows, separate 3min among the comparative example 2, when disengaging time reaches 30min, three groups capture rate all is improved, particularly the capture rate of the nanometer magnetic bead group of bacillus cereus specific antibody modification improves the most obvious, this shows the capture rate that can improve the nanometer magnetic bead group by time expand widely, but it still is lower than the capture rate of short period of time co-modified multi-arm well star polymkeric substance group of bacillus cereus antibody and long-chain vitamin H when separating (3min).This shows technical solution of the present invention (3min) high efficiency separation enrichment bacillus cereus at short notice.
Embodiment 4
Aseptic meat is pulverized, made testing sample solution in the usual way, add bacillus cereus and regulate bacterium colony concentration to 10
4Cfu/mL is standby.
The bacillus cereus antibody and the co-modified dendrimer (0.1 mg) of long-chain vitamin H that prepare are joined respectively in the sample solution, place on the mixing instrument, with rotating speed incubated at room 15 min of 30 rpm.Add the nanometer magnetic bead (0.1 mg) be modified with Streptavidin then, place on the mixing instrument, with the rotating speed of 30 rpm incubated at room 15 min again.At last, conventional magnetic force frame separates 3 min.After magnetic separates, supernatant liquor is poured in the aseptic centrifuge tube, separated the immunomagnetic beads of catching bacillus cereus and then clean twice with PBST, mix, and with the resuspended immunomagnetic beads of the aseptic PBS solution of 1 mL.Capture rate such as embodiment 2 methods obtain, and all the other are with embodiment 2.The results are shown in Table 1, show the bacillus cereus in this programme energy efficiently concentrating sample separation.
Embodiment 5
Germ-free milk is the sample testing sample solution, adds bacillus cereus and regulates bacterium colony concentration to 10
4Cfu/mL.All the other are with embodiment 4.
Embodiment 6
Aseptic grates vegetables is made testing sample solution in the usual way, adds bacillus cereus 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 bacillus cereus and regulates bacterium colony concentration to 10
4Cfu/mL.All the other are with implementing 4.
The comparison of bacillus cereus separating effect in the different actual samples of table 1
| Actual sample | The multi-arm well star polymkeric substance group capture rate that bacillus cereus antibody and long-chain vitamin H are co-modified |
| Embodiment 4 meats | 84.1% |
| Embodiment 5 milk | 84.5% |
| Embodiment 6 vegetables | 85.3% |
| Embodiment 7 whole bloods | 80.6% |
The above only is preferred embodiment of the present invention, not in order to limiting the present invention, all any modifications of doing within the spirit and principles in the present invention, is equal to and replaces and improvement etc., all should be included within protection scope of the present invention.
Claims (3)
1. the sharp separation novel method of bacillus cereus, it is characterized in that may further comprise the steps: (1) gets 2.8 mg multi-arm well star polymer dissolution in 2 mL, 0.02 M, pH 6.5 phosphoric acid buffer PBS, add 0.6 mg N-maloyl imines NHSS, 0.4 carbodiimide hydrochloride EDC mg ethyl 3-(3-dimethylamino), room temperature places on the mixing instrument and stirs, and activates 15 min; Get 6 mg bacillus cereus specific antibodies and add in the above-mentioned reaction solution, room temperature places and stirs 30 min on the mixing instrument; Decompression is spin-dried for solvent, deionized water dissolving, dialysis 1 d in PBS and deionized water; Dialysis finishes the solution lyophilize that obtains is got multi-arm well star polymkeric substance-antibody complex; (2) get 15 mg long-chain vitamin Hs, 3.6 mg NHSS, 2.4 mg EDC are dissolved in 2 mL PBS(0.02 M, and pH 6.5) in the damping fluid; Add 0.55 mg multi-arm well star polymkeric substance-antibody complex, room temperature places and stirs 30 min on the mixing instrument; Decompression is spin-dried for solvent, deionized water dissolving, dialysis 1 d in PBS and deionized water; Dialysis finishes the solution lyophilize that obtains is got long-chain vitamin H-multi-arm well star polymkeric substance-antibody complex; (3) get 1 mL testing sample solution, adding the co-modified multi-arm well star polymkeric substance of 0.1 mg bacillus cereus antibody and long-chain vitamin H is step (2) long-chain vitamin H-multi-arm well star polymkeric substance-antibody complex, place on the mixing instrument, with rotating speed incubated at room 15 min of 30 rpm; Add 0.1 mg and be modified with the nanometer magnetic bead of Streptavidin, place on the mixing instrument, with the rotating speed of 30 rpm incubated at room 15 min again, conventional magnetic force frame separates 3 min; (4) after the magneticseparation, after the 0.1%PBST washing, with the resuspended nanometer magnetic bead-Streptavidin-long-chain vitamin H-multi-arm well star polymkeric substance-antibody-bacillus cereus antigenic compound of catching bacillus cereus that namely gets of PBS damping fluid.
2. method according to claim 1 is characterized in that described multi-arm well star polymkeric substance is multi-arm well star polyamide-amide, and its molecular weight is 70000 Da.
3. method according to claim 1 is characterized in that the described nanometer magnetic bead particle diameter of having modified Streptavidin is 20-50 nm, is preferably 30 nm.
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| CN106967709A (en) * | 2017-02-20 | 2017-07-21 | 南昌大学 | The method that the magnetic nano-particle fast enriching of antibiotics modification separates Listeria monocytogenes |
| CN110308277A (en) * | 2019-06-14 | 2019-10-08 | 珠海市德灏生物科技有限公司 | The quick magnetism separate method of universal pathogenic microorganism and kit |
| CN115058359A (en) * | 2022-05-18 | 2022-09-16 | 南昌大学第一附属医院 | Method for enriching bacillus cereus through magnetic separation |
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