CN101435824A - Method for enriching avian influenza virus in sample of antibody coupling magnetic nano granule - Google Patents
Method for enriching avian influenza virus in sample of antibody coupling magnetic nano granule Download PDFInfo
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- CN101435824A CN101435824A CNA2008102396737A CN200810239673A CN101435824A CN 101435824 A CN101435824 A CN 101435824A CN A2008102396737 A CNA2008102396737 A CN A2008102396737A CN 200810239673 A CN200810239673 A CN 200810239673A CN 101435824 A CN101435824 A CN 101435824A
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Abstract
The invention discloses a method for the antibody coupling of avian influenza virus in a magnetic nanometer particle enriched sample and belongs to the field of inspection and quarantine. The method for the antibody coupling of avian influenza virus in the magnetic nanometer particle enriched sample comprises the following steps: (1) magnetic nanometer particles, a polyclonal antibody of avian influenza virus and a coupling agent are mixed and react for 2 hours at room temperature to obtain a magnetic nanometer particle solution coupled with the polyclonal antibody of avian influenza virus; (2) a coupling product is purified and is removed with an unreacted antibody; (3) the coupling product and a solution which is to be tested react and are enriched with magnet; and (4) an enriched product is detected. The method has the advantages that the method does not lose virus activity and has high speed, high efficiency, high sensitivity and strong specificity. The method is rapid and simple and has simple equipment and low cost.
Description
Technical field
The present invention relates to the method for avian influenza virus in the magnetic nanoparticle enriched sample of antibody coupling, more specifically the say so method of a kind of magnetic nanoparticle and antibody coupling and purifying, the coupling antibody that obtains is applicable to the enrichment of pathogenic microorganisms such as avian influenza virus, thereby can fast detecting go out avian influenza virus from water or in other environmental sample, belong to inspection and quarantine field.
Background technology
Bird flu (Bird Flu or Avian Influenza) is a kind of acute infectious disease that is caused by avian influenza virus.By the end of in April, 2008 (beginning in 2003), the World Health Organization (WHO) reports highly pathogenic human avian influenza confirmed cases 378 examples altogether, and from 14 countries, mortality ratio is up to 63% respectively.Because the high mortality of bird flu and the mankind generally lack immunity to avian influenza virus, the task of research avian influenza virus is very arduous.Detection for avian influenza virus has at present had many methods, for example viral separation, RT-PCR, ELISA, fluorescence RT-PCR etc., but all there is the problem of recall rate undoubtedly in these methods, if viral level does not reach the detection lower limit of this detection method in the sample, just detect not come out, therefore this area presses for a kind of method of enrichment avian influenza virus, has fast sensitive and equipment characteristics simple, with low cost simultaneously, so as to diagnose better faster, treatment and birds flu-preventing.
Nanometer technology is one of major fields of at present the most forward-looking in fundamental research and the hi-tech development in the world, drive property.Nano material is because of having unique physicochemical property, the specific surface area that it is huge, special reactivity and nano effect make it differ from traditional material greatly, in development fast, have great potentiality aspect high sensitivity, high accuracy detection technique and the sensing means.China emerges in an endless stream in design, research and development, sign and the potential application achievement of nano meter biomaterial.Study and utilize the characteristic of novel nano biomaterial, in conjunction with existing detection technique, realize quick, high special, the high responsive check purpose in port, exploitation has cause of disease enrichment, nanometer and the bioactivator mark and the detection technique of China's independent intellectual property right, and the commanding elevation of capturing nano material action oriented research field is had the important strategic meaning.
Magnetic nanoparticle is a kind of novel nano-functional material of at first being developed in 1978 by Senyei A E.Its inside is magnetic nuclear, thereby externally under the effect in magnetic field, microballoon can directedly move; The outside is one deck macromolecule layer, and surface distributed many reactive groups, can with biochemical reagents generation couplings such as cell, protein, nucleic acid, enzyme, and then under the effect in magnetic field, realize to separate.Magnetic nanoparticle is from being born, and it has just received the concern of researcher, and the application of succeeding at biochemical analysis field.Utilize magnetic nanoparticle isolated protein, enzyme, antibody, cell, simple, the easy and simple to handle while velocity of separation of equipment needed thereby is fast, the efficient height, and the biologically active that can keep sample, therefore in recent years in fields such as biological medicines, magnetic nanoparticle has been widely used in a plurality of fields such as fixing of immunoassay, foranalysis of nucleic acids, cell separation, enzyme.
The magnetic nanoparticle bag by last specific antibody, acceptor, single stranded DNA, is used for the target body of separate complex sample, obtains immense success.Compare with traditional separation method, magnetic nanoparticle is used for the separation of the biological sample of complex component, can realize separating and enrichment is carried out simultaneously, improved velocity of separation and bioaccumulation efficiency greatly, the while also makes the sensitivity of analyzing and testing improve greatly.At present, there has been relevant article to report magnetic nanoparticle has been applied to trace microorganism or some active chemistry in the testing environment sample, obtained good effect.But at present, nano material and protein coupling are global problems.Main difficult point comprises the water miscible problem of nano material; The activation of nano-material surface group; The covalent bond of antibody molecule and nano material; Coupling condition must be able to guarantee that nano material and antibody activity can not be impaired; Outside also require can effectively remove the antibody molecule of not coupling after the coupling.
Technology contents
First technical matters that the present invention will solve is: the method that avian influenza virus in a kind of magnetic nanoparticle enriched sample of antibody coupling is provided, pass through reaction condition optimization, realize the coupling of specific antibody and magnetic nanoparticle, and the product purification method studied, thereby set up the method for magnetic nanoparticle and antibody coupling and purifying.
Another technical matters that will solve of the present invention is: the magnetic nanoparticle coupling antibody of purifying is used for the enrichment of pathogenic microorganisms such as avian influenza virus, and adopt method for quick that concentration effect is verified, set up that a kind of new specific avian influenza virus separates fast, the method for enrichment.
For achieving the above object, the present invention is by the following technical solutions:
The method of avian influenza virus comprises the steps: in the magnetic nanoparticle enriched sample of antibody coupling
(1) magnetic nanoparticle, avian influenza virus polyclonal antibody and coupling agent are mixed, room temperature reaction 2 hours obtains containing the solution that coupling has the magnetic nanoparticle of avian influenza virus polyclonal antibody;
(2) purifying coupled product is removed unreacted antibody;
(3) behind coupled product and the solution reaction to be measured, use the magnet enrichment;
(4) detection of enriched product.
Described step (1) is mixed magnetic nanoparticle, avian influenza virus polyclonal antibody and coupling agent, obtain containing the solution that coupling has the magnetic nanoparticle of avian influenza virus polyclonal antibody, be meant magnetic nanoparticle, coupling agent and avian influenza virus polyclonal antibody are mixed in the ratio of 1-2 μ mol:1-6mg:0.625-5mg.
Described " magnetic nanoparticle " is meant the magnetic nanoparticle of superparamagnetism.
" coupling agent " of indication of the present invention is meant and can makes antibody and magnetic nanoparticle carry out covalently bound reagent.Because the architectural feature and the magnetic nanoparticle of antibody are modified carboxyl, the coupling agent of selection should meet a kind of of following condition: the energy activated carboxyl, make the carboxyl and the reaction of the amino on the antibody of activation, and form peptide bond.The coupling agent that the present invention uses is ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDC) or N-hydroxy thiosuccinimide (Sulfo-NHS) or the two mixing.Be preferably independent use ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDC).
Described " avian influenza virus polyclonal antibody " be meant by avian influenza virus antigen in the mammal hypodermic injection, carry out repeatedly immunity after, collect the serum of immunized animal and the purified avian influenza virus polyclonal antibody that obtains.
The method one of described step (2) purifying coupled product is the sieve method purifying, and molecular sieve is sephadex G100.
The method two of described step (2) purifying coupled product is the centrifuge method purifying, and centrifugal condition is 4 ℃, 3000-12000r/min, and centrifugal 10-20min discards supernatant after centrifugal, and the gained precipitation is the magnetic nanoparticle of coupling antibody.
Described step (4) detects the method for enriched product for to detect with avian influenza virus fluorescent RT-PCR detection reagent box.
The present invention passes through magnetic nanoparticle and antibody molecule The Characteristic Study, by selecting suitable coupling agents kind and concentration, water miscible magnetic nanoparticle and specific antibody can be carried out covalent coupling, obtain the magnetic nanoparticle of biological functional, this biological functional magnetic nanoparticle combines by immunologic opsonin with virus in the solution, again by the needed virus of magnet enrichment.The present invention is optimized various coupling conditions, has set up the method that preparation and purifying have immunocompetent antibody coupling magnetic nanoparticle, lays a good foundation for further developing coherent detection reagent.
In view of the above, the invention provides a kind of method of new enrichment avian influenza virus, its velocity of separation is fast, efficient is high, and required equipment is simple, with low cost.
Advantage of the present invention is:
(1) method provided by the invention is not lost virus activity;
(2) method speed provided by the invention is fast, efficient is high;
(3) highly sensitive, the high specificity of method provided by the invention.
(4) method provided by the invention is quick, easy, and equipment is simple, with low cost.
Description of drawings
Fig. 1 detects the forward and backward viral level testing result of enrichment with the fluorescence RT-PCR method.
Fig. 2 detects the forward and backward viral level testing result of enrichment with the fluorescence RT-PCR method.
Embodiment
Embodiment 1: coupling, the purifying of magnetic nanoparticle and avian influenza virus polyclonal antibody, the enrichment of avian influenza virus and detection
1, material
The borate buffer of pH8.0: 0.05M borax 30% (V/V), 0.2M boric acid 70% (V/V), the pH value is 8.
Magnetic nanoparticle (available from Tianjin times Si Le company product), EDC (ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate), Sulfo-NHS (N-hydroxy thiosuccinimide), monoethanolamine, 1M glycocoll, 0.02MNaH
2PO
4, 0.1M NaCl, 1% Sodium azide (mass percent), Sephadex G100 (available from the BIO-RAD product), DEAE Sepharose Fast Flow (available from Pharmacia company).
Avian influenza virus antigen (available from Harbin veterinary institute country bird flu reference laboratory).
Freund's complete adjuvant and incomplete Freund (available from Beijing's biological products assay institute).
TRIZOL reagent (INVIOTROGEN).
Avian influenza virus fluorescent RT-PCR detection reagent box (available from Shenzhen basic company product).
Avian influenza virus polyclonal antibody (6.25mg/ml), self-control; Its preparation method is for to divide immune new zealand white rabbit 4 times with avian influenza virus antigen, the 1st immunity: get 2.0mL (250 μ g/mL) avian influenza virus antigen and equal-volume Freund's complete adjuvant, fully be ground to complete emulsification, the subcutaneous multi-point injection of rabbit nape portion; Carry out the 2nd immunity after 3 weeks at interval: get 2.0mL (150 μ g/mL) avian influenza virus antigen and equal-volume incomplete Freund, fully be ground to complete emulsification, the subcutaneous multi-point injection of rabbit nape portion; Carry out the 3rd immunity after 3 weeks at interval: get 2.0mL (150 μ g/mL) Brucella soluble antigen, the rabbit ear vein injection.After 20 days, arteria carotis bloodletting results antiserum.
Get the above-mentioned serum of 20ml, add physiological saline 20ml, dropwise add (NH again
4)
2SO
4Saturated solution 10ml makes into 20% (NH
4)
2SO
4Solution, the limit edged stirs, and after fully mixing, leaves standstill 30min.4 ℃, the centrifugal 20min of 3000r/min discards precipitation, defibrinates to remove.In supernatant, add (NH again
4)
2SO
4Saturated solution 30ml makes into 50% (NH
4)
2SO
4Solution fully mixes, and leaves standstill 30min.4 ℃, the centrifugal 20min of 3000r/min abandons supernatant.In precipitation, add 20ml physiological saline, make it dissolving, add (NH again
4)
2SO
4Saturated solution 10ml makes into 33% (NH4)
2SO
4Solution after fully mixing, leaves standstill 30min.4 ℃, the centrifugal 20min of 3000r/min abandons supernatant, to remove albumin.With 10ml physiological saline solution precipitation, the bag filter of packing into.The desalination of dialysing in 4 ℃ of refrigerators, dialysed overnight in ordinary water, again in physiological saline in 4 ℃ of dialysis 24h, liquid is changed 5 times in the centre.4 ℃, the centrifugal 10min of 3000r/min goes precipitation, and supernatant is and slightly carries the avian influenza virus polyclonal antibody.
Slightly carry the ion-exchange resin purification of avian influenza virus polyclonal antibody:
1) getting volume fraction is 20% alcohol-pickled DEAE Sepharose Fast Flow filler 20mL filling pillar (112cm * 20cm Millipore, available from BIO-RAD company), earlier wash post with the 200mL pure water with 1.5mL/min, the Buffer A (10mmol/L Tris-HCl pH8.0) that uses 100mL again is with 1.5mL/min balance pillar.
2) the avian influenza virus polyclonal antibody sample with first pure back gained utilizes bag filter to be replaced with Buffer A damping fluid, with sample on the 1.0mL/min flow velocity, opens Ultraviolet Detector simultaneously, opening entry.
3) after upward sample finishes, at first use Buffer A wash-out, elution speed is 1.0mL/min, and after collecting eluting peak; Carry out the constant gradient wash-out with containing 0.1mol/L NaCl Buffer A solution subsequently, collect eluting peak 1, be labeled as P1, the P1 sample is the avian influenza virus polyclonal antibody of purifying, preserves in 4 ℃ of refrigerators.
2. method
(1) coupling of magnetic nanoparticle and antibody: concentration is that the magnetic nanoparticle suspension of 0.01M is got 100 μ l and added borate buffer and mend to 800 μ l, adds EDC 2mg and Sulfo-NHS 4mg, room temperature reaction 15 minutes.Add 10 μ l monoethanolamine cancellation EDC then.Add 100 μ l avian influenza virus polyclonal antibodies (6.25mg/ml), room temperature reaction 2 hours.Add 100 μ l 1M glycocoll, the unreacted carboxyl on the sealing magnetic nanoparticle.Add 10 μ l, 1% Sodium azide at last, be put in 2 ℃ of refrigerators and preserve.
(2) separate the magnetic nanoparticle and antibody (separating) of not coupling with Sephadex G100 molecular sieve: get 10gSephadex G100 dry powder, add an amount of 0.1M NaCl, 100 ℃ of heating 3 hours, be cooled to room temperature after, in the chromatographic column of packing into.After adjusting flow velocity, use 200ml NaH
2PO
4Wash-out.Use UV-detector to detect the 280nm absorption value of eluent.Collect each component, top component should be the coupled product of required magnetic nanoparticle and antibody.
(3) coupled product enrichment avian influenza virus
1. antigen-antibody reaction: get each 1000 μ l of avian influenza virus antigen that n (the avian influenza virus antigen number of n=dilution) 1.5ml centrifuge tube adds dilution in proportion respectively, in every pipe, add 100 μ l couplings again the magnetic nanoparticle suspension of antibody is arranged, cover lid, vortex fully mixes or the mixing of vibrating under the room temperature, and wet box was hatched 60 minutes in 37 ℃ of incubators;
2. magnet enrichment: centrifuge tube taken out from 37 ℃ of incubators place on the magnetic separator frame, room temperature left standstill 45 minutes.Sucking-off supernatant carefully.Take out centrifuge tube from the magnetic separator frame, (0.02M, pH7.2), vortex mixed promptly obtains the sample after the enrichment to add 200 μ l PBS.
(4) detection of enriched product: sample after the above-mentioned enrichment and supernatant are extracted RNA with TRIZOL reagent, detect with avian influenza virus fluorescent RT-PCR detection reagent box, concrete grammar sees " H5 subtype avian influenza virus fluorescent RT-PCR method for detecting " GB/T 19438.2-2004 for details.
3. result
As shown in Figure 1, the low more viral level of Ct value is high more, and the high more viral level of Ct value is low more.With the viral level after the magnetic nanoparticle enrichment of coupling antibody (top curve A) obviously than viral level height with (following curve C) before viral level (curve B of centre) after the magnetic nanoparticle enrichment of coupling antibody not and the enrichment.
Embodiment 2: coupling, the purifying of magnetic nanoparticle and avian influenza virus polyclonal antibody, the enrichment of avian influenza virus and detection
1. material
The borate buffer of pH8.0: 0.05M borax 30% (V/V), 0.2M boric acid 70% (V/V), dissolving back pH is 8.
Magnetic nanoparticle (available from Tianjin times Si Le company product), EDC (ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate), monoethanolamine, 1M glycocoll, 0.02M NaH
2PO
4, 0.1M NaCl, 1% Sodium azide (mass percent), DEAE Sepharose Fast Flow (available from Pharmacia company).
Avian influenza virus polyclonal antibody (6.25mg/ml) is with the preparation method of embodiment 1.
Avian influenza virus antigen (available from Harbin veterinary institute country bird flu reference laboratory).
Freund's complete adjuvant and incomplete Freund (available from Beijing's biological products assay institute).
TRIZOL reagent (INVIOTROGEN).
Avian influenza virus fluorescent RT-PCR detection reagent box (available from Shenzhen basic company product).
2. method
(1) coupling of magnetic nanoparticle and antibody: getting concentration is 0.01M magnetic nanoparticle suspension 200 μ l and 800 μ l avian influenza virus polyclonal antibodies (6.25mg/ml), adds EDC 2mg room temperature reaction 2 hours.Add 100 μ l 1M glycocoll, the unreacted carboxyl on the sealing magnetic nanoparticle.Add 10 μ l, 1% Sodium azide at last, be put in 2 ℃ of refrigerators and preserve.
(2) separate the magnetic nanoparticle and the antibody of not coupling: the coupling reaction products therefrom is centrifugal, centrifugal condition is 4 ℃, 3000-12000r/min, centrifugal 10-20min, discard supernatant after centrifugal gently, the gained precipitation is the magnetic nanoparticle of coupling antibody, will precipitate then with phosphate buffer (PBS, 0.02M pH7.2) suspension is standby.
(3) coupled product enrichment avian influenza virus
1. antigen-antibody reaction: get each 1000 μ l of avian influenza virus antigen that n (the avian influenza virus antigen number of n=dilution) 1.5ml centrifuge tube adds dilution in proportion respectively, in every pipe, add 100 μ l couplings again the magnetic nanoparticle suspension of antibody is arranged, cover lid, vortex fully mixes or the mixing of vibrating under the room temperature, and wet box was hatched 60 minutes in 37 ℃ of incubators;
2. magnet enrichment: centrifuge tube taken out from 37 ℃ of incubators place on the magnetic separator frame, room temperature left standstill 45 minutes.Sucking-off supernatant carefully.Take out centrifuge tube from the magnetic separator frame, (0.02M, pH7.2), vortex mixed promptly obtains the sample after the enrichment to add 200 μ l PBS.
4) detection of enriched product: sample after the above-mentioned enrichment and supernatant are extracted RNA with TRIZOL reagent, detect with avian influenza virus fluorescent RT-PCR detection reagent box, concrete grammar sees " H5 subtype avian influenza virus fluorescent RT-PCR method for detecting " GB/T 19438.2-2004 for details.
3. result
As shown in Figure 2, the low more viral level of Ct value is high more, and the high more viral level of Ct value is low more.With the viral level after the magnetic nanoparticle enrichment of coupling antibody (top curve A) obviously than viral level height with (following curve C) before viral level (curve B of centre) after the magnetic nanoparticle enrichment of coupling antibody not and the enrichment.
Claims (9)
1. the method for avian influenza virus in the magnetic nanoparticle enriched sample of antibody coupling comprises the steps:
(1) magnetic nanoparticle, avian influenza virus polyclonal antibody and coupling agent are mixed, room temperature reaction 2 hours obtains containing the solution that coupling has the magnetic nanoparticle of avian influenza virus polyclonal antibody;
(2) purifying coupled product is removed unreacted antibody and magnetic nanoparticle;
(3) behind coupled product and the solution reaction to be measured, use the magnet enrichment;
(4) detection of enriched product.
2. the method for avian influenza virus in the magnetic nanoparticle enriched sample of antibody coupling according to claim 1, it is characterized in that: described step (1) is mixed magnetic nanoparticle, avian influenza virus polyclonal antibody and coupling agent, obtain containing the solution that coupling has the magnetic nanoparticle of avian influenza virus polyclonal antibody, be meant magnetic nanoparticle, coupling agent and avian influenza virus polyclonal antibody are mixed in the ratio of 1-2 μ mol:1-6mg:0.625-5mg.
3. the method for avian influenza virus in the magnetic nanoparticle enriched sample of antibody coupling according to claim 1, it is characterized in that: described magnetic nanoparticle is the magnetic nanoparticle of superparamagnetism.
4. the method for avian influenza virus in the magnetic nanoparticle enriched sample of antibody coupling according to claim 1 is characterized in that: described coupling agent is that ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate or N-hydroxy thiosuccinimide or said two devices mix arbitrarily.
5. the method for avian influenza virus in the magnetic nanoparticle enriched sample of antibody coupling according to claim 4, it is characterized in that: described coupling agent is ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate.
6. the method for avian influenza virus in the magnetic nanoparticle enriched sample of antibody coupling according to claim 1, it is characterized in that: the method for described step (2) purifying coupled product is the sieve method purifying, and molecular sieve is sephadexG100.
7. the method for avian influenza virus in the magnetic nanoparticle enriched sample of antibody coupling according to claim 1, it is characterized in that: the method for described step (2) purifying coupled product is the centrifuge method purifying, centrifugal condition is 4 ℃, 3000-12000r/min, centrifugal 10-20min, discard supernatant after centrifugal, the gained precipitation is the magnetic nanoparticle of coupling antibody.
8. the method for avian influenza virus in the magnetic nanoparticle enriched sample of antibody coupling according to claim 1, it is characterized in that: described avian influenza virus polyclonal antibody is that avian influenza virus antigen is in the mammal hypodermic injection, after carrying out immunity, collect the serum and the purified avian influenza virus polyclonal antibody that obtains of immunized animal.
9. the method for avian influenza virus in the magnetic nanoparticle enriched sample of antibody coupling according to claim 1 is characterized in that: described step (4) detects the method for enriched product for to detect with avian influenza virus fluorescent RT-PCR detection reagent box.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103275940A (en) * | 2013-05-08 | 2013-09-04 | 中国人民解放军军事医学科学院军事兽医研究所 | Method for enriching influenza virus through utilizing asialofetuin-containing magnetic bead |
| CN103983771A (en) * | 2014-05-23 | 2014-08-13 | 广东海洋大学 | Preparation and application of immune magnetic bead indirect competition enzyme-linked immuno sorbent assay (ELISA) kit for detecting hidden state T-2 toxin |
| CN109843790A (en) * | 2016-09-29 | 2019-06-04 | 生物辐射实验室股份有限公司 | Protein-nano particle conjugate purification method |
| CN111944871A (en) * | 2020-08-22 | 2020-11-17 | 南京健邦锦源医疗科技有限公司 | Magnetite virus extracting solution and using method thereof |
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2008
- 2008-12-15 CN CNA2008102396737A patent/CN101435824A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103275940A (en) * | 2013-05-08 | 2013-09-04 | 中国人民解放军军事医学科学院军事兽医研究所 | Method for enriching influenza virus through utilizing asialofetuin-containing magnetic bead |
| CN103983771A (en) * | 2014-05-23 | 2014-08-13 | 广东海洋大学 | Preparation and application of immune magnetic bead indirect competition enzyme-linked immuno sorbent assay (ELISA) kit for detecting hidden state T-2 toxin |
| CN103983771B (en) * | 2014-05-23 | 2016-08-17 | 广东海洋大学 | The preparation of a kind of immunomagnetic beads indirect competitive ELISA kit for detecting hidden state T-2 toxin and application |
| CN109843790A (en) * | 2016-09-29 | 2019-06-04 | 生物辐射实验室股份有限公司 | Protein-nano particle conjugate purification method |
| CN109843790B (en) * | 2016-09-29 | 2023-11-21 | 生物辐射实验室股份有限公司 | Protein-nanoparticle conjugate purification method |
| CN111944871A (en) * | 2020-08-22 | 2020-11-17 | 南京健邦锦源医疗科技有限公司 | Magnetite virus extracting solution and using method thereof |
| CN111944871B (en) * | 2020-08-22 | 2023-06-23 | 南京健邦锦源医疗科技有限公司 | Magnetic bead virus extracting solution and use method thereof |
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Application publication date: 20090520 |