CN105842439A - Oriented immunomagnetic beads of fumonisins and preparation method and application of oriented immunomagnetic beads - Google Patents
Oriented immunomagnetic beads of fumonisins and preparation method and application of oriented immunomagnetic beads Download PDFInfo
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Abstract
The invention relates to oriented immunomagnetic beads of fumonisins and a preparation method and application of the oriented immunomagnetic beads. The oriented immunomagnetic beads are prepared by coupling protein G or protein A to magnetic beads, coupling a fumonisins antibody to the protein G or protein A located on magnetic beads; and utilizing a cross-linking agent to cross-link the protein G or protein A magnetic beads combined with the fumonisins antibody. According to the preparation method, the strong affinity between protein G/protein A and the antibody and the specific binding between protein G/protein A and an Fc segment of the antibody are fully utilized. The immunomagnetic beads are mainly used for binding and purifying fumonisins in grain, food, feed, milk, blood samples, and other multiple samples. The immunomagnetic beads can be used for pre-treatment of a sample before detection of fumonisins, and detection and purification of fumonisins in a sample.
Description
Technical field
Orientation immunomagnetic beads that the present invention relates to a kind of fumonisin and its production and use, belongs to fumonisin purification and detection field.
Background technology
Fumonisin (Fumon isins) be one group mainly by fusarium moniliforme (Fusarium
Moniliforme) the produced mycotoxin of breeding under uniform temperature and damp condition.Fumonisin is widely distributed in nature, and hazardness is big, gradually causes the great attention of Chinese scholars.At present, it has been found that fumonisin analog be broadly divided into A, B, C, P tetra-class.Research confirms that fumonisin can cause horse cerebral white matter malacosis (EL-EM), nerve poisoning and show the symptoms such as disturbance of consciousness, blind and movement disorder, severe patient even causes death.Pig is caused pulmonary edema syndrome (PPE), and liver and esophageal injury can be caused.Fumonisin also can cause the atherosclerosis sample of primate to change, and induces rat liver cancer.Generation with mankind's esophageal carcinoma is the most closely related.International cancer research institution and California Environmental protection mechanism have announced that fumonisin is the potential carcinogen (2B level carcinogen) of the mankind.Mainly Semen Maydis and the goods thereof that fumonisin pollutes, in addition with some such as food such as rice, Sorghum vulgare Pers., Semen setariae, milk, medicated beer.The report polluted about fumonisin the most all over the world is more, and wherein the pollution of Semen Maydis and goods thereof account for major part.The fumonisin of high-load all has been reported that in the Semen Maydis and corn product of a lot of countries and regions, such as Korea S, China, Brazil, Nigeria, South Africa, Britain, French and Italian.Also there is the investigation of fumonisin pollution condition in the provinces such as Sichuan Province China, Zhejiang, Henan, and positive rate is the highest.
Pollution range based on fumonisin is relatively wide, and toxic action also relatively pollution range based on fumonisin is relatively wide, and toxic action is the biggest, and many countries and regions have carried out systematic study to fumonisin.But limitation and detection method for food with fumonisins in fodder there is no unified standard the most in the world.Sweden specifies that in human foods, the limitation of fumonisin is 1mg/kg, FDA Food and Drug Administration (FDA) has issued the highest limitation directiveness bulletin of the Semen Maydis for human consumption and corn product fumonisin, it is stipulated that in human consumption Semen Maydis, the highest limitation of fumonisin is 2mg/kg.Meanwhile, the highest limitation directiveness bulletin of fumonisin in animal feed has also been issued in the herding medical center (CVM) of FDA, it is stipulated that its limitation scope is 1 ~ 50mg/kg.About regulation in the meeting (February calendar year 2001) of food additive in FAO/WHO community, the threshold limit values of human body is reruned FB1, FB2, FB3 amount less than 2ug/kg for the amount of daily intaking by body by fumonisin.This relevant laws and regulations are the most formulated by China.In inspection and quarantining for import/export recommended industry standard, liquid chromatography is 0.05mg/kg to the mensuration lower bound of fumonisin B1, B2.
The detection method of fumonisin has thin layer chromatography, high performance liquid chromatography (HPLC), enzyme linked immunosorbent assay (ELISA), enzyme-linked immunosorbent assay etc. at present.
Wherein thin layer chromatography is that to use the earliest be also the method for most widely used detection fumonisin, and its advantage is suitable for not through the human users of special training, and low cost, it is not necessary to expensive instrument,.But thin layer chromatography is loaded down with trivial details to sample treatment, experimentation is complicated, and the required detection cycle is longer, is easily subject to the interference of impurity.With range estimation sxemiquantitative during mensuration, subjective impact is relatively big, and sensitivity is the most high, can not meet the most far away modern measure requirement.
The detection of elisa (ELISA) detection method is special, quick, highly sensitive and cost is relatively low.The feature of low cost, it is adaptable to the screening of a large amount of sample of infrastructure and generaI investigation, can be greatly saved time and expense, the most increasingly be welcome by Inspection Unit of basic unit.The subject matter of ELISA method is to easily cause false positive.Therefore the examination detection of basic unit it is mainly used in.
High performance liquid chromatography (HPLC) has that accuracy is high, susceptiveness strong, can the advantage such as microdetermination, be the method being usually used in the detection of alimentary toxicosis element at present.But because it is higher to the requirement of toxin purity in sample, cause testing cost height, cycle long, it is impossible to meet the needs of batch samples rapid screening, being restricted so using.Immune affinity column-the high performance liquid chromatography (IAC-HPLC) grown up this year) immunoaffinity purification technology is combined with high performance liquid chromatography.The use making HPLC is more extensive.Immune affinity column, as a kind of new purification techniques, is applied more and more extensive in the analysis of mycotoxin detects.It is to be attached to specific antibody on the solid phase carrier of activation fill form.When sample extracting solution is by pillar, antigen therein and antibodies, other impurity are then washed off by aqueous solution, then are got off by antigen i.e. toxin eluent with organic solvent, so that the toxin in sample is purified.
Due to the difference of Cleaning Principle, high performance liquid chromatography requires height to the process of sample.After the steps such as the Sample pretreatment method mainly common practice for commonly using at present is all first to pass through to pulverize by sample to be checked, filtration, utilize immune affinity column that sample carries out the affine purification of immunity of fumonisin.Then eluted product is carried out high performance liquid chromatography detection.Owing to sample to be checked is more complicated, although early stage have passed through the steps such as filtration, but the phenomenon that affinity column blocks can occur with immune affinity column when.Affinity column blocking can reduce toxin adhesion, slows down the percent of pass of reaction solution, and then affects the purification efficiency of affinity column.Additionally, due to the feature of immune affinity column self, can cause and be similar to column plate effect, the gel being positioned at affinity column top is combined more with fumonisin, and gets over bottom affinity column, and toxin combines the fewest.The gel bottom affinity column is caused to tend not to the fumonisin being effectively combined in sample.
Fumonisin in sample is purified by the orientation immunomagnetic beads if, with fumonisin, requirement to sample will be relaxed, have only to the sample after pulverizing mix with magnetic bead in certain solution, fumonisin in sample just can be adsorbed by the antibody above magnetic bead, after magnetic bead utilizes magnetic principles adherent, remaining part is removed.It is incorporated into the fumonisin above orientation immunomagnetic beads the most again to elute.The most simple to operate, but also the fumonisin in sample can be enriched with.The sample of low content can be detected.Additionally, due to carry out fumonisin purification under liquid-phase condition completely, each magnetic bead is identical for the binding ability of toxin.
The most common orientation immunomagnetic beads is all that antibody is directly coupled to magnetic bead surfaces, but owing to antibody molecule is relatively big, coupling is uncontrolled, and antibody may be coupled on magnetic bead in any direction.Some antibody coupling direction can cause when antigen molecule combines owing to sterically hindered reason cannot be in conjunction with, and more in some cases, the antigen binding regions of antibody is coupled on magnetic bead cause this antigen binding regions to be closed.Owing to fumonisin molecule is the least, the epitope carried only has 1-2, and the closing of an antigen binding regions this may result in the antibody of this part and loses antigen binding capacity.Thus influence whether the antigen joint efficiency of whole orientation immunomagnetic beads.Utilize protein A or Protein G can the feature of specific binding antibody constant region (Fc district), by protein A or Protein G, antibody is fixed on magnetic bead surfaces.Outside the antigen binding regions (Fab district) making antibody is all exposed to, considerably increase the antigen binding capacity of orientation immunomagnetic beads, we term it orientation immunomagnetic beads.
Summary of the invention
It is an object of the invention to provide a kind of fumonisin orientation immunomagnetic beads and its production and use, in order to achieve the above object, in the present invention, make use of the function of Protein G or protein A, Protein G is the albumen on a kind of G type streptococcal cell wall, can combine at the specific Fc position with many animals antibody, and there is the highest affinity.Protein A is a kind of albumen on aureus cell wall, similar with Protein G., there is antibody binding capacity, by Fc region and the antibodies of antibody, microbe-derived Protein G and this characteristic of protein A with it for many years evolve obtain host escape and self-protection ability closely related.We have cloned the gene order of wild Protein G and protein A, and after its codon is optimized, is recombinated, and having gone out at expression in escherichia coli has gene recombinant protein G and the protein A of high-affinity with antibody.After the Protein G of gene recombinaton or protein A are coupled on magnetic bead, then by fumonisin antibody coupling to Protein G or protein A, prepare fumonisin Protein G/protein A magnetic bead, then with cross-linking agent, carrier has been cross-linked.It is the formation of the fumonisin orientation immunomagnetic beads of high-affinity.This magnetic bead is easy and simple to handle, and purification fumonisin efficiency is high.Sample can be carried out purification after simple process, obtains the fumonisin that purity is the highest.Detect for high performance liquid chromatography.
Present invention utilizes the feature that Protein G is combined with Ig antibody specificity with protein A, IgG antibody is made up of two heavy chains and two light chains, and antibody is divided into Fab district and Fc district, and wherein, Fab district is the region that antigen combines.Protein G is a kind of special albumen on streptococcal cell wall, has specific binding ability with the Fc region of antibody.After Protein G and antibodies, the Fab region of antibody is free outside, does not affect the antigen binding capacity of antibody.Protein G after our gene optimization restructuring, the Protein G of 1 molecule can have the highest affinity of antibody in conjunction with the IgG antibody of 3 molecules.Similar with Protein G, protein A is a kind of special albumen on aureus cell wall, has specific binding ability with the Fc region of antibody.After protein A and antibodies, the Fab region of antibody is free outside, does not affect the antigen binding capacity of antibody.Protein A after our gene optimization restructuring, the Protein G of 1 molecule can have the highest affinity of antibody in conjunction with the IgG antibody of 6 molecules.
It is good that the orientation immunomagnetic beads prepared based on this Protein G or protein A has specificity, and fumonisin binding capacity is big, the feature that purification efficiency is high.
Fumonisin orientation immunomagnetic beads and preparation method thereof is described as follows:
1. by the magnetic bead 50mM containing 0.01% SDS
PH6.0 MES buffer hangs.
2. in magnetic bead, add 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide (EDC) to final concentration 5-20mg/ml, add sulfuration-N-hydroxy-succinamide (NHS) (Sulfo-NHS) to final concentration 10-25mg/ml.
3. room temperature reaction 15-60 minute.
The most reacted magnetic bead 50mM MES buffer solution ph 6.0 washs 3 times, is then resuspended in 50mM
In the MES buffer of PH6.0.
5. Protein G or protein A are added resuspended after magnetic bead in, make protein content reach 5-10:1 with the mol ratio of magnetic bead surfaces aglucon.
6. room temperature reaction 2-4 hour.
The most reacted magnetic bead 50mM MES buffer solution ph 6.0 washs 3 times.
The ethanolamine room temperature of the most reacted magnetic bead 1M is closed.
9. magnetic bead 20mM PBS PH7.4 washs 3 times, is then resuspended in 20mM PBS PH7.4 buffer.
10. in magnetic bead, add anti-fumonisin antibody, make amount of antibody reach 3-5:1. with Protein G amount mol ratio in magnetic bead
The magnetic bead adding antibody is reacted 10-40 minute by 11. at 37 DEG C.
12. magnetic beads are with 0.1 M borate buffer
PH9.0 washs 3 times, and then magnetic bead is resuspended in 1M
In the borate buffer of PH9.0.
Fumonisin antibody protein G/ protein A magnetic bead carrier is added the triethanolamine of final concentration 0.2M and the dimethyl pimelate (DMP) of 20mM, PH8.3. room temperature reaction 1-2 hour by 13..
14. terminate reaction with the ethanolamine of 50mM PH9.0, wash fumonisin antibody protein G/ protein A magnetic bead 3 times with the 10mMPBS PH7.4 containing 0.01% thimerosal.
Magnetic bead is resuspended in the 10mMPBS PH7.4 containing 0.01% thimerosal by 15..It is put in 4 DEG C of preservations.
Compared with prior art, present invention have the advantage that
The most sufficiently make use of the characteristic that Protein G is specific binding with protein A and IgG antibody, make antibody be coupled on carrier by Protein G/protein A.And outside the Fab fragment of IgG antibody is sufficiently exposed to, the capturing ability of fumonisin being greatly improved, the purification efficiency of fumonisin also effectively improves.
2. present invention uses the Protein G after genetic modification and protein A, by the optimization of codon and the optimization of IgG binding domain gene, making the antibody binding capacity of Protein G and protein A increase substantially, and then improve the purification efficiency of fumonisin.
3. using purification fumonisin of the present invention easy and simple to handle, several steps can be obtained by the fumonisin that purity is higher, and more convenient operator uses.
4. use the fumonisin purity that obtains of the present invention the highest, follow-up need not do other purification process again and be just used directly for high performance liquid chromatography detection or fluoroscopic examination, save time and the expense of operator.
Accompanying drawing explanation
Fig. 1, the fumonisin orientation immunomagnetic beads structural representation of Protein G coupling.
Fig. 2, Semen Maydis sample horse second of the three ten-day periods of the hot season content of toxins detection HPLC collection of illustrative plates.
Fig. 3, feedstuff sample horse second of the three ten-day periods of the hot season content of toxins detection HPLC collection of illustrative plates.
A in figure: magnetic bead, B: Protein G, C: fumonisin antibody.
Specific embodiment
Embodiment 1: utilize gene recombinant protein G to prepare fumonisin orientation immunomagnetic beads
The preferred embodiment that the present invention prepares fumonisin orientation immunomagnetic beads is as follows:
1. magnetic bead activation
Take the nanometer magnetic bead of 5mg carboxyl modified, with containing 0.01%
The 50mM PH6.0 MES buffer solution of SDS 3 times, then hangs the 50mM PH6.0 MES buffer of magnetic bead 5ml.
2. in magnetic bead, add 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide (EDC) 100mg, add sulfuration-N-hydroxy-succinamide (NHS) (Sulfo-NHS) 100mg, room temperature reaction 15 minutes.
The most reacted magnetic bead 50mM MES buffer solution ph 6.0 washs 3 times, is then resuspended in the MES buffer of 5ml 50mM PH6.0.
4. in magnetic bead, add 20mg Protein G, room temperature reaction 4 hours.
The most reacted magnetic bead 50mM MES buffer solution ph 6.0 washs 3 times.
The ethanolamine room temperature of the most reacted magnetic bead 20ml 1M is closed 2 hours.
7. magnetic bead 50ml 20mM PBS PH7.4
Wash 3 times, be then resuspended in 5ml
In 20mM PBS PH7.4 buffer.
8. in magnetic bead, add 200mg anti-fumonisin antibody, the magnetic bead adding antibody reacted 30 minutes at 37 DEG C.
9. magnetic bead 20mM PBS PH7.4
Wash 3 times, be then resuspended in 20mM
In PBS PH7.4 buffer.
10. magnetic bead washs 3 times with 0.1 M borate buffer PH9.0, during then magnetic bead is resuspended in the borate buffer of 5ml 0.1M PH9.0.
11. weigh in the PBS that 26mg dimethyl pimelate (DMP) adds containing fumonisin antibody protein G magnetic bead, and the triethanolamine being subsequently adding 100ul 2M is adjusted to PH8.3, room temperature reaction 1 hour.
12. terminate reaction with the ethanolamine of 25ml 50mM PH9.0.
13. wash fumonisin antibody protein G magnetic bead 3 times with the 10mMPBS PH7.4 containing 0.01% thimerosal, are then resuspended in this buffer of 5ml by magnetic bead, are put in 4 DEG C of preservations.
Embodiment 2: utilize gene recombinant protein A to prepare fumonisin orientation immunomagnetic beads
The preferred embodiment that the present invention prepares fumonisin orientation immunomagnetic beads is as follows:
1. magnetic bead activation.
2. take the nanometer magnetic bead of 5mg carboxyl modified, with the 50mM PH6.0 MES buffer solution containing 0.01% SDS 3 times, then the 50mM PH6.0 MES buffer of magnetic bead 5ml is hanged.
3. in magnetic bead, add 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide (EDC) 100mg, add sulfuration-N-hydroxy-succinamide (NHS) (Sulfo-NHS) 100mg, room temperature reaction 20 minutes.
The most reacted magnetic bead 50mM MES buffer solution ph 6.0 washs 3 times, is then resuspended in 5ml
In the MES buffer of 50mM PH6.0.
5. in magnetic bead, add 30mg protein A, room temperature reaction 4 hours.
The most reacted magnetic bead 50mM MES buffer solution ph 6.0 washs 3 times.
The ethanolamine room temperature of the most reacted magnetic bead 20ml 1M is closed 2 hours.
8. magnetic bead 50ml 20mM PBS PH7.4
Wash 3 times, be then resuspended in 5ml
In 20mM PBS PH7.4 buffer.
9. in magnetic bead, add 300mg anti-fumonisin antibody, the magnetic bead adding antibody is reacted 30 minutes at 37 DEG C.
10. magnetic bead 20mM PBS PH7.4 washs 3 times, is then resuspended in 20mM PBS PH7.4 buffer.
11. magnetic beads wash 3 times with 0.1 M borate buffer PH9.0, during then magnetic bead is resuspended in the borate buffer of 5ml 0.1 M boron PH9.0.
12. weigh in the PBS that 26mg dimethyl pimelate (DMP) adds containing fumonisin antibody protein A magnetic bead, and the triethanolamine being subsequently adding 100ul 2M is adjusted to PH8.3, room temperature reaction 1 hour.
13. terminate reaction with the ethanolamine of 25ml 50mM PH9.0.
14. wash fumonisin antibody protein A magnetic bead 3 times with the 10mMPBS PH7.4 containing 0.01% thimerosal, are then resuspended in this buffer of 5ml by magnetic bead, are put in 4 DEG C of preservations.
Embodiment 3: the fumonisin orientation immunomagnetic beads purification utilizing Protein G coupling and the fumonisin detected in Semen Maydis sample
1., by broken for corn sample steel pulverizing, cross 2mm sub-sieve.
2. take 10g sieve pulverize sample add 50mL 20mM PH7.4
PBS solution mixes.
3. adding the flavacin orientation immunomagnetic beads 10ml of 1mg/ml Protein G coupling, after mixing, room temperature is placed 30 minutes.
4. reacted magnetic bead mixed liquor is put into magnetic frame upper 2 minute, treats that magnetic bead is adherent, remove remaining buffer with suction pipe.
5. in magnetic bead, add 50ml 20mM PH7.4 PBS wash magnetic bead, then magnetic bead is put in magnetic frame after magnetic bead is adherent, remove buffer with suction pipe.
6. repeated washing step 3 time.
7. in magnetic bead, add the glycine buffer of 5ml 0.1M PH3.0, mixing.The fumonisin on magnetic bead is made to elute.
8. being put into by magnetic bead makes magnetic bead adherent in magnetic frame, be fetched in another test tube by the buffer of eluting, adds 500ul PH9.0 1M at once
Tris buffer, is sufficiently mixed.
9. the buffer high performance liquid chromatography (HPLC) after eluting detects its content.
10. according to HPLC detection collection of illustrative plates, high performance liquid chromatography testing result is as in figure 2 it is shown, can show that this Semen Maydis sample horse second of the three ten-day periods of the hot season content of toxins is 3.31ppm.
Embodiment 4: the fumonisin orientation immunomagnetic beads purification utilizing protein A coupling and the fumonisin detected in feedstuff sample
1., by broken for Feed Sample steel pulverizing, cross 2mm sub-sieve.
2. take 10g sieve pulverize sample add 50mL 100mM PH8.0
Tris buffer soln mixes.
3. adding the flavacin orientation immunomagnetic beads 10ml of 1mg/ml protein A coupling, after mixing, room temperature is placed 30 minutes.
4. reacted magnetic bead mixed liquor is put into magnetic frame upper 2 minute, treats that magnetic bead is adherent, remove remaining buffer with suction pipe.
5. in magnetic bead, add 50ml 100mM PH8.0 Tris buffer solution magnetic bead, then magnetic bead is put in magnetic frame after magnetic bead is adherent, remove buffer with suction pipe.
6. repeated washing step 3 time.
7. in magnetic bead, add the citrate buffer solution of 5ml 0.1M PH5.0, mixing, make the fumonisin on magnetic bead elute.
8. being put into by magnetic bead makes magnetic bead adherent in magnetic frame, be fetched in another test tube by the buffer of eluting, and the buffer high performance liquid chromatography (HPLC) after eluting detects its content.
9. according to HPLC detection collection of illustrative plates, high performance liquid chromatography testing result is as it is shown on figure 3, can show that this feedstuff sample horse second of the three ten-day periods of the hot season content of toxins is 4.87ppm.
Protein G gene order
1 efnkygvsdy
yknlinnakt vegvkdlqaq vvesakkari seatdglsdf lksqtpaedt
61 vksielaeak vlanreldky gvsdyhknli nnaktvegvk dlqaqvvesa kkariseatd
121 glsdflksqt paedtvksie laeakvlanr eldkygvsdy yknlinnakt vegvkalide
181 ilaalpktdt yklilngktl kgettteavd aataekvfkq yandngvdge wtyddatktf
241 tvtekpevid aseltpavtt yklvingktl kgettteavd aataekvfkq yandngvdge
301 wtyddatktf tvtekpevid aseltpavtt yklvingktl kgetttkavd aetaekafkq
361 yandngvdgv wtyddatktf tvtemvtevp gdaptepekp easiplvplt patpiakdda
421 kkddtkkeda kkpeakkeda kkaetlpttg egsnpfftaa alavmagaga lavaskrked
GenBank:
CAA27638.1
Protein A gene order
1 mkkkkiysir
klgvgiasvt lgtllisggv tpaanaaqhd eaqqnafyqv lnmpnlnadq
61 rngfiqslkd dpsqsanvlg eaqklndsqa pkadaqqnkf nkdqqsafye ilnmpnlnee
121 qrngfiqslk ddpsqstnvl geakklnesq apkadnnfnk eqqnafyeil nmpnlneeqr
181 ngfiqslkdd psqsanllae akklndaqap kadnkfnkeq qnafyeilhl pnlteeqrng
241 fiqslkddps vskeilaeak klndaqapke ednnkpgked gnkpgkedgn kpgkednkkp
301 gkedgnkpgk ednkkpgked gnkpgkedgn kpgkedgnkp gkedgnkpgk edgngvhvvk
361 pgdtvndiak angttadkia adnkladknm ikpgqelvvd kkqpanhada nkaqalpetg
421 eenpfigttv fgglslalga allagrpspn yknkqyttid iilskpilty ir
GenBank:
AAB05743.1
Claims (10)
1. the orientation immunomagnetic beads of a fumonisin, it is characterised in that include magnetic bead carrier, Protein G or protein A and fumonisin antibody.
2. according to the orientation immunomagnetic beads described in claim 1, it is characterized in that Protein G or protein A are coupled on carrier by covalent bond, then fumonisin antibody is combined with Protein G, then with cross-linking agents, forms the coupling carrier of magnetic bead-Protein G/protein A-fumonisin antibody formation.
3. orient immunomagnetic beads according to the fumonisin described in claim 1, it is characterised in that Protein G used is the Protein G of natural Protein G or DNA recombinant expression.
4. orient immunomagnetic beads according to the fumonisin described in claim 1, it is characterised in that protein A used is the protein A of natural protein A or DNA recombinant expression.
5. according to the Protein G described in claim 3, including the Protein G expressed according to sequence 1, and the Protein G expressed after sequence optimisation.
6. according to the protein A described in claim 4, including the protein A expressed according to sequence 2, and the protein A expressed after sequence optimisation.
7. according to the carrier described in claim 1, including nanometer magnetic bead and diameter more than the common magnetic bead of 1 μm.
8. according to the anti-fumonisin antibody described in claim 2, it is characterised in that include monoclonal IgG antibody and polyclonal antibody.
9. the orientation immunomagnetic beads preparation method of a purification fumonisin, it is characterised in that:
1) magnetic bead 50mM PH6.0 MES buffer containing 0.01% SDS is hanged；
2) in magnetic bead, addition EDC, to final concentration 5-20mg/ml, adds Sulfo-NHS to final concentration 10-25mg/ml；
3) room temperature reaction 15-60 minute；
4) reacted magnetic bead 50mM MES buffer solution ph 6.0 washs 3 times, is then resuspended in the MES buffer of 50mM PH6.0；
5) Protein G or protein A are added resuspended after magnetic bead in, make protein content reach 5-10:1 with the mol ratio of magnetic bead surfaces aglucon；
6) room temperature reaction 2-4 hour；
7) reacted magnetic bead 50mM MES buffer solution ph 6.0 washs 3 times；
8) the ethanolamine room temperature of reacted magnetic bead 1M is closed；
9) magnetic bead 20mM PBS PH7.4 washs 3 times, is then resuspended in 20mM PBS PH7.4 buffer；
10) in magnetic bead, add anti-fumonisin antibody, make amount of antibody reach 3-5:1 with Protein G amount mol ratio in magnetic bead；
11) magnetic bead adding antibody is reacted 10-40 minute at 37 DEG C；
12) magnetic bead washs 3 times with 0.1 M borate buffer PH9.0, during then magnetic bead is resuspended in the borate buffer of 1M PH9.0；
13) fumonisin antibody protein G/ protein A magnetic bead carrier is added triethanolamine and the DMP of 20mM, PH8.3. room temperature reaction 1-2 hour of final concentration 0.2M；
14) reaction is terminated, with the 10mMPBS containing 0.01% thimerosal with the ethanolamine of 50mM PH9.0
PH7.4 washing fumonisin antibody protein G/ protein A magnetic bead 3 times；
15) magnetic bead is resuspended in the 10mMPBS containing 0.01% thimerosal
In PH7.4, it is put in 4 DEG C of preservations.
10. the orientation immunomagnetic beads of a fumonisin, its purposes is to include the isolated and purified of fumonisin in grain, feedstuff, milk and milk product, Aquatic product, blood, urine, water, and fumonisin after purification may be used for high performance liquid chromatography and the detection of other method.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110261603A (en) * | 2019-07-05 | 2019-09-20 | 中国科学院长春应用化学研究所 | The application of magnetic bead of modification and preparation method thereof and quantitative detection related antigen is passed through on a kind of surface |
| CN117664953A (en) * | 2024-01-31 | 2024-03-08 | 云南伦扬科技有限公司 | Method for rapidly detecting fumonisin B1 and mercury by using Au-Ag Janus@Au NPs with SERS and nano enzyme activities |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110261603A (en) * | 2019-07-05 | 2019-09-20 | 中国科学院长春应用化学研究所 | The application of magnetic bead of modification and preparation method thereof and quantitative detection related antigen is passed through on a kind of surface |
| CN117664953A (en) * | 2024-01-31 | 2024-03-08 | 云南伦扬科技有限公司 | Method for rapidly detecting fumonisin B1 and mercury by using Au-Ag Janus@Au NPs with SERS and nano enzyme activities |
| CN117664953B (en) * | 2024-01-31 | 2024-05-17 | 云南伦扬科技有限公司 | Method for rapidly detecting fumonisin B1 and mercury by using Au-Ag Janus@Au NPs with SERS and nano enzyme activities |
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