CN104174185A - Malachite green immunoaffinity column, and making method and use thereof - Google Patents
Malachite green immunoaffinity column, and making method and use thereof Download PDFInfo
- Publication number
- CN104174185A CN104174185A CN201310188359.1A CN201310188359A CN104174185A CN 104174185 A CN104174185 A CN 104174185A CN 201310188359 A CN201310188359 A CN 201310188359A CN 104174185 A CN104174185 A CN 104174185A
- Authority
- CN
- China
- Prior art keywords
- protein
- malachite green
- antibody
- affinity column
- immune affinity
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention relates to a malachite green immunoaffinity column, and a making method and a use thereof. The immunoaffinity column is coupled to an agarose gel carrier by using a protein G, and an anti-malachite green antibody is used to couple with the protein G on agarose. A cross-linking agent is used to cross-link the above obtained anti-malachite green antibody combed protein G-agarose gel carrier. The obtained cross-linked carrier is used to make the immunoaffinity column. The above purification column is mainly used for purifying malachite green in foods, aquatic products and other various samples, and is in favor of realizing later stage high performance liquid chromatography (HPLC) and fluorescence detection of malachite green in the samples.
Description
Technical field:
The present invention relates to immune affinity column of a kind of malachite green and its production and use.Belong to food safety detection field.
Background technology:
Malachite green (Malachite green, MG) belong to Synthesis of diaminodiphenyl, be a kind of dye class bactericide, the parasite, fungi or the bacterium that can be used for treating fish or fish-egg infect, and once in the aquaculture disease prevention and cure of a plurality of countries and regions, are used widely.Yet MG enters the cheek and the skin epithelial cell mild inflammation that can cause fish after fish body.Its main metabolites in aquatic organism is procrypsis malachite green (Leucomalachite green, LMG).In recent years, the side effects such as the high poison of MG and LMG, high residue and carcinogenic, teratogenesis, mutagenesis expose gradually, and the mankind are edible for a long time the residual aquatic products of MG, easily causes sub-chronic or slow poisoning.Thereby since the nineties in 20th century, many countries are all classified as the forbidden drugs of aquaculture.According to the bill 2002/675/EC of European Union regulation, in animal derived food, the residual total amount of MG and LMG is restricted to 2 μ g/kg; The positive list of Japan is also clearly defined in that in import aquatic products, must not to detect MG residual.China classifies MG as forbidden drugs at agricultural industry mark.But because of its good anti-bacterial effect, low price, still have indivedual raisers at Misuse.In China's aquatic products and environment, the residual phenomenon that exceeds standard of MG and LMG often has generation, as the countries and regions such as European Union, Japan, Hong Kong all once detected from the eel of China mainland import, it was residual, this has not only brought certain ecological environment problem, and has become the key factor that affects China's fish quality and competitiveness in the international market.Therefore, particularly important to MG and the residual detecting & monitoring of LMG.
The detection method of malachite green is conventional chromatography, immunological method, Raman spectroscopy etc.
Immunological method comprises Enzyme Linked Immunoadsorbent Assay ELISA and collaurum detection method.Mainly to utilize AI in the antibody of malachite green and sample.By chromogenic reaction, detect the malachite green in sample.The method is easy to use, and detection sensitivity is high.But complicated owing to detecting sample, matrix is more, and the false positive probability of detection is very high.And immunological method is difficult to distinguish malachite green and concealed malachite green.
Raman scattering is the inelastic scattering phenomenon that a kind of molecule is formed by photon excitation, and it can be used for characterizing the molecular structure property of chemical substance, also can be used for identifying the functional group existing in molecule, realizes the fingerprint of chemical molecular and debates knowledge.Utilize the method detect malachite green have without sample preparation, can be without wound fast detecting.But Raman spectrum detects, need special equipment, only in minority scientific research report, occur the method at present.Large-scale practical application not yet.
Chromatography method is to utilize chromatogram that the malachite green in sample or concealed malachite green are carried out to separation and reallocation.Then detect.Can carry out quantitative analysis, be most widely used also the most approved detection method so far.China has formulated the examination criteria (GB/T19857-2005, GB/T20361-2006) of two cover MG residual quantities in succession in 2005 and 2006, regulation adopts MG in Liquid Chromatography-Tandem Mass Spectrometry and aquatic products by HPLC.
Chromatography for malachite green detects, and sample collection and pre-treatment are the bottlenecks of present analysis method, often the main source of evaluated error.Therefore, the pretreatment technology of sample is one of the difficult point of current MG and LMG analysis and research and focus.In general, sample pre-treatments is divided extraction and is purified 2 steps, experiment adopts solid-phase extraction column to purify [Mitrowska K to the MG in sample extracting solution and LMG mostly, Deng J Chromatogr A, 2005,1089 (1/2): 187] in recent years, application molecularly imprinted polymer SPE (MISE) method separated and purifying MG is studied widely.As 2010, Mart í nez etc. [Mart í nez B M J AnalChim Acta, 2010,665 (1): 47-54.] reported the method that adopts MISE separation and purifying MG, and average recovery rate can reach 98%; In the present invention, we prepare malachite green immune affinity column and utilize malachite green specific antibody to treat the malachite green of sample in this to carry out separation and purification.Simple to operate, purification efficiency is higher.
Summary of the invention:
The object of the present invention is to provide a kind of malachite green immune affinity column and its production and use
In order to achieve the above object, in the present invention, utilized the function of Protein G, Protein G is the albumen on a kind of G type streptococcus cell membrane, can specificly combine with the Fc position of many animals antibody.And there is very high affinity.We have cloned the gene order of Protein G, and after its codon is optimized, is recombinated, have gone out the gene recombinant protein G that has high-affinity with antibody at expression in escherichia coli.After the Protein G of genetic recombination is coupled on carrier, then by malachite green antibody coupling on Protein G, prepared malachite green-Protein G-carrier, then with crosslinking agent, carrier be cross-linked.After carrier dress post after crosslinked, just formed the malachite green immune affinity column of high-affinity.This affinity column is easy and simple to handle, and purifying malachite green efficiency is high.Sample just can carry out purifying after simple processing, obtains the malachite green that purity is very high.For high performance liquid chromatography, detect and luminoscope detection.
Concrete operations are as follows:
The advantage of maximum of the present invention is exactly to have utilized the feature of Protein G and Ig antibody specific binding, and IgG antibody consists of two heavy chains and two light chains, and antibody is divided into HeFc district, Fab district, and wherein, Fab district is the region of antigen combination.
Protein G is a kind of special albumen on streptococcus cell membrane, has specific binding ability with the Fc region of antibody.After Protein G is combined with antibody, the Fab regional tour of antibody, from outside, does not affect the antigen binding capacity of antibody.Protein G after our gene optimization restructuring, the Protein G of 1 molecule can, in conjunction with the IgG antibody of 3 molecules, have very high affinity of antibody.And only have antibody capable to be combined with Protein G, all the other albumen all can not be combined with Protein G, and specificity is very high.Take this Protein G, to be that the immune affinity column of basis preparation has specificity good, and malachite green binding capacity is large, the feature that purification efficiency is high.
Malachite green immune affinity column and preparation method thereof is described as follows:
1. carrier activation
Select agarose carrier, sepharose4B, activates with Epichlorohydrin activation method.
Get 2% preswollen Ago-Gel sepharose4B, with the distilled water of 20 times of volumes, fully rinse, wash away remaining ethanol, with funnel, filter out moisture.
Take 5 grams of wet gels after elimination moisture content, add the NaOH of 7.5 milliliters of 0.8M, 2 milliliters of 30% epoxychloropropane, the sodium borohydride NaBH4 of 2mg/ml, 5 milliliters, at 25 ℃, shaking table reaction is 8 hours.
After reaction, with a large amount of distilled water washings, remove the epoxychloropropane mixing in gel.
2. by the coupling of Protein G and activated carrier sepharose4B
By the Ago-Gel having activated coupling buffer (NaHCO3 of 0.1M, 0.8MNaCl, PH8.9) washing 3 times for sepharose4B.The Protein G that adds 2mg/ml, room temperature coupling 4 hours.
By the good agarose carrier 20mM of coupling, the phosphate buffer PBS of PH7.4 washing 3 times.Coupling has the agarose carrier sepharose called after Protein G-sepharose of Protein G.
3. anti-malachite green antibody is connected with the Protein G on Protein G-sepharose carrier
Protein G and antibody have specific affinity, under certain reaction condition, coupling are had the Ago-Gel of Protein G be connected with antibody.Antibody is connected on agarose.Because the binding site of Protein G and antibody is the Fc region of antibody, the antigen binding domain of antibody is unaffected, has fully guaranteed the antigen binding capacity of antibody.
Anti-malachite green antibody is dissolved in the PBS of 20mM, PH7.4 to final concentration 2mg/ml.Antibody is added in the washed Protein G-sepharose of PBS buffer solution with 20Mm PH7.4.Room temperature was in conjunction with 30 minutes.
In conjunction with after carrier with PBS, wash.Be connected with the carrier called after antibody-Protein G-sepharose of antibody.
4. carrier is crosslinked
Antibody-Protein G-sepharose is washed 3 times with the borate buffer of 0.1M PH9.0.Then the borate buffer that adds 0.1M PH9.0, in buffer solution, add crosslinking agent as heptan two imidic acid dimethyl ester (DMP) to final concentration 20mM.Normal temperature crosslinked 1 hour.
Add 50mM, the monoethanolamine cessation reaction of PH9.0.And seal 10 minutes with the monoethanolamine of 50mM.
Antibody-Protein G-sepharose carrier 20mM after crosslinked, the PBS washing of PH7.4 3 times.
5. fill post
Antibody-agarose carrier after crosslinked is packed in chromatographic column as required, can prepare as required the malachite green affinity purification post of different capabilities.The structure of chromatographic column is as shown in Figure 1:
Compared with prior art, tool of the present invention has the following advantages:
1) utilized fully the characteristic of Protein G and IgG antibody specific binding, antibody is coupled on carrier by Protein G.And outside the Fab fragment of IgG antibody is exposed to fully, increased substantially the capturing ability of malachite green, the purification efficiency of malachite green also effectively improves.。
2) the present invention has used the Protein G after genetic modification, by the optimization in conjunction with domain gene to the optimization of codon and IgG.The antibody binding capacity of Protein G is increased substantially, and then improved the purification efficiency of malachite green.
3) use purifying malachite green of the present invention easy and simple to handle, several steps just can obtain the malachite green that purity is higher.More convenient operator uses.
4) the malachite green purity of using the present invention to obtain is very high, and follow-up other purification process of need not doing again just can be directly used in high performance liquid chromatography detection or fluoroscopic examination.Operator's time and expense have been saved.
Accompanying drawing explanation
Fig. 1: malachite green immune affinity column structural representation
A: injection port; B: cylinder; C: sieve plate; D: aflatoxin antibody protein G-sepharose filler
E: sieve plate; F: outlet
Fig. 2: malachite green standard items detect HPLC collection of illustrative plates
LMG: malachite green peak; LGV: crystal violet peak
Fig. 3: flesh of fish Malachite Green testing result
LMG: malachite green peak; LGV: crystal violet peak
The specific embodiment
Embodiment 1: the preparation of malachite green immunoaffinity purification post
The preferred embodiment that the present invention prepares malachite green immunoaffinity purification post is as follows:
1. Ago-Gel activation
Get 2% Ago-Gel sepharose2B, with the distilled water of 20 times of volumes, fully rinse, wash away remaining ethanol.With funnel, filter out moisture.Take 5 grams of wet gels after elimination moisture content, add the NaOH of 7.5 milliliters of 0.8M, 2 milliliters of 30% epoxychloropropane, the sodium borohydride NaBH4 of 2mg/ml, 5 milliliters, at 25 ℃, shaking table reaction is 8 hours.
After reaction, with 50 ml distilled water washings, remove the epoxychloropropane mixing in gel.
2. the coupling of the Ago-Gel of Protein G and activation
Get 1 gram of Ago-Gel after activation, with the coupling buffer (NaHCO of 0.1M
3, 0.8M NaCl, PH8.9) wash 3 times.The Protein G 20ml that adds 2mg/ml, room temperature coupling 4 hours.
By the good agarose carrier 20mM of coupling, the phosphate buffer PBS of PH7.4 washing 3 times.Coupling has the agarose carrier sepharose called after Protein G-sepharose of Protein G.
3. anti-malachite green IgG monoclonal antibody is combined with Protein G-sepharose
Anti-malachite green antibody is dissolved in the PBS of 20mM, PH7.4 to final concentration 2mg/ml, cumulative volume 10ml.Protein G-sepharose is washed three times to each 30ml with the PBS buffer solution of 20Mm PH7.4.The malachite green antibody being dissolved in PBS is added in washed Protein G-sepharose.Room temperature was in conjunction with 30 minutes.
In conjunction with after the PBS washing of 20Mm PH7.4 three times for carrier, each 30ml.Be connected with the carrier called after antibody-Protein G-sepharose of antibody.
4. crosslinked in conjunction with the agarose sepharose of IgG
Antibody-Protein G-sepharose is washed 3 times to each 30ml with the borate buffer of 0.1M PH9.0.
By wet gel solid, add the borate buffer 20ml of 0.1M PH9.0, in buffer solution, add crosslinking agent imidic acid dimethyl ester (DMP) in heptan two to final concentration 20mM.Normal temperature crosslinked 1 hour.
Add 100mM, the monoethanolamine 20ml cessation reaction of PH9.0.And seal 10 minutes with the monoethanolamine 20ml of 50mM.
Antibody-Protein G-sepharose carrier 20mM after crosslinked, the PBS washing of PH7.4 3 times, each 30ml.
5. fill post
Sepharose after crosslinked is resuspended with 10 milliliters of 20mM PBS PH7.4, then pack in empty affinity purification post cylinder.
Embodiment 2: utilize malachite green immune affinity column to reclaim malachite green standard items
1. the preparation of malachite green standard items
1) get malachite green standard items, with the PBS buffer solution of 0.01M PH7.4, dissolve, and be diluted to 3ppb.
2) get the malachite green standard items 10ml having diluted, all sample detection.
2. by malachite green immunoaffinity purification post equilibrium at room temperature 30 minutes.
3. take out immune affinity column, injection port is connected with injector syringe, and syringe is linked on gas control crosshead.
4. with deionized water washing affinity column 3 times, each 10 milliliters, regulate pore crosshead air pump pressure, liquid is flowed out with the flow velocity of 3 drops/sec.
5. sample is added in purification column, adjust flux is to 1-2 drop/sec.Until sample all flows out purification column.
6. with distilled water, wash purification column 3 times, each 5 milliliters.
7. add 1ml methyl alcohol, collect eluted product.
8. eluted product detects with high-efficient liquid phase chromatogram HPLC.
9. high performance liquid chromatography testing result is as accompanying drawing 2:
According to detecting collection of illustrative plates, the malachite green standard items rate of recovery is 75.2%.
Embodiment 3: utilize malachite green immune affinity column purifying and detect the malachite green in the flesh of fish
1. oppress sample treatment
1) flesh of fish is removed non-edible part, with the equal quality sample of tissue refiner;
2) take 2.0 ± 0.05g homogenate sample to 50ml centrifuge tube;
3) add successively 0.4ml (0.25g/ml) hydrochloric acid hydroxylammonium solution, 0.8ml (1mol/L) p-methyl benzenesulfonic acid solution, 0.8ml ammonium acetate buffer and 18ml acetonitrile, mix with vortex instrument or homogenizer vibration, sonic oscillation extracts 2min, 1500r/min mechanical shaking extraction 3min;
4) the centrifugal 3min of 4000g under room temperature, carefully takes out the new centrifuge tube of 5ml upper strata liquid to one, and 50 ℃ of nitrogen dry up (after drying up, taking out immediately);
5) by whole loadings after the redissolution of 10ml PBS (0.01M PH7.4) buffer solution.
2. by malachite green immunoaffinity purification post equilibrium at room temperature 30 minutes.
3. take out immune affinity column, injection port is connected with injector syringe, and syringe is linked on gas control crosshead.
4. with deionized water washing affinity column 3 times, each 10 milliliters, regulate pore crosshead air pump pressure, liquid is flowed out with the flow velocity of 3 drops/sec.
5. sample is added in purification column, adjust flux is to 1-2 drop/sec.Until sample all flows out purification column.
6. with distilled water, wash purification column 3 times, each 5 milliliters.
7. add 1ml methyl alcohol, collect eluted product.
8. eluted product detects with high-efficient liquid phase chromatogram HPLC.
9. high performance liquid chromatography testing result is shown in accompanying drawing 3 in Table 1, HPLC collection of illustrative plates:
Table 1: flesh of fish sample malachite green residue detection result
According to HPLC collection of illustrative plates, can find out, after malachite green immune affinity column purifying, HPLC detects and to obtain containing in this part of flesh of fish sample concealed malachite green and two components of crystal violet.Total malachite green content is 4.13147ng/ul.
Claims (11)
1. an immune affinity column for malachite green, is characterized in that including carrier, Protein G and anti-malachite green antibody.
2. according to the immune affinity column described in claim 1, it is characterized in that Protein G is coupled on carrier by chemical bond, malachite green antibody is combined rear crosslinked with crosslinking agent with Protein G.
3. according to the immune affinity column described in claim 1, it is characterized in that Protein G is the Protein G of DNA recombinant expression.
4. according to the immune affinity column described in claim 2, it is characterized in that described Protein G comprises the Protein G of expressing according to GenBank CAA27638.1 sequence, and entered the Protein G of expressing after sequence optimisation.
5. according to the immune affinity column described in claim 3, the Protein G that it is characterized in that described sequence optimisation, comprises that the Protein G codon carrying out for Bacillus coli expression is optimized, Protein G is carried out to antibody calmodulin binding domain CaM carries out sequence change to increase affinity and to increase in Protein G gene order antibody calmodulin binding domain CaM quantity to improve antibody affinity.
6. according to the immune affinity column described in claim 1, it is characterized in that described carrier is Ago-Gel.
7. according to the immune affinity column described in claim 6, it is characterized in that described Ago-Gel is including but not limited to the Ago-Gel of cyanogen bromide-activated, crosslinked Ago-Gel, polyacrylic acid Ago-Gel, polyacrylamide Ago-Gel, sephadex and cellulose.
8. according to the immune affinity column described in claim 6, it is characterized in that described anti-malachite green antibody comprises monoclonal IgG antibody and polyclonal antibody.
9. an immune affinity column preparation method for purifying malachite green, is characterized in that
1) agarose sepharose4B, adds the NaOH of 0.8M, 30% epoxychloropropane, and the sodium borohydride NaBH4 of 2mg/ml,, at 25 ℃, shaking table reaction activates for 8 hours.Or buy the sepharose4B of cyanogen bromide-activated.
2) sepharose4B of every gram of activation adds the Protein G of 30-200nmol, at 0.1M NaHCO3, and in the buffer solution of 0.5M NaCl PH8.8, room temperature coupling 2 hours.Or 4 ℃ of couplings are spent the night.
3) coupled product is used 1M Tris.HCl PH8.0 room temperature reaction 2 hours.
4) coupled good sepharose-Protein G, with the PBS washing of 20mM PH7.4.
5) malachite green antibody is dissolved in the PBS of same 20mM PH7.4, every gram of Protein G-sepharose adds 200nmol-1.2umol antibody.Room temperature reaction 30 minutes.
6) by 20mM PBS PH7.4 washing for malachite green antibody-Protein G-sepharose carrier, then add the triethanolamine of final concentration 0.2M and the dimethyl pimelate (DMP) of 20mM, PH8.3. room temperature reaction 1 hour.
7) by the monoethanolamine cessation reaction of 20mM PH7.4, with the 10mMPBS PH7.4 that contains 0.01% thimerosal, wash malachite green antibody-Protein G-sepharose carrier.Dress post, is put in 4 ℃ of preservations.
10. immune affinity column according to claim 1 detects the purposes of malachite green.
11. purposes according to claim 10, is characterized in that: including but not limited to the separation and purification of grain, aquatic products, food Malachite Green.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310188359.1A CN104174185A (en) | 2013-05-21 | 2013-05-21 | Malachite green immunoaffinity column, and making method and use thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310188359.1A CN104174185A (en) | 2013-05-21 | 2013-05-21 | Malachite green immunoaffinity column, and making method and use thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104174185A true CN104174185A (en) | 2014-12-03 |
Family
ID=51955699
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310188359.1A Pending CN104174185A (en) | 2013-05-21 | 2013-05-21 | Malachite green immunoaffinity column, and making method and use thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104174185A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106140099A (en) * | 2015-04-17 | 2016-11-23 | 北京美正生物科技有限公司 | A kind of immune affinity column of isolated and purified lactoferrin and its production and use |
| CN106669638A (en) * | 2017-01-22 | 2017-05-17 | 中国农业科学院饲料研究所 | Protein G coated Sundan red I immunoaffinity column and preparation method thereof |
| CN107050926A (en) * | 2017-01-22 | 2017-08-18 | 中国农业科学院饲料研究所 | A kind of coating protein G lemon yellow immune affinity column and preparation method thereof |
| CN107449899A (en) * | 2017-08-14 | 2017-12-08 | 天津科技大学 | A kind of malachite green immunoaffinity gel detection column and preparation method thereof |
| CN110208441A (en) * | 2019-01-17 | 2019-09-06 | 广西科技大学 | The rapidly extracting and detection method of concealed malachite green in fresh-water fishes |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102115493A (en) * | 2009-12-30 | 2011-07-06 | 本元正阳基因技术有限公司 | Preparation method for recombinant protein G and application of same |
-
2013
- 2013-05-21 CN CN201310188359.1A patent/CN104174185A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102115493A (en) * | 2009-12-30 | 2011-07-06 | 本元正阳基因技术有限公司 | Preparation method for recombinant protein G and application of same |
Non-Patent Citations (1)
| Title |
|---|
| 孙兴荣 等: "抗黄曲霉素M1免疫亲和柱的制备", 《现代食品科技》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106140099A (en) * | 2015-04-17 | 2016-11-23 | 北京美正生物科技有限公司 | A kind of immune affinity column of isolated and purified lactoferrin and its production and use |
| CN106140099B (en) * | 2015-04-17 | 2018-10-16 | 北京美正生物科技有限公司 | A kind of immune affinity column and its preparation method and application isolating and purifying lactoferrin |
| CN106669638A (en) * | 2017-01-22 | 2017-05-17 | 中国农业科学院饲料研究所 | Protein G coated Sundan red I immunoaffinity column and preparation method thereof |
| CN107050926A (en) * | 2017-01-22 | 2017-08-18 | 中国农业科学院饲料研究所 | A kind of coating protein G lemon yellow immune affinity column and preparation method thereof |
| CN107449899A (en) * | 2017-08-14 | 2017-12-08 | 天津科技大学 | A kind of malachite green immunoaffinity gel detection column and preparation method thereof |
| CN110208441A (en) * | 2019-01-17 | 2019-09-06 | 广西科技大学 | The rapidly extracting and detection method of concealed malachite green in fresh-water fishes |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104117229B (en) | A kind of Aspergillus flavus toxin immuno-affinity column and its production and use | |
| CN104174185A (en) | Malachite green immunoaffinity column, and making method and use thereof | |
| US6358692B1 (en) | High speed, automated, continuous flow, multi-dimensional molecular selection and analysis | |
| CN105777896A (en) | Method for purifying acidic peaks of antibodies | |
| CN106706829B (en) | The method that affine in immunity purification-Liquid Chromatography-Tandem Mass Spectrometry measures research of diarrhetic shellfish poisons in shellfish | |
| CN102407098A (en) | Preparation method of immunoaffinity chromatography medium and application in tetraodotoxin purification | |
| CN104931597A (en) | Method capable of simultaneously detecting varieties of pesticide residues in aquatic product | |
| Armutcu et al. | Aspartic acid incorporated monolithic columns for affinity glycoprotein purification | |
| CN106669638A (en) | Protein G coated Sundan red I immunoaffinity column and preparation method thereof | |
| CN104117227B (en) | A kind of deoxynivalenol immune affinity column and its production and use | |
| CN104784972B (en) | A kind of preparation and its application of aurantiamarin para-immunity affinity column | |
| CN104117228B (en) | A kind of 6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone immune affinity column and its production and use | |
| CN102955008B (en) | Method for detecting sulfonamide residue in eel by pressurized capillary electrochromatography | |
| CN102382178B (en) | Separation method of sialoglycopeptide | |
| CN106399432B (en) | Method for preparing N-linked glycopeptide from monoclonal antibody and N-linked glycopeptide | |
| CN104181300B (en) | A kind of fumonisin immune affinity column and its production and use | |
| CN108663440B (en) | Method for constructing UPLC fingerprint spectrum of callicarpa nudiflora medicinal material and standard fingerprint spectrum | |
| CN104345114B (en) | A kind of method of reverse phase separation derivatization leucine and isoleucine | |
| CN1325516C (en) | Biomimetic affinity purification method of vitellus immune globulin | |
| CN109908879B (en) | Method for detecting tetracycline antibiotics | |
| CN103852542B (en) | Method for detecting complete degradation product of low molecular weight heparin based on post-column derivatization | |
| CN104415572A (en) | Citrinin immuno-affinity column and preparation method thereof | |
| CN108570118B (en) | Affinity chromatography purification method of placenta-like chondroitin sulfate A or derivative thereof | |
| CN108178810A (en) | The preparation and its application of a kind of reverse phase/anion exchange mixed mode polymer | |
| CN114894934B (en) | Construction method and application of scorpion medicinal material and formula particle UPLC characteristic spectrum thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20141203 |
|
| RJ01 | Rejection of invention patent application after publication |