CN103333818A - Method for separating staphylococcus aureus (SA) - Google Patents
Method for separating staphylococcus aureus (SA) Download PDFInfo
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Abstract
The invention discloses a method for enriching and separating staphylococcus aureus (SA), provides a good basis to the subsequent research on a target bacterium, and relates to the technical field of biology. The method comprises the following steps: performing covalent coupling on a dendrimer and an antibody; coating the antibody modified dendrimer with a long-chain biotin molecule; capturing a target bacterium in a sample liquid by the antibody and long-chain biotin comodified dendrimer; identifying and coupling the long-chain biotin dendrimer in the sample liquid by using streptavidin modified nano-magnetic beads; and carrying out separation and weight suspending on the captured bacterium, wherein the weight suspending solution can be directly used for subsequent analysis. Compared with a traditional bacterium magnetic separation method, the method is more suitable for magnetically separating bacterium in a complex matrix, so that the target bacterium separation efficiency in a sample can be 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
The food-borne pathogenic fungi pollution is one of significant problem of China's food safety.According to the WHO statistics, developed country approximately has 1/3rd people to infect food origin disease every year, and the whole world has 2,200,000 people to die because suffering from food origin disease every year.In China, annual food poisoning number of cases is 20～400,000 people, and except mishap, major part causes by food-borne pathogens.Streptococcus aureus
(Staphylococcus aureus, SA)as one of common food-borne pathogens, be the unique permission of China Ministry of Health pathogenic bacterium that Permissible limit exists in quick-frozen brand food.Yet the poisoning caused by it happens occasionally, and harm is serious, be mainly manifested in and produce pathogenic enterotoxin, can cause that purulent infects, as caused local suppurative infection, pneumonia, pseudomembranous enteritis, pericarditis etc., the even systemic infection such as septicemia, Sepsis.Simultaneously, streptococcus aureus is also the Main Pathogenic Bacteria that causes nosocomial infection, as the apparatus articles for use by polluting cause pyogenic infection, injection site pyogenic infection, bronchitis, pneumonia etc.Therefore, no matter be to detect or aspect clinical bacteria check in early days at food-borne pathogens, development fast, the technology of streptococcus aureus is all that the utmost point 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 object bacteria in this technology efficient capture, concentrated enrichment liquid improves pathogenic microbes detect sensitivity.In recent years, the immunomagnetic separation based on magnetic micro-beads (IMS) is connected to object bacteria antibody on magnetic bead, the magnetic bead that then will be connected with antibody drop in sample liquid to object bacteria caught, enrichment, magnetic separates (concrete principle is shown in Fig. 2 A).Yet, should the isolation technique based on the micron order immunomagnetic beads there be many limitation at present: 1) relative nano level magnetic bead, the specific surface area of micron magnetic bead is less, has reduced magnetic capture efficiency; 2), due to the particle properties of micron magnetic bead self, between itself and bacterial cell, by heterogeneous reaction (multiphase reaction) combination, usually need to go specificity to catch the bacterial cell in food substrate the longer time; 3) micron magnetic bead monodispersity is poor, and precipitation easily occurs self to assemble or form in food substrate liquid; 4) traditional immune magnetic separation technique, by antibody molecule, directly be coupled on immunomagnetic beads often, this process often can cause antibody activity to reduce significantly and the change of antibody direction in space, has increased the space steric effect between antibody, thereby has reduced the capture rate of antibody; 5) food substrate character is complicated and wherein non-purpose pathogenic bacterium (miscellaneous bacteria) concentration is large, and a micron magnetic bead easily produces non-specific adsorption, is difficult to realize the specific isolation to purpose bacterium in 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, and cell is squeezed and even breaks) of bacterial cell, causes the failure separated; (7), during magnetic bead coupling antibody, generally adopt hydrophobic absorption or chemical coupling mode that the activated antibody of tool is connected to magnetic bead surfaces.If antibody and magnetic bead surfaces distance are too near, because of the hydrophobic of magnetic bead nature and remained on surface thereof or strong hydrophilicity group, easily cause that the antibody space conformation changes, cause the antibody biological activity to descend.
Summary of the invention
For the defect of prior art, the purpose of this invention is to provide high, the easy disengaging time of a kind of capture rate short, the method for purpose bacterium (streptococcus aureus) specificity sharp separation in the food substrate of (being less than 30 T/m) complexity under low gradient magnetic.Comprise the steps:
The method that streptococcus aureus separates comprises the following steps:
(1) often take 1.0 mg dendrimers, be suspended in 4 mL 0.01 mol/L, in pH 8.0 PBS phosphoric acid buffers, the glutaraldehyde water solution 545 μ L of agitation and dropping 25%, the final concentration that makes glutaraldehyde is 3%, room temperature reaction 3.5 h under the rotating speed of shaking table 150 r/min; Drip streptococcus aureus
sAspecific antibody 1 mL, make its final concentration reach 3 mg/mL left and right; Room temperature reaction 24 h under the rotating speed of shaking table 150 r/min; Decompression is spin-dried for solvent, deionized water dissolving, dialysis 1 d in PBS and deionized water; Dialysis finishes the solution lyophilize obtained is obtained to dendrimer-antibody complex; (2) get 15 mg long-chain vitamin Hs, be suspended in 4 mL 0.01 mol/L, in pH 8.0 PBS phosphoric acid buffers, the glutaraldehyde water solution 545 μ L of agitation and dropping 25%, the final concentration that makes glutaraldehyde is 3%; Room temperature reaction 3.5 h under the rotating speed of shaking table 150 r/min; Add 0.53 mg step (1) gained dendrimer-antibody complex, room temperature reaction 24 h under the rotating speed of shaking table 150 r/min; Decompression is spin-dried for solvent, deionized water dissolving, dialysis 1 d in PBS and deionized water; Dialysis finishes the solution lyophilize obtained is obtained to long-chain vitamin H-dendrimer-antibody complex; (3) get 1 mL testing sample solution, add 0.1 mg
sAantibody and the co-modified dendrimer of long-chain vitamin H are step (2) long-chain vitamin H-dendrimer-antibody complex, be placed on blending instrument, rotating speed incubated at room 15 min with 30 rpm, add 0.1 mg to be modified with the nanometer magnetic bead of Streptavidin, be placed on blending instrument, with the rotating speed of 30 rpm at incubated at room 15 min; Conventional magnetic frame separates 3 min; (4) after deionized water cleans gently, with the PBS damping fluid mixed resuspended obtain nanometer magnetic bead-Streptavidin of being enriched with streptococcus aureus-long-chain vitamin H-dendrimer-antibody-
sAmixture.
Described dendrimer is amidized polyamide-amide type dendrimer PAMAM-G4, and its molecular weight is 14215 Da.
The described nanometer magnetic bead particle diameter of having modified Streptavidin is 20-50nm, is preferably 30 nm.
Dendrimer by amino and
sAthe carboxyl of specific antibody is realized the covalent coupling of dendrimer and antibody.
Dendrimer, by the carboxyl of amino and long-chain biotin molecule, is realized the covalent coupling of dendrimer and long-chain vitamin H; Add excessive long-chain vitamin H to guarantee exposed amino sites on the sealing dendrimer.
Concrete principle is shown in 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 product and leftovers, fried egg, glutinous rice cakes and bean jellies etc. such as fresh vegetables, meat, seafood and milks.Sample preparation gets final product according to conventional treatment method, after sample is pulverized, makes solution to be measured.
Adopt technical solution of the present invention to there is following beneficial effect:
1, the present invention is by the cascade scale effect of dendrimer, magnetic bacterium signal exponentially level is enlarged, just can realize the separation of magnetic bacterium under lower magneticstrength, and within the identical time, than routine immunization magnetic bead separation method, compare, be separated to purpose bacterium ability stronger, be specially adapted to the separation of complex sample, as food samples, whole blood sample etc., magnetic field slow for the purpose bacterium speed in the 20-50 nm immunomagnetic beads separate complex matrix sample after simple employing antibody modification requires high defect, adopt dendrimer 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 under low gradient magnetic (being less than 30 T/m) complicated food substrate.
2, this programme, for antibody molecule is coupled on dendrimer, has been avoided in the ordinary method antibody molecule is coupled to magnetic bead surfaces and has been caused antibody activity to reduce and sterically hindered large shortcoming.
3, the present invention adopts dendrimer, 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 dendrimer, the nanometer magnetic bead that can modify in conjunction with Streptavidin, thus make on dendrimer 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, after replacing the micron order magnetic particle with nanometer magnetic bead (20-50 nm), because the nanometer magnetic bead particle diameter is little, specific surface area is large, the steric hindrance of being combined with bacterium 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 dispersiveness and stability preferably in complex matrices, so the use of nanometer magnetic bead can overcome above-mentioned all defects owing to using the micron magnetic bead to cause.
5, the present invention is in sepn process, introduced tree-shaped high-polymer molecular, be connected with a large amount of long-chain biotin molecules on dendrimer, can be special and high-affinity ground be dispersed in coupling in matrix solution the identification of Streptavidin nanometer magnetic bead arranged, thereby make on dendrimer in conjunction with a large amount of nanometer magnetic beads, greatly increase the magnetic bead quantity of target bacteria surface bonding, realized the target bacteria that sharp separation is caught under magnetic field.With traditional bacterial magnetic separation method, comparing, is nanometer magnetic bead more stable in matrix because of what add, and the method is more suitable in complex matrices carries out the magnetic separation to bacterium, 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 that the activated antibody of tool is connected to magnetic bead surfaces.If antibody and magnetic bead surfaces distance are too near, because of the hydrophobic of magnetic bead nature and remained on surface thereof or strong hydrophilicity group, easily cause that the antibody space conformation changes, cause the antibody biological activity to descend.Yet this experimental program is introduced dendrimer, dendritic polymer in coupling process, it has certain space size (4-6nm), thereby makes antibody molecule away from magnetic bead and magnetic bead surfaces, has avoided the disadvantageous effect of magnetic bead nature and surperficial antagonist molecule.Simultaneously, the dendrimer, dendritic polymer of introducing but can not affect the antibody space conformation, thereby has played the bioactive effect of protection antibody molecule.
The accompanying drawing explanation
The structural representation of Fig. 1 PAMAM: solid space structure (A) and orthographic plan (B).
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 explain the present invention, is not intended to limit 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 dendrimer is amidized polyamide-amide type dendrimer PAMAM-G4, and its molecular weight is 14215 Da, purchased from Weihai Chen Yuan new chemical materials company limited.
Conventional magnetic frame separates magneticstrength and is less than 30 T/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 800 mL distilled water, with 5 M NaOH, adjust pH to 7.4, then be settled to 1000 mL and obtain 0.01 M PBS.Volume ratio with 1/1000 (V/V) adds Tween 20 again, obtains 0.1%PBST.
Embodiment 1
1, dendrimer-antibody complex, preparation in accordance with the following steps:
(1) take the amidized dendrimer of 1.0 mg, be suspended in 4 mL phosphoric acid buffers (pH 8.0 for PBS, 0.01 mol/L), the glutaraldehyde water solution 545 μ L of agitation and dropping 25%, the final concentration that makes glutaraldehyde is 3%.Room temperature reaction 3.5 h under the rotating speed of shaking table 150 r/min;
(2) drip streptococcus aureus to above-mentioned solution
sAspecific antibody 1 mL, make its final concentration reach 3 mg/mL left and right.Room temperature reaction 24 h under the rotating speed of shaking table 150 r/min;
(3) above-mentioned solution decompression is spin-dried for to solvent, deionized water dissolving, dialysis 1 d in PBS and deionized water; Dialysis finishes the solution lyophilize obtained.
2, prepared in accordance with the following steps by long-chain vitamin H-dendrimer-antibody complex:
(1) get 15 mg long-chain vitamin Hs, be suspended in 4 mL phosphoric acid buffers (pH 8.0 for PBS, 0.01mol/L), the glutaraldehyde water solution 545 μ L of agitation and dropping 25%, the final concentration that makes glutaraldehyde is 3%.Room temperature reaction 3.5 h under the rotating speed of shaking table 150 r/min;
(2) 0.53 mg dendrimer-antibody complex is joined in above-mentioned solution to room temperature reaction 24 h under the rotating speed of shaking table 150 r/min;
(3) above-mentioned solution decompression is spin-dried for to solvent, deionized water dissolving, dialysis 1 d in PBS and deionized water; Dialysis finishes the solution lyophilize obtained.
3. enrichment is caught: get testing sample solution 1 mL, add 0.1 mg long-chain vitamin H-dendrimer-antibody complex, be placed on blending instrument, with rotating speed incubated at room 15 min of 30 rpm form long-chain vitamin H-dendrimers-antibody-
sAantigenic compound; Add 0.1 mg to be modified with the nanometer magnetic bead of Streptavidin, be placed on blending instrument, with the rotating speed of 30 rpm incubated at room 15 min again, centrifuge tube is inserted to conventional magnetic frame and separate 3 min;
4., after deionized water cleans gently, with mixed resuspended the obtaining of PBS damping fluid, be enriched with streptococcus aureus
sAmixture nanometer magnetic bead-Streptavidin-vitamin H-dendrimer-antibody-
sAantigen.
Embodiment 2 concentration effect experiments
(1) getting 1 mL concentration is 10
4cfu/mL's
sAin the aseptic centrifuge tube of 1.5 mL, centrifugal 5 min of 12000 rpm, abandon supernatant, resuspended with the aseptic PBS solution of equal-volume.
(2) enrichment is caught: arrange respectively the technical solution of the present invention group (
sAthe dendrimer group that antibody and long-chain vitamin H are co-modified),
sAthe nanometer magnetic bead group that specific antibody is modified,
sAthe micron magnetic bead group enrichment purpose bacterium that specific antibody is modified.
(3) after magnetic separates, supernatant liquor is poured in aseptic centrifuge tube, caught and separate
sAimmunomagnetic beads with PBST, clean twice, 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 to gradient dilution, with dull and stereotyped, to each gradient counting, calculate the capture rate of object bacteria by the capture rate formula, test triplicate at every turn.Each calculation formula of organizing capture rate is as follows: (total number of bacterial colony of being adsorbed by enrichment/all total plate count) * 100%.
Describedly respectively organize enrichment to catch the scheme of purpose bacterium as follows:
A. the technical solution of the present invention group (
sAthe dendrimer group that antibody and long-chain vitamin H are co-modified) enrichment is caught purpose bacterium scheme as embodiment 1, specific as follows:
By 0.1 mg
sAthe co-modified dendrimer of antibody and vitamin H is that vitamin H-dendrimer-antibody complex joins containing in the object bacteria centrifuge tube, is placed on blending instrument, with rotating speed incubated at room 15 min of 30 rpm
.then add 0.1 mg to be modified with the nanometer magnetic bead of Streptavidin, be placed on blending instrument, with the rotating speed of 30 rpm incubated at room 15 min again.Finally, centrifuge tube is inserted to conventional magnetic frame and separate 3 min.
B.
sAit is specific as follows that purpose bacterium scheme is caught in the nanometer magnetic bead group enrichment that specific antibody is modified:
0.1 mg is prepared
sAthe nanometer magnetic bead that specific antibody is modified joins containing in the 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 to conventional magnetic frame and separate 3 min.
Described
sAthe nanometer magnetic bead preparation that specific antibody is modified: (1) is got 10 mg nanometer magnetic beads (30 nm do not have the coupling Streptavidin) and is used successively dehydrated alcohol, 1 M NaOH, each washing of 1 M HCl 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 ℃ of activation 2 h.(2) magnetic frame reclaims magnetic bead, and PBS(0.02 M, pH 4.0) wash three times after, magnetic bead is resuspended in aseptic PBS, by every mg magnetic bead, adds 80 μ g
sAspecific antibody, be placed in 37 ℃ of coupling 2 h on blending instrument.(3) add the thanomin room temperature to seal 2 h.Magnet stand reclaims magnetic bead, PBS washing three times, and 10 ml PBS(are containing 0.05% NaN
3, 0.5% BSA, pH 7.4) resuspended immunomagnetic beads standby in 4 ℃ of Refrigerator stores.
C.
sAit is specific as follows that purpose bacterium scheme is caught in the micron magnetic bead group enrichment that specific antibody is modified:
0.1 mg is prepared
sAthe micron magnetic bead that specific antibody is modified joins containing in the 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 to conventional magnetic frame and separate 3 min.
The micron magnetic bead preparation that described SA specific antibody is modified: (1) is got 10 mg micron magnetic beads (1150 nm do not have the coupling Streptavidin) and is used successively dehydrated alcohol, 1 M NaOH, each washing of 1 M HCl 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 ℃ of activation 2 h.(2) magnetic frame reclaims magnetic bead, and PBS(0.02 M, pH 4.0) wash three times after, magnetic bead is resuspended in aseptic PBS, by every mg magnetic bead, adds 80 μ g
sAspecific antibody, be placed in 37 ℃ of coupling 2 h on blending instrument.(3) add the thanomin room temperature to seal 2 h.Magnet stand reclaims magnetic bead, PBS washing three times, and 10 ml PBS(are containing 0.05% NaN
3, 0.5% BSA, pH 7.4) resuspended immunomagnetic beads standby in 4 ℃ of Refrigerator stores.
It is as follows that each organizes capture rate:
| SAThe micron magnetic bead group that specific antibody is modified is caught Obtain rate | SAThe nanometer magnetic bead group capture rate that specific antibody is modified | SAThe dendrimer group capture rate that antibody and long-chain vitamin H are co-modified |
| 56.9% | 20.12% | 90.9% |
Experimental result shows,
sAthe capture rate of the micron magnetic bead group that specific antibody is modified is apparently higher than the capture rate of nanometer magnetic bead group, and this explanation contrasts nanometer magnetic bead group, because micron magnetic bead volume is large, magnetic is strong, and 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 again
sAthe micron magnetic bead group that specific antibody is modified, this shows that technical solution of the present invention can increase object bacteria nano surface magnetic bead fraction of coverage by dendrimer, thereby magnetic is improved greatly, and then realized (3min) high efficiency separation Concentration of Gold staphylococcus aureus at short notice.
Experiment is caught in embodiment 3 enrichments
Conventional magnetic frame disengaging time is 30 min, and all the other are with embodiment 2.
It is as follows that each organizes capture rate:
| SAThe micron magnetic bead group capture rate that specific antibody is modified | SAThe nanometer magnetic bead group capture rate that specific antibody is modified | SAThe dendrimer group capture rate that antibody and long-chain vitamin H are co-modified |
| 60.71% | 38.1% | 91.8% |
Experimental result shows, separates 3 min in comparative example 2, and when disengaging time reaches 30 min, the capture rate of three groups all is improved, particularly
sAthe capture rate of the nanometer magnetic bead group that specific antibody is modified improves the most obvious, and this shows can improve widely by time expand the capture rate of nanometer magnetic bead group, but when it still separates (3 min) lower than the short period of time
sAthe capture rate of the dendrimer group that antibody and long-chain vitamin H are co-modified.This shows technical solution of the present invention (3 min) high efficiency separation Concentration of Gold staphylococcus aureus at short notice.
Embodiment 4
Aseptic meat is pulverized, made in the usual way testing sample solution, add
sAregulate bacterium colony concentration to 10
4cfu/mL is standby.
By what prepare
sAthe co-modified dendrimer (0.1 mg) of antibody and long-chain vitamin H joins respectively in sample solution, 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) be 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 separates 3 min.Magnetic is poured supernatant liquor in aseptic centrifuge tube into after separating, and separate, has caught
sAimmunomagnetic beads with PBST, clean twice, mix, and with the resuspended immunomagnetic beads of the aseptic PBS solution of 1 mL.Capture rate obtains as embodiment 2 methods, and all the other are with embodiment 2.The results are shown in Table 1, show in this programme energy efficiently concentrating sample separation
sA.
Embodiment 5
Germ-free milk is the sample testing sample solution, adds
sAregulate bacterium colony concentration to 10
4cfu/mL.All the other are with embodiment 4
Embodiment 6
Aseptic grates vegetables, make testing sample solution in the usual way, adds
sAregulate bacterium colony concentration to 10
4cfu/mL.All the other are with embodiment 4.
Embodiment 7
Testing sample is aseptic whole blood, adds
sAregulate bacterium colony concentration to 10
4cfu/mL.All the other are with embodiment 4.
In the different actual samples of table 1
sAthe comparison of separating effect
| Actual sample | SAThe dendrimer group capture rate that antibody and long-chain vitamin H are co-modified |
| Embodiment 4 meats | 81.0% |
| Embodiment 5 milk | 82.1% |
| Embodiment 6 vegetables | 83.1% |
| Embodiment 7 whole bloods | 82.9% |
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any modifications of doing within the spirit and principles in the present invention, be equal to and replace and improvement etc., within all should being included in protection scope of the present invention.
Claims (3)
1. streptococcus aureus
sAthe method of separating, it is characterized in that comprising the following steps: often take 1.0 mg dendrimers, be suspended in 4 mL 0.01 mol/L, in pH 8.0 PBS phosphoric acid buffers, the glutaraldehyde water solution 545 μ L of agitation and dropping 25%, the final concentration that makes glutaraldehyde is 3%, room temperature reaction 3.5 h under the rotating speed of shaking table 150 r/min; Drip streptococcus aureus
sAspecific antibody 1mL, make its final concentration reach 3 mg/mL left and right; Room temperature reaction 24 h under the rotating speed of shaking table 150 r/min; Decompression is spin-dried for solvent, deionized water dissolving, dialysis 1 d in PBS and deionized water; Dialysis finishes the solution lyophilize obtained is obtained to dendrimer-antibody complex; (2) get 15 mg long-chain vitamin Hs, be suspended in 4 mL 0.01 mol/L, in pH 8.0 PBS phosphoric acid buffers, the glutaraldehyde water solution 545 μ L of agitation and dropping 25%, the final concentration that makes glutaraldehyde is 3%; Room temperature reaction 3.5 h under the rotating speed of shaking table 150 r/min; Add 0.53 mg step (1) gained dendrimer-antibody complex, room temperature reaction 24 h under the rotating speed of shaking table 150 r/min; Decompression is spin-dried for solvent, deionized water dissolving, dialysis 1 d in PBS and deionized water; Dialysis finishes the solution lyophilize obtained is obtained to long-chain vitamin H-dendrimer-antibody complex; (3) get 1 mL testing sample solution, add 0.1 mg
sAantibody and the co-modified dendrimer of long-chain vitamin H are step (2) long-chain vitamin H-dendrimer-antibody complex, be placed on blending instrument, rotating speed incubated at room 15 min with 30 rpm, add 0.1 mg to be modified with the nanometer magnetic bead of Streptavidin, be placed on blending instrument, with the rotating speed of 30 rpm at incubated at room 15 min; Conventional magnetic frame separates 3 min; (4) after deionized water cleans gently, with the PBS damping fluid resuspended obtain nanometer magnetic bead-Streptavidin of being enriched with streptococcus aureus-long-chain vitamin H-dendrimer-antibody-
sAmixture.
2. method claimed in claim 1, is characterized in that described dendrimer is amidized polyamide-amide type dendrimer PAMAM-G4, and its molecular weight is 14215 Da.
3. method according to claim 1, is characterized in that the described nanometer magnetic bead particle diameter of having modified Streptavidin is 20-50nm, is preferably 30 nm.
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| CN106978412A (en) * | 2017-02-20 | 2017-07-25 | 南昌大学 | The new method of the coated Beads enrichment staphylococcus aureus of vancomycin combination bovine serum albumin(BSA) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102645536A (en) * | 2012-04-19 | 2012-08-22 | 沈鹤柏 | Method for detecting staphylococcus aureus |
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| CN102645536A (en) * | 2012-04-19 | 2012-08-22 | 沈鹤柏 | Method for detecting staphylococcus aureus |
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| Title |
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| 司宝财: "树枝状大分子复合磁性颗粒的制备与表征", 《中国优秀硕士学位论文全文数据库工程科技I辑》 * |
| 王程程 等: "基于免疫磁珠快速分离金黄色葡萄球菌的研究", 《食品工业科技》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106978412A (en) * | 2017-02-20 | 2017-07-25 | 南昌大学 | The new method of the coated Beads enrichment staphylococcus aureus of vancomycin combination bovine serum albumin(BSA) |
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