CN103333817B - Method for magnetically separating yersinia enterocolitica (YE) - Google Patents
Method for magnetically separating yersinia enterocolitica (YE) Download PDFInfo
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Abstract
The invention relates to the technical field of biology and discloses a method for enriching and separating yersinia enterocolitica (YE). 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 bacteria 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
Food-borne pathogenic fungi pollution is one of significant problem of China's food safety.According to WHO statistics, developed country about has the people of 1/3rd 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.Yersinia entero-colitica (
yersinia enterocolitica, YE) be a class zoonosis pathogenic bacteria, be found in New York, United States the earliest.In last century, once there be twice large outbreak of epidemic in the mid-80 China, caused people more than 500 to infect, and it is proved and is food pollution yersinia entero-colitica and causes.As the food-borne pathogens that a class is main, it causes except bowel symptoms, also can cause respiratory tract, cardiovascular systems, bone, reticular tissue and general disease, and when there is septicemia, case fatality rate reaches more than 30%.Therefore, countries in the world are all classified as the conventional sense project of import and export food.Development is quick, the technology of efficiently concentrating sample separation Virulence of Yersinia is all very necessary.
Immunity magnetic separation technique is one of important component part of food-borne pathogens rapid screening technology, and this technology can efficient capture, object bacteria in concentrated enrichment liquid, improves pathogenic microbes detect sensitivity.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 microorganism 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 often can cause antibody activity to reduce significantly and the change of antibody direction in space, increases the space steric effect between antibody, thus reduces the capture rate of antibody; 5) food substrate character is complicated and miscellaneous bacteria concentration that is wherein non-object pathogenic bacterium is large, and 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 to magnetic bead surfaces by general hydrophobic adsorbent or the chemical coupling mode of adopting.If antibody and magnetic bead surfaces distance too near, because of the hydrophobic or strong hydrophilicity group of magnetic bead nature and remained on surface thereof, easily cause antibody space conformation to change, cause antibody bioactive to decline.
Summary of the invention
For the defect of prior art, the object of this invention is to provide high, the easy disengaging time of a kind of capture rate short, the method for object bacterium yersinia entero-colitica specificity sharp separation in the food substrate that (to be less than 30 T/m) under low gradient magnetic complicated.Comprise the steps:
(1) often take the amidized dendrimer of 1.1 mg, be suspended in 4 mL 0.01 mol/L, in pH 8.0 PBS phosphoric acid buffer, the glutaraldehyde water solution 545 μ L of agitation and dropping 25%, makes the final concentration of glutaraldehyde be 3%; Room temperature reaction 3.5 h under the rotating speed of shaking table 150 r/min; Yersinia entero-colitica is added to above-mentioned solution
yEspecific antibody 12 mg, makes its final concentration reach about 3 mg/mL; Room temperature reaction 24 h under the rotating speed of shaking table 150 r/min; Decompression is spin-dried for solvent, deionized water dissolving, and dialyse 1 d in PBS and deionized water; Dialysis terminates, by the solution lyophilize obtained, to obtain dendrimer-antibody complex;
(2) often get 15 mg long-chain biological elements, be suspended in 4 mL 0.01 mol/L, in pH 8.0 PBS phosphoric acid buffer, the glutaraldehyde water solution 545 μ L of agitation and dropping 25%, makes the final concentration of glutaraldehyde be 3%; Room temperature reaction 3.5 h under the rotating speed of shaking table 150 r/min; 0.55 mg step (1) gained dendrimer-antibody complex is joined in above-mentioned solution, room temperature reaction 24 h under the rotating speed of shaking table 150 r/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-dendrimer-antibody complex;
(3) get testing sample solution 1 mL, add 0.1 mg long-chain biological element-dendrimer-antibody complex, be placed on blending instrument, with rotating speed incubated at room 15 min of 30 rpm formed long-chain biological element-dendrimer-antibody-
yEantigenic compound; 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, with PBS damping fluid mix resuspended namely obtain mixture nanometer magnetic bead-Streptavidin-vitamin H-dendrimer-antibody of being enriched with yersinia entero-colitica-
yEantigen.
Dendrimer is amidized polyamidoamine dendrimer PAMAM-G4, and its molecular weight is 14215 Da.
Described nanometer magnetic bead particle diameter is 20-50 nm, is preferably 30nm.
Dendrimer by amino and
yEthe carboxyl of specific antibody realizes the covalent coupling of dendrimer and antibody.
Dendrimer, by carboxyl that is amino and long-chain biological element molecule, realizes the covalent coupling of dendrimer and long-chain biological element; Add excessive long-chain biological element to ensure amino sites exposed on closed 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 freeze cutting, as products such as meat, seafood and milks.Sample preparation is treatment process conveniently, as will be made solution to be measured after sample comminution.
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 dendrimer, 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, dendrimer 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 dendrimer 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 dendrimer, 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, dendrimer 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 dendrimer 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 high-polymer molecular, dendrimer 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 dendrimer 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 dendrimer, dendritic polymer in coupling process, it has certain space size (4-6 nm), 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 dendrimer, dendritic 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 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 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 dendrimer is amidized polyamidoamine dendrimer PAMAM-G4, and its molecular weight is 14215 Da, purchased from Weihai Chen Yuan new chemical materials company limited.
Conventional magnetic frame is separated magneticstrength and is less than 30 T/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, dendrimer-antibody complex, prepare in accordance with the following steps:
(1) take the amidized dendrimer of 1.1 mg, be suspended in 4 mL phosphate buffered saline buffers (PBS, 0.01mol/L, pH 8.0), the glutaraldehyde water solution 545 μ L of agitation and dropping 25%, makes the final concentration of glutaraldehyde be 3%.Room temperature reaction 3.5 h under the rotating speed of shaking table 150 r/min;
(2) yersinia entero-colitica is dripped to above-mentioned solution
yEspecific antibody 12 mg, makes its final concentration reach about 3 mg/mL.Room temperature reaction 24 h under the rotating speed of shaking table 150 r/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-dendrimer-antibody complex is prepared in accordance with the following steps:
(1) often get 15 mg long-chain biological elements, be suspended in 4 mL phosphoric acid buffers (PBS, 0.01mol/L, pH 8.0), the glutaraldehyde water solution 545 μ L of agitation and dropping 25%, makes the final concentration of glutaraldehyde be 3%.Room temperature reaction 3.5 h under the rotating speed of shaking table 150 r/min;
(2) 0.55 mg dendrimer-antibody complex is joined in above-mentioned solution, room temperature reaction 24 h under the rotating speed of shaking table 150 r/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 1 mL, adds 0.1 mg long-chain biological element-dendrimer-antibody complex, is placed on blending instrument, with rotating speed incubated at room 15 min of 30 rpm formed long-chain biological element-dendrimer-antibody-
yEantigenic compound; 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 resuspended namely obtaining with PBS damping fluid and be enriched with yersinia entero-colitica
yEmixture nanometer magnetic bead-Streptavidin-vitamin H-dendrimer-antibody-
yEantigen.
Embodiment 2 concentration effect is tested
(1) getting 1 mL concentration is 10
4cfu/mL's
yEin 1.5 mL sterile centrifugation tube, centrifugal 5 min of 12000 rpm, abandon supernatant, resuspended by the aseptic PBS solution of equal-volume.
(2) enrichment is caught: arrange respectively technical solution of the present invention group (
yEantibody and the plain co-modified dendrimer group of long-chain biological),
yEspecific antibody modify nanometer magnetic bead group,
yEthe micron magnetic bead group enrichment object bacterium that specific antibody is modified.
(3) after Magneto separate, supernatant liquor is poured in sterile centrifugation tube, and separate and caught
yEimmunomagnetic 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 (
yEantibody and the co-modified dendrimer group of long-chain biological element) enrichment catches object bacterium scheme as embodiment 1, specific as follows:
By 0.1 mg
yEantibody and the co-modified dendrimer of vitamin H and vitamin H-dendrimer-antibody complex join containing in object bacteria centrifuge tube, are 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.
yEit is specific as follows that object bacterium scheme is caught in the nanometer magnetic bead group enrichment that specific antibody is modified:
0.1 mg is prepared
yEthe nanometer magnetic bead that specific antibody 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.
Described
yEthe nanometer magnetic bead preparation that 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 by every mg magnetic bead
yEspecific antibody, 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.
yEit is specific as follows that object bacterium scheme is caught in the micron magnetic bead group enrichment that specific antibody is modified:
0.1 mg is prepared
yEthe micron magnetic bead that specific antibody 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.
Described
yEthe micron magnetic bead preparation that 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 by every mg magnetic bead
yEspecific antibody, 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:
| YEThe micron magnetic bead group capture rate that specific antibody is modified | YEThe nanometer magnetic bead group capture rate that specific antibody is modified | YEAntibody and the plain co-modified dendrimer group capture rate of long-chain biological |
| 59.0% | 22.7% | 91.1% |
Experimental result shows,
yEthe 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 illustrates contrast nanometer magnetic bead group, because micron magnetic bead volume is large, magnetic 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
yEthe 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, thus magnetic is improved greatly, and then achieve (3 min) high efficiency separation enrichment yersinia entero-colitica at short notice.
Experiment is caught in embodiment 3 enrichment
Conventional magnetic frame disengaging time is 30 min, and all the other are with embodiment 2.
Each group of capture rate is as follows:
| YEThe micron magnetic bead group that specific antibody is modified is caught Obtain rate | YEThe nanometer magnetic bead group capture rate that specific antibody is modified | YEAntibody and the plain co-modified dendrimer group capture rate of long-chain biological |
| 63.7% | 38.6% | 93.8% |
Experimental result shows, is separated 3 min in comparative example 2, and when reaching 30 min when disengaged, the capture rate of three groups is obtained for raising, particularly
yEthe capture rate of the nanometer magnetic bead group that specific antibody is modified improves the most obvious, and this shows to improve the capture rate of nanometer magnetic bead group widely by time expand, but its still lower than short period of time separation (3 min) time
yEthe capture rate of antibody and the plain co-modified dendrimer group of long-chain biological.This shows that technical solution of the present invention can (3 min) high efficiency separation enrichment yersinia entero-colitica at short notice.
Embodiment 4
Aseptic meat is pulverized, makes testing sample solution in the usual way, add
yEregulate bacterium colony concentration to 10
4cfu/mL is for subsequent use.
By what prepare
yEantibody and the co-modified dendrimer (0.1 mg) of long-chain biological element join in sample solution respectively, are 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, and separate and caught
yEimmunomagnetic 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 in this programme energy efficiently concentrating sample separation
yE.
Embodiment 5
Germ-free milk is sample testing sample solution, adds
yEregulate bacterium colony concentration to 10
4cfu/mL.All the other are with embodiment 4.
Embodiment 6
Aseptic oyster is pulverized, and makes testing sample solution in the usual way, adds
yEregulate bacterium colony concentration to 10
4cfu/mL.All the other are with embodiment 4.
Embodiment 7
Testing sample is aseptic whole blood, adds
yEregulate bacterium colony concentration to 10
4cfu/mL.All the other are with embodiment 4.
In the different actual sample of table 1
yEthe comparison of separating effect
| Actual sample | YEAntibody and the plain co-modified dendrimer group capture rate of long-chain biological |
| Embodiment 4 meat | 82.2% |
| Embodiment 5 milk | 83.5% |
| Embodiment 6 oyster | 83.3% |
| Embodiment 7 whole blood | 81.6% |
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. Magneto separate yersinia entero-colitica
yEmethod, it is characterized in that comprising the following steps: (1) often takes 1.1 mg dendrimers, be suspended in 4 mL 0.01 mol/L, in pH 8.0 PBS phosphoric acid buffer, the glutaraldehyde water solution 545 μ L of agitation and dropping 25%, the final concentration of glutaraldehyde is made to be 3%, room temperature reaction 3.5 h under the rotating speed of shaking table 150 r/min; Yersinia entero-colitica is added to above-mentioned solution
yEspecific antibody 12 mg, makes its final concentration reach about 3 mg/mL; Room temperature reaction 24 h under the rotating speed of shaking table 150 r/min; Decompression is spin-dried for solvent, deionized water dissolving, and dialyse 1 d in PBS and deionized water; Dialysis terminates, by the solution lyophilize obtained, to obtain dendrimer-antibody complex; (2) often get 15 mg long-chain biological elements, be suspended in 4 mL 0.01 mol/L, in pH 8.0 PBS phosphoric acid buffer, the glutaraldehyde water solution 545 μ L of agitation and dropping 25%, makes the final concentration of glutaraldehyde be 3%; Room temperature reaction 3.5 h under the rotating speed of shaking table 150 r/min; 0.55 mg step (1) gained dendrimer-antibody complex is joined in above-mentioned solution, room temperature reaction 24 h under the rotating speed of shaking table 150 r/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-dendrimer-antibody complex; (3) get testing sample solution 1 mL, add 0.1 mg long-chain biological element-dendrimer-antibody complex, be placed on blending instrument, with rotating speed incubated at room 15 min of 30 rpm formed long-chain biological element-dendrimer-antibody-
yEantigenic compound; 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, centrifuge tube is inserted conventional magnetic frame and be separated 3 min; Described nanometer magnetic bead particle diameter is 20-50 nm; (4) after Magneto separate, after deionized water cleans gently, with PBS damping fluid resuspended namely obtain mixture nanometer magnetic bead-Streptavidin-vitamin H-dendrimer-antibody of being enriched with YE-
yEmixture.
2. method according to claim 1, it is characterized in that described dendrimer is amidized polyamidoamine dendrimer PAMAM-G4, its molecular weight is 14215 Da.
3. method according to claim 1, is characterized in that described nanometer magnetic bead particle diameter is 30 nm.
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| WO2003035897A2 (en) * | 2001-10-24 | 2003-05-01 | Michael Burton | Enzyme substrates for detecting beta-d-ribofuranosidase activity |
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| WO2003035897A2 (en) * | 2001-10-24 | 2003-05-01 | Michael Burton | Enzyme substrates for detecting beta-d-ribofuranosidase activity |
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