CN103833845A - Method for synthesis of chlorothalonil artificial antigen - Google Patents
Method for synthesis of chlorothalonil artificial antigen Download PDFInfo
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- CN103833845A CN103833845A CN201310644040.5A CN201310644040A CN103833845A CN 103833845 A CN103833845 A CN 103833845A CN 201310644040 A CN201310644040 A CN 201310644040A CN 103833845 A CN103833845 A CN 103833845A
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- 239000000427 antigen Substances 0.000 title claims abstract description 37
- 102000036639 antigens Human genes 0.000 title claims abstract description 37
- 108091007433 antigens Proteins 0.000 title claims abstract description 37
- CRQQGFGUEAVUIL-UHFFFAOYSA-N chlorothalonil Chemical compound ClC1=C(Cl)C(C#N)=C(Cl)C(C#N)=C1Cl CRQQGFGUEAVUIL-UHFFFAOYSA-N 0.000 title claims abstract description 25
- 238000000034 method Methods 0.000 title claims abstract description 18
- 230000015572 biosynthetic process Effects 0.000 title abstract description 5
- 238000003786 synthesis reaction Methods 0.000 title abstract description 5
- 239000005747 Chlorothalonil Substances 0.000 title abstract 4
- 238000006243 chemical reaction Methods 0.000 claims abstract description 20
- 230000008878 coupling Effects 0.000 claims abstract description 17
- 238000010168 coupling process Methods 0.000 claims abstract description 17
- 238000005859 coupling reaction Methods 0.000 claims abstract description 17
- 230000003053 immunization Effects 0.000 claims abstract description 10
- 239000003814 drug Substances 0.000 claims abstract description 9
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims abstract description 8
- 229940098773 bovine serum albumin Drugs 0.000 claims abstract description 7
- 101000609762 Gallus gallus Ovalbumin Proteins 0.000 claims abstract description 6
- 102000014914 Carrier Proteins Human genes 0.000 claims abstract description 5
- 108010078791 Carrier Proteins Proteins 0.000 claims abstract description 5
- 238000004458 analytical method Methods 0.000 claims abstract description 4
- 229940079593 drug Drugs 0.000 claims abstract description 4
- 239000007788 liquid Substances 0.000 claims description 45
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 21
- 238000000502 dialysis Methods 0.000 claims description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
- 239000011259 mixed solution Substances 0.000 claims description 12
- 238000002360 preparation method Methods 0.000 claims description 12
- 238000012797 qualification Methods 0.000 claims description 10
- 238000005516 engineering process Methods 0.000 claims description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- 238000009835 boiling Methods 0.000 claims description 6
- 239000008367 deionised water Substances 0.000 claims description 6
- 229910021641 deionized water Inorganic materials 0.000 claims description 6
- 230000003203 everyday effect Effects 0.000 claims description 6
- 238000001556 precipitation Methods 0.000 claims description 6
- 238000002203 pretreatment Methods 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
- 239000003643 water by type Substances 0.000 claims description 6
- 239000002253 acid Substances 0.000 claims description 5
- RAABOESOVLLHRU-UHFFFAOYSA-N diazene Chemical compound N=N RAABOESOVLLHRU-UHFFFAOYSA-N 0.000 claims description 5
- 238000010189 synthetic method Methods 0.000 claims description 5
- YOETUEMZNOLGDB-UHFFFAOYSA-N 2-methylpropyl carbonochloridate Chemical compound CC(C)COC(Cl)=O YOETUEMZNOLGDB-UHFFFAOYSA-N 0.000 claims description 3
- 229910000071 diazene Inorganic materials 0.000 claims description 3
- 238000004090 dissolution Methods 0.000 claims description 3
- 239000000376 reactant Substances 0.000 claims description 3
- 238000002390 rotary evaporation Methods 0.000 claims description 3
- IMFACGCPASFAPR-UHFFFAOYSA-N tributylamine Chemical class CCCCN(CCCC)CCCC IMFACGCPASFAPR-UHFFFAOYSA-N 0.000 claims description 3
- XINQFOMFQFGGCQ-UHFFFAOYSA-L (2-dodecoxy-2-oxoethyl)-[6-[(2-dodecoxy-2-oxoethyl)-dimethylazaniumyl]hexyl]-dimethylazanium;dichloride Chemical compound [Cl-].[Cl-].CCCCCCCCCCCCOC(=O)C[N+](C)(C)CCCCCC[N+](C)(C)CC(=O)OCCCCCCCCCCCC XINQFOMFQFGGCQ-UHFFFAOYSA-L 0.000 claims description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 2
- 101710145634 Antigen 1 Proteins 0.000 claims 1
- SLXKOJJOQWFEFD-UHFFFAOYSA-N 6-aminohexanoic acid Chemical compound NCCCCCC(O)=O SLXKOJJOQWFEFD-UHFFFAOYSA-N 0.000 abstract 1
- 229960002684 aminocaproic acid Drugs 0.000 abstract 1
- 238000010364 biochemical engineering Methods 0.000 abstract 1
- 150000001718 carbodiimides Chemical class 0.000 abstract 1
- 239000011248 coating agent Substances 0.000 abstract 1
- 238000000576 coating method Methods 0.000 abstract 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 abstract 1
- 238000002649 immunization Methods 0.000 abstract 1
- 238000001514 detection method Methods 0.000 description 4
- 230000031700 light absorption Effects 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 2
- 108010058846 Ovalbumin Proteins 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 229940092253 ovalbumin Drugs 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- 208000031888 Mycoses Diseases 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 150000001793 charged compounds Chemical class 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 230000003628 erosive effect Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 239000003547 immunosorbent Substances 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 238000003822 preparative gas chromatography Methods 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/77—Ovalbumin
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2430/00—Assays, e.g. immunoassays or enzyme assays, involving synthetic organic compounds as analytes
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Abstract
The invention discloses a method for synthesis of a chlorothalonil artificial antigen and belongs to the technical field of biochemical engineering. A chlorothalonil raw drug and 6-aminocaproic acid undergo a reaction to produce hapten CTNH as a hydroxy product, the CTNH is coupled with carrier protein bovine serum albumin (BSA) by a carbodiimide method so that a conjugate CTNH-BSA is obtained and is used as an immunizing antigen, the CTNH is coupled with carrier protein chicken egg albumin (OVA) so that a conjugate CTNH-OVA is obtained and is used as a coating antigen, and conjugate coupling ratios are determined by an ultraviolet method. The method realizes successfully synthesis of the chlorothalonil artificial antigen, has simple and effective synthesis steps, can be completely used for immunization analysis and provides a necessary artificial antigen for later research.
Description
Technical field
A synthetic method for m-tetrachlorophthalodinitrile artificial antigen, belongs to technical field of biochemical industry.
Background technology
M-tetrachlorophthalodinitrile is a kind of wide spectrum organochlorine sterilant, and the fungal disease of various farm crop and various greening lawns etc. is had to prophylactic effect.M-tetrachlorophthalodinitrile does not have interior suction conduction, but once after being sprayed onto on plant materials, just can there is good tackyness at its body surface, be difficult for being fallen by rain drop erosion, therefore the drug effect phase longer, add it and be widely used, therefore, m-tetrachlorophthalodinitrile and meta-bolites thereof have larger residual at Plants and Soils, even detect in Arctic region.The method that detects at present m-tetrachlorophthalodinitrile is mainly the instrumental method such as vapor-phase chromatography (GC) and liquid phase chromatography (HPLC), but there is complex operation in these methods, consuming time, the shortcomings such as expense is somewhat expensive, can not realize the rapid detection of a large amount of samples, therefore set up a kind of fast and convenient m-tetrachlorophthalodinitrile detection method significant.Euzymelinked immunosorbent assay (ELISA) (ELISA) is that one is very efficient, responsive, detection method fast, but the monoclonal antibody that obtains high-affinity and high specific is the prerequisite of immunology detection, and wherein the synthetic of artificial antigen is a wherein important step.
Summary of the invention
The object of this invention is to provide the synthetic method of a kind of m-tetrachlorophthalodinitrile artificial antigen.Prepared product is for the research of m-tetrachlorophthalodinitrile immune analysis method, for people's research from now on provides essential artificial antigen.
Technical scheme of the present invention: the synthetic method of a kind of m-tetrachlorophthalodinitrile artificial antigen, the former medicine of m-tetrachlorophthalodinitrile reacts the haptens CTNH that obtains having carboxyl with 6-aminocaprolc acid, with carbodlimide method, CTNH and carrier proteins bovine serum albumin BSA coupling are obtained to conjugate CTNH-BSA as immunizing antigen again, CTNH and carrier proteins chicken ovalbumin OVA coupling are obtained to conjugate CTNH-OVA as envelope antigen, measure the coupling ratio of conjugate with ultraviolet method; Step is:
(1) haptenic synthetic: to take the former medicine 500mg(1.9mmol of m-tetrachlorophthalodinitrile), KOH 106.6mg(1.9mmol), 6-aminocaprolc acid 262mg(2mmol), these three kinds of medicines are dissolved in to 20mL ethanol, 60 DEG C of reaction 12h, at 90 DEG C of reaction 12h, by reactant rotary evaporation, then use 20mL water dissolution again, with hydrochloric acid tune pH to 4-5, produce precipitation, precipitation is dried and is haptens CTNH, identify and analysis with liquid matter technology used in conjunction;
(2) preparation of immunizing antigen CTNH-BSA
Preparation A liquid: take 35.8mg CTNH, 1-ethyl-3-(3-dimethylaminopropyl) phosphinylidyne diimine (EDC) 56.9mg, N-maloyl imines (NHS) 35.6mg, dissolve with 1mL anhydrous dimethyl formamide (DMF), stirring at room temperature reaction 8h, obtains A liquid;
Preparation B liquid: take bovine serum albumin (BSA) 112mg and be dissolved in the 0.01mol/L PBS of 20mL pH7.2, obtain B liquid;
At ambient temperature, dropwise A liquid is joined in B liquid, room temperature reaction spends the night, and obtains conjugate CTNH-BSA mixed solution;
Dialysis tubing pre-treatment: get the dialysis tubing of 10cm, boil 5min in boiling water, then use the deionized water rinsing 3min of 60 DEG C, be kept in 4 DEG C of deionized waters for subsequent use;
Conjugate CTNH-BSA mixed solution is put into dialysis tubing and dialyse 3 days in the PBS of 0.01mol/L, change liquid three every day; CTNH-BSA is as immunizing antigen;
(3) preparation of envelope antigen CTNH-OVA
Preparation C liquid: take 40mg CTNH, be dissolved in 1mL dry DMF, drip successively the isobutyl chlorocarbonate of 32 μ L tri-n-butylamines and 17.5 μ L, 4 DEG C of stirring reaction 1h, obtain C liquid;
Preparation D liquid: take chicken ovalbumin (OVA) 166mg and be dissolved in the PBS of 30mL, obtain D liquid; At 4 DEG C, dropwise C liquid is joined in D liquid, 4 DEG C of reaction 5h, obtain conjugate CTNH-OVA mixed solution;
Dialysis tubing pre-treatment: get the dialysis tubing of 10cm, boil 5min in boiling water, then use the deionized water rinsing 3min of 60 DEG C, be kept in 4 DEG C of deionized waters for subsequent use;
Conjugate CTNH-OVA mixed solution is put into dialysis tubing and dialyse 3 days in the PBS of 0.01mol/L, change liquid three every day, and CTNH-OVA is as envelope antigen;
(4) qualification of m-tetrachlorophthalodinitrile artificial antigen
1. adopt liquid matter technology used in conjunction qualification haptens;
2. artificial antigen adopts ultraviolet method to identify its coupling result, according to the concentration of conjugate small molecular and albumen, calculates its coupling ratio.
The qualification of m-tetrachlorophthalodinitrile artificial antigen
Coupling ratio is measured: be the method for estimating the ratio (coupling ratio) of two kinds of molecules coupled in conjugate, although measuring method kind is a lot, be all to set up according to the principle that detects two kinds of molecule contents (or relative content) coupled in conjugate.Ultraviolet method is to determine coupling ratio according to the small molecules concentration in synthetic artificial antigen with the ratio of protein concentration.
Conjugate determination of protein concentration: the content that the quality of albumen before reaction can be obtained to albumen in conjugate divided by the volume of the rear conjugate of dialysis.
The mensuration of conjugate small molecular concentration: concentration is that the small molecules of 10 μ g/mL is at the light absorption value A1=0.57826 at characteristic peak 355nm place, conjugate is at the light absorption value A2=0.35219 at 355nm place, micromolecular molecular weight is 360.61g/mol, so, concentration=(0.35219 × 10 μ g/mL)/0.57826 of conjugate small molecular
=6.09 μ g/mL; Coupling ratio=[(6.09 μ g/mL/360.61)/(3.33mg/mL/68000)] × 12(extension rate)=4.
Beneficial effect of the present invention: the present invention successfully synthesizes m-tetrachlorophthalodinitrile artificial antigen, and synthesis step is succinct, effectively, can be used in immunoassay completely, for people's research later provides approach easily.
Brief description of the drawings
The synthetic haptenic liquid matter of Fig. 1 is used in conjunction (LC/MS) qualification figure.
1-a: can see when the time is 4.16 minutes having one stronger to go out peak in total ion figure.
The molecular weight 359 of 1-b:CTNH, under mass spectrum negative ion scan pattern, molecular ion peak is 358 (M-1).
The ultraviolet qualification figure of the synthetic immunizing antigen CTNH-BSA of Fig. 2.
The ultraviolet qualification figure of the synthetic envelope antigen CTNH-OVA of Fig. 3.
Embodiment
(1) haptenic synthetic: to take the former medicine 500mg(1.9mmol of m-tetrachlorophthalodinitrile), KOH 106.6mg(1.9mmol), 6-aminocaprolc acid 262mg(2mmol), these three kinds of medicines are dissolved in to 20mL ethanol, 60 DEG C of reaction 12h, then at 90 DEG C of reaction 12h.By reactant rotary evaporation, then use 20mL water dissolution, adjust pH to 4-5 with hydrochloric acid, produce precipitation, precipitation is dried and is haptens CTNH.With liquid matter technology used in conjunction identify with analyze.
(2) preparation of conjugate CTNH-BSA
Take 35.8mg CTNH, 1-ethyl-3-(3-dimethylaminopropyl) phosphinylidyne diimine (EDC) 56.9mg, N-maloyl imines (NHS) 35.6mg, with 1mL anhydrous dimethyl formamide (DMF) dissolving (being called A liquid), stirring at room temperature reaction 8h.Take in the 0.01mol/L PBS that bovine serum albumin (BSA) 112mg is dissolved in 20mL pH7.2 (being called B liquid), at room temperature condition, dropwise A liquid is joined in B liquid, room temperature reaction spends the night, and obtains conjugate CTNH-BSA mixed solution;
Dialysis tubing pre-treatment: get the dialysis tubing of 10cm, boil 5min in boiling water, then use the deionized water rinsing 3min of 60 DEG C, be kept in 4 DEG C of deionized waters for subsequent use;
Conjugate CTNH-BSA mixed solution is put into dialysis tubing and dialyse 3 days in the PBS of 0.01mol/L, change liquid three every day.CTNH-BSA is as immunizing antigen;
(3) preparation of conjugate CTNH-OVA
Take 40mg CTNH, be dissolved in 1mL dry DMF, drip successively the isobutyl chlorocarbonate (being called C liquid) of 32 μ L tri-n-butylamines and 17.5 μ L, 4 DEG C of stirring reaction 1h.Take in the PBS that chicken ovalbumin (OVA) 166mg is dissolved in 30mL (being called D liquid).At 4 DEG C, dropwise C liquid is joined in D liquid, 4 DEG C of reaction 5h, obtain conjugate CTNH-OVA mixed solution;
Dialysis tubing pre-treatment: get the dialysis tubing of 10cm, boil 5min in boiling water, then use the deionized water rinsing 3min of 60 DEG C, be kept in 4 DEG C of deionized waters for subsequent use;
Conjugate CTNH-OVA mixed solution is put into dialysis tubing and dialyse 3 days in the PBS of 0.01mol/L, change liquid three every day.CTNH-OVA is as envelope antigen;
The qualification of m-tetrachlorophthalodinitrile artificial antigen
Adopt liquid matter technology used in conjunction qualification haptens; Artificial antigen adopts ultraviolet method to identify its coupling result, according to the concentration of conjugate small molecular and albumen, calculates its coupling ratio.
The tiring and suppress of table 1 immune mouse
In table, numerical value is the light absorption value at 450nm place.
Claims (1)
1. the synthetic method of a m-tetrachlorophthalodinitrile artificial antigen, it is characterized in that: the former medicine of m-tetrachlorophthalodinitrile reacts the haptens CTNH that obtains having carboxyl with 6-aminocaprolc acid, with carbodlimide method, CTNH and carrier proteins bovine serum albumin BSA coupling are obtained to conjugate CTNH-BSA as immunizing antigen again, CTNH and carrier proteins chicken ovalbumin OVA coupling are obtained to conjugate CTNH-OVA as envelope antigen, measure the coupling ratio of conjugate with ultraviolet method; Step is:
(1) haptenic synthetic: to take the former medicine 1.9mmol of m-tetrachlorophthalodinitrile, KOH 1.9mmol, 6-aminocaprolc acid 2mmol, is dissolved in 20mL ethanol by these three kinds of medicines, 60 DEG C of reaction 12h, at 90 DEG C of reaction 12h, by reactant rotary evaporation, then use 20mL water dissolution again, with hydrochloric acid tune pH to 4-5, produce precipitation, precipitation is dried and is haptens CTNH, identify and analysis with liquid matter technology used in conjunction;
(2) preparation of immunizing antigen CTNH-BSA
Preparation A liquid: take 35.8mg CTNH, 1-ethyl-3-(3-dimethylaminopropyl) phosphinylidyne diimine EDC 56.9mg, N-maloyl imines NHS 35.6mg, with 1mL anhydrous dimethyl formamide, DMF dissolves, stirring at room temperature reaction 8h, obtains A liquid;
Preparation B liquid: take in the 0.01mol/L PBS that bovine serum albumin BSA 112mg is dissolved in 20mL pH7.2, obtain B liquid;
At ambient temperature, dropwise A liquid is joined in B liquid, room temperature reaction spends the night, and obtains conjugate CTNH-BSA mixed solution;
Dialysis tubing pre-treatment: get the dialysis tubing of 10cm, boil 5min in boiling water, then use the deionized water rinsing 3min of 60 DEG C, be kept in 4 DEG C of deionized waters for subsequent use;
Conjugate CTNH-BSA mixed solution is put into dialysis tubing and dialyse 3 days in the PBS of 0.01mol/L, change liquid three every day; CTNH-BSA is as immunizing antigen;
(3) preparation of envelope antigen CTNH-OVA
Preparation C liquid: take 40mg CTNH, be dissolved in 1mL dry DMF, drip successively the isobutyl chlorocarbonate of 32 μ L tri-n-butylamines and 17.5 μ L, 4 DEG C of stirring reaction 1h, obtain C liquid;
Preparation D liquid: take chicken ovalbumin OVA 166mg and be dissolved in the PBS of 30mL, obtain D liquid; At 4 DEG C, dropwise C liquid is joined in D liquid, 4 DEG C of reaction 5h, obtain conjugate CTNH-OVA mixed solution;
Dialysis tubing pre-treatment: get the dialysis tubing of 10cm, boil 5min in boiling water, then use the deionized water rinsing 3min of 60 DEG C, be kept in 4 DEG C of deionized waters for subsequent use;
Conjugate CTNH-OVA mixed solution is put into dialysis tubing and dialyse 3 days in the PBS of 0.01mol/L, change liquid three every day, and CTNH-OVA is as envelope antigen;
(4) qualification of m-tetrachlorophthalodinitrile artificial antigen
1. adopt liquid matter technology used in conjunction qualification haptens;
2. artificial antigen adopts ultraviolet method to identify its coupling result, according to the concentration of conjugate small molecular and albumen, calculates its coupling ratio.
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Cited By (4)
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| CN104020290A (en) * | 2014-06-26 | 2014-09-03 | 江南大学 | Chlorothalonil enzyme-linked immunosorbent assay method |
| CN104672332A (en) * | 2015-03-09 | 2015-06-03 | 武汉市畜牧兽医科学研究所 | Antibody for testing free gossypol, ELISA (enzyme-linked immune sorbent assay) method and kit |
| CN107022023A (en) * | 2017-04-25 | 2017-08-08 | 江南大学 | A kind of synthetic method of 6 benzyl aminoadenine artificial antigen |
| CN114316027A (en) * | 2020-10-10 | 2022-04-12 | 中国农业大学 | Flunixin artificial antigen and preparation method and application thereof |
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| CN107022024A (en) * | 2017-04-25 | 2017-08-08 | 江南大学 | A kind of synthetic method of iprodione artificial antigen |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104020290A (en) * | 2014-06-26 | 2014-09-03 | 江南大学 | Chlorothalonil enzyme-linked immunosorbent assay method |
| CN104672332A (en) * | 2015-03-09 | 2015-06-03 | 武汉市畜牧兽医科学研究所 | Antibody for testing free gossypol, ELISA (enzyme-linked immune sorbent assay) method and kit |
| CN104672332B (en) * | 2015-03-09 | 2018-10-09 | 武汉市畜牧兽医科学研究所 | Antibody, ELISA method and kit for detecting free gossypol |
| CN107022023A (en) * | 2017-04-25 | 2017-08-08 | 江南大学 | A kind of synthetic method of 6 benzyl aminoadenine artificial antigen |
| CN107022023B (en) * | 2017-04-25 | 2020-09-29 | 江南大学 | Synthetic method of 6-benzylamino adenine artificial antigen |
| CN114316027A (en) * | 2020-10-10 | 2022-04-12 | 中国农业大学 | Flunixin artificial antigen and preparation method and application thereof |
| CN114316027B (en) * | 2020-10-10 | 2023-09-05 | 中国农业大学 | Fluoronii Xin Rengong antigen and preparation method and application thereof |
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