CN103130712A - Paraquat hapten, paraquat complete antigen and preparation method of paraquat hapten and paraquat complete antigen - Google Patents
Paraquat hapten, paraquat complete antigen and preparation method of paraquat hapten and paraquat complete antigen Download PDFInfo
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- CN103130712A CN103130712A CN2011103968932A CN201110396893A CN103130712A CN 103130712 A CN103130712 A CN 103130712A CN 2011103968932 A CN2011103968932 A CN 2011103968932A CN 201110396893 A CN201110396893 A CN 201110396893A CN 103130712 A CN103130712 A CN 103130712A
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Abstract
The invention discloses paraquat hapten, paraquat complete antigen and a preparation method of the paraquat hapten and the paraquat complete antigen. Midbody 4,4-bipyridine of paraquat is utilized to combined analogue N-methyl-N'-valeric acid group-bipyridine-bibromide, carrier protein bovine serum albumin (BSA) is coupled through a mixed anhydride method, and paraquat artificial complete antigen can be successfully combined after ultraviolet spectrum identification and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) identification. The preparation method is simple and effective and low in cost. The combined paraquat artificial complete antigen can be applied to preparing peculiarly identifying efficient antibody of chemical pesticide paraquat and developing immune quick test kits remained by quick detection of paraquat to provide convenient methods for detection research of the materials.
Description
Technical field
The present invention relates to the preparation method of a kind of Paraquat haptens, Paraquat complete antigen, Paraquat haptens preparation method and Paraquat complete antigen, belong to technical field of biochemical industry, can the residual immune quick testing reagent box of rapid detection Paraquat for the preparation of the efficient antibody of specific recognition chemical pesticide Paraquat and development.
Background technology
Paraquat (Paraquat, PQ) have another name called Gramoxone, paraquat, chemistry effective constituent is 1, 1,-dimethyl-4, 4,-bipyridine cation salt, because it has wide spectrum, quick-acting, the characteristics such as safety, be widely used in the gardens weeding, the bare place chemical weed control, can kill most of Gramineae and broadleaf weeds, since nineteen fifty-five Britain ICI Crop protection company finds Paraquat, its commercialization industrial scale enlarges year by year, more than 130 countries and regions promotion and application in the world, especially in Asia and South and North America, usage quantity is in further expansion at present, China is the production of Paraquat maximum and uses one of state.
Paraquat all has stronger toxicity to people and animals, is a kind of high poisonous substance under the absorption condition, and the rat acute LD50 of passing through mouth is 57～150mg/kg, and its mouse oral LD50 is 110～150mg/kg, and the hare LD50 of passing through mouth is 236～325mg/kg, is 50～70mg/kg to ox.Rat acute is 500mg/kg through skin LD50, and rabbit is 235mg/kg.Paraquat is all toxic to each internal organs of health, and lungs are target organs of its poisoning rear main infringement, and the damage of patients with acute intoxication pulmonary capillary, alveolar edema are pulmonary fibrosis at last.The external report 40%～50% of its mortality ratio, domestic indivedual reports are up to more than 80%.Due to the widespread use of Paraquat, can enter into grain and oil crop and some food raw materials by number of ways, even cause under harmful level non-, also can produce potential hazard to HUMAN HEALTH.Therefore, set up the residual detection method of Paraquat and have very urgent and necessary realistic meaning.
The Accurate Determining method of Paraquat comprises gas chromatography (GC), high performance liquid chromatography (HPLC), MS, high performance capillary electrophoresis (HPCE), tlc (TLC) etc. at present, although TLC is easy and simple to handle, but operator must directly contact standard substance in testing process, it is relatively poor that it measures accuracy, detection sensitivity is not high yet, therefore can only be used for semi-quantitative analysis.GC, HPLC and mass spectroscopy etc. all can be carried out the accurate quantitative analysis analysis to the Paraquat in sample, but sample pretreatment process is complicated, complex operation, and all need the operator of expensive plant and instrument and specialty, are not suitable for on-the-spot rapid detection.The advantages such as it is quick, easy, sensitive, portable that immunological method detects that Paraquat possesses, be fit to on-the-spot rapid detection, and the basis of immunological detection method is the antibody of corresponding high specific, high-affinity, and white grass is withered as the small molecules haptens, itself does not possess immunogenicity, therefore, haptenic structure of modification and holoantigen are synthetic is to set up one of the key of immune fast survey technology and difficult point.
Summary of the invention
The purpose of this invention is to provide a kind of Paraquat haptens.
Second purpose of the present invention provides a kind of Paraquat haptenic preparation method.
The 3rd purpose of the present invention provides a kind of Paraquat complete antigen.
The 4th purpose of the present invention provides a kind of preparation method of Paraquat complete antigen.
Technical scheme of the present invention is summarized as follows:
A kind of Paraquat haptens has formula I structure:
Its chemical name is N-methyl-N '-valeric acid base-two pyridine dibromide.
The haptenic preparation method of Paraquat shown in formula I is comprised of following steps:
(1) under the logical nitrogen condition of the synthetic lucifuge of N-methyl-two pyridine iodide, 4,4 '-two pyridines are dissolved in acetone, stir and are cooled to 4 ℃, and methyl iodide is added drop-wise in mentioned solution.Keep 4 ℃ of stirrings, approximately the 6h reaction reaches balance.Filter and use washing with acetone, drain to get yellow needle crystal, the recrystallization final vacuum is dry.
(2) synthesis step (1) the gained compound of N-methyl-N '-Valeric acid ethylester base-two pyridine iodine bromide is dissolved in dimethyl formamide (DMF), and heating slowly drips 5-bromine Valeric acid ethylester, backflow 4h, and naturally cooling, room temperature is placed.Cooling crystallization.Filter, drain, vacuum-drying gets red plate crystal.
(3) synthesis step (2) the gained compound of N-methyl-N '-valeric acid base-two pyridine dibromide adds the 40% bromine hydracid aqueous solution, mixes reflux 1h.Evaporated under reduced pressure on rotatory evaporator obtains oily matter.After cooling, adding a small amount of acetone is that the adularescent solid is separated out.Filter, drain vacuum-drying.
A kind of Paraquat complete antigen has formula II structure:
The preparation method of a kind of Paraquat complete antigen of formula II structure is comprised of following steps:
9mg Paraquat haptens is dissolved in 500ul dimethyl sulfoxide (DMSO) (DMSO), adds the 15ul tri-n-butylamine, and then stirring at room 5min adds the 8ul isobutyl chlorocarbonate, stirring at room 30min.Carrier proteins is dissolved in the borate buffer solution of 2ml.Paraquat haptens solution is slowly splashed in carrier proteins solution.4 ℃ of stirrings are spent the night, and with PBS dialysis three days, change one time dialyzate in every four hours.
The concentration that described Paraquat haptens is dissolved in dimethyl sulfoxide (DMSO) is 15-20mg/mL.
Described carrier proteins is BSA or OVA, KLH etc.
Beneficial effect of the present invention: the present invention successfully synthesizes the Paraquat artificial complete antigen, synthesis step is succinctly effective, whole preparation process need not main equipment, with low cost, complete antigen of the present invention has kept the physicochemical property such as the chemical structure, charge distribution, space structure of former compound preferably, utilize this complete antigen to carry out immunity to laboratory animal, can obtain good, the highly sensitive antibody of specificity, for the detection of this material later on provides approach easily, can satisfy the needs of research.
Description of drawings
The haptenic nucleus magnetic hydrogen spectrum figure of Fig. 1 Paraquat
The SDS-PAGE of Fig. 2 Paraquat artificial complete antigen analyzes, wherein 1,3,5,8:BSA; 2,4,6,7,9:PQ-BSA
Fig. 3 PQ-BSA ultraviolet is identified figure
Fig. 4 PQ-BSA Mass Spectrometric Identification figure
This figure is the comparison diagram of BSA and PQ-BSA, and wherein A figure is the mass spectrum of BSA, and the mass-to-charge ratio of surveying is 66291.8594; B figure is the mass spectrum of PQ-BSA, and its mass-to-charge ratio is 67967.6797, has increased 1675.8203 compared to its molecular weight of peak of BSA.Because the haptenic molecular weight of its coupling is 272, therefore the haptens in its coupling is about 6, i.e. coupling ratio BSA/PQ=1/6.Can prove conclusively Paraquat complete antigen coupling success based on above mass spectrum result.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment if no special instructions, is ordinary method.Reagent and material used in following embodiment if no special instructions, all can be bought from conventional reagent company and obtain.
Technical scheme of the present invention: the preparation of the haptenic preparation of a kind of Paraquat and Paraquat complete antigen:
Select the intermediate (4,4-, two pyridines) of Paraquat, synthetic Paraquat haptens N-methyl-N '-valeric acid base-two pyridines-dibromide utilizes mixed anhydride method and carrier proteins BSA to carry out coupling, preparation Paraquat artificial antigen.
One, the haptenic preparation of Paraquat
Paraquat haptens synthetic route is as follows:
Concrete building-up process:
(1) the synthetic of N-methyl-two pyridine iodide is fixed in iron stand with three mouthfuls of round-bottomed flasks of 250ml, and lucifuge is led to nitrogen.4,4 '-two pyridine 808mg are dissolved in 8ml acetone, stir and are cooled to 4 ℃, and methyl iodide 400 μ l are added drop-wise in mentioned solution.Keep 4 ℃ of stirrings.(developping agent is methyl alcohol: glacial acetic acid=5: 2) to control reaction end with TLC.Approximately the 6h reaction reaches balance.Filter, use washing with acetone, drain, get yellow needle crystal.This crystallization dehydrated alcohol recrystallization.Vacuum-drying.
(2) synthetic 0.85mgN-methyl-two pyridine compounds of N-methyl-N '-Valeric acid ethylester base-two pyridine iodine bromide is dissolved in 10ml dimethyl formamide (DMF), logical nitrogen stirring and dissolving, and control temperature to 120 ℃, slowly drip 1.7ml 5-bromine Valeric acid ethylester, backflow 4h, and carry out simultaneously TLC monitor to the reactant primitive reaction complete, stopped reaction.Naturally cooling, room temperature is placed.Coolingly separate out red plate crystal.Filter, drain, vacuum-drying obtains red plate crystal.
(3) the synthetic 500mg N-methyl-N ' of N-methyl-N '-valeric acid base-two pyridine dibromide-Valeric acid ethylester base-two pyridine compounds in logical nitrogen four-hole bottle, adds 40% bromine hydracid aqueous solution 18ml, mixes reflux 1h.TLC Indicator Reaction thing all is hydrolyzed.Stopped heating.Excessive acid and the water of pressure reducing and steaming on rotatory evaporator keeps bath temperature to be no more than 60 ℃, and evaporate to dryness obtains oily matter.After cooling, adding a small amount of acetone is that the adularescent solid is separated out.Filter, drain vacuum-drying.
Two, the haptenic evaluation of Paraquat
The product that step 1 is prepared carries out nucleus magnetic hydrogen spectrum, and nucleus magnetic hydrogen spectrum figure sees Fig. 1.By above evaluation as can be known, the molecular structural formula of purpose product is that chemical name is suc as formula the Paraquat haptens shown in II: N-methyl-N '-valeric acid base-two pyridine dibromide.
One. the preparation of Paraquat complete antigen
The synthetic route of Paraquat complete antigen is as follows:
Concrete building-up process: 9mg Paraquat haptens is dissolved in 500ul dimethyl sulfoxide (DMSO) (DMSO), adds the 15ul tri-n-butylamine, and then stirring at room 5min adds the 8ul isobutyl chlorocarbonate, stirring at room 30min.15mg BSA is dissolved in the borate buffer solution of 2ml pH8.7.Paraquat haptens solution is slowly splashed in BSA solution.4 ℃ of stirrings are spent the night, and with PBS dialysis three days, change one time dialyzate in every four hours.Be sub-packed in the EP pipe-20 ℃ of preservations.
Two. the evaluation of Paraquat complete antigen
Molecular weight according to the molecular weight ratio carrier proteins of complete antigen after coupling increases, and adopts the SDS-PAGE gel electrophoresis to identify coupling result (Fig. 2), and PQ-BAS place swimming lane band is compared BSA and obviously lagged behind, and molecular weight increases.
Carry out the scanning of ultraviolet continuous wavelength, coupling protein BSA, complete antigen PQ-BSA, small molecules agricultural chemicals Paraquat PQ, its maximum absorption wavelength obviously different (Fig. 3).
Mass Spectrometric Identification, the mass spectrum of comparison BSA and PQ-BSA, compared to the peak of BSA, complete antigen PQ-BSA molecular weight has increased 1675.8203, can prove conclusively Paraquat complete antigen coupling success based on above mass spectrum result.
Claims (6)
1. Paraquat haptens has formula I structure:
Its chemical name is N-methyl-N '-valeric acid base-two pyridine dibromide.
2. the haptenic preparation method of the described Paraquat of claim 1 is comprised of following steps:
(1) the synthetic of N-methyl-two pyridine iodide is fixed in iron stand with three mouthfuls of round-bottomed flasks of 250ml, and lucifuge is led to nitrogen.4,4 '-two pyridine 808mg are dissolved in 8ml acetone, stir and are cooled to 4 ℃, and methyl iodide 400 μ l are added drop-wise in mentioned solution.Keep 4 ℃ of stirrings.Approximately the 6h reaction reaches balance.Filter, use washing with acetone, drain, get yellow needle crystal.This crystallization dehydrated alcohol recrystallization.Vacuum-drying.
(2) synthetic 0.85mgN-methyl-two pyridine compounds of N-methyl-N '-Valeric acid ethylester base-two pyridine iodine bromide is dissolved in 10ml dimethyl formamide (DMF), logical nitrogen stirring and dissolving, and control temperature to 120 ℃, slowly drip 1.7ml 5-bromine Valeric acid ethylester, backflow 4h, and carry out simultaneously TLC monitor to the reactant primitive reaction complete, stopped reaction.Naturally cooling, room temperature is placed.Coolingly separate out red plate crystal.Filter, drain, vacuum-drying obtains red plate crystal.
(3) the synthetic 500mgN-methyl-N ' of N-methyl-N '-valeric acid base-two pyridine dibromide-Valeric acid ethylester base-two pyridine compounds in logical nitrogen four-hole bottle, adds 40% bromine hydracid aqueous solution 18ml, mixes reflux 1h.TLC Indicator Reaction thing all is hydrolyzed.Stopped heating.Excessive acid and the water of pressure reducing and steaming on rotatory evaporator keeps bath temperature to be no more than 60 ℃, and evaporate to dryness obtains oily matter.After cooling, adding a small amount of acetone is that the adularescent solid is separated out.Filter, drain vacuum-drying.
3. Paraquat complete antigen has formula II structure:
4. the preparation method of the described Paraquat complete antigen of claim 3 is comprised of following steps:
9mg Paraquat haptens is dissolved in 500ul dimethyl sulfoxide (DMSO) (DMSO), adds the 15ul tri-n-butylamine, and then stirring at room 5min adds the 8ul isobutyl chlorocarbonate, stirring at room 30min.Carrier proteins is dissolved in the borate buffer solution of 2ml.Paraquat haptens solution is slowly splashed in carrier proteins solution.4 ℃ of stirrings are spent the night, and with PBS dialysis three days, change one time dialyzate in every four hours.
5. to be dissolved in the concentration of dimethyl sulfoxide (DMSO) be 15-20mg/mL to the described Paraquat haptens of claim 4.
6. the described carrier proteins of claim 4 can be bovine serum albumin (BSA), oralbumin (OVA), human serum albumin (HAS) or keyhole azurin (KLH) etc.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108251381A (en) * | 2018-04-04 | 2018-07-06 | 江南大学 | A kind of paraquat monoclonal antibody hybridoma cell strain and its application |
| CN109796396A (en) * | 2019-02-18 | 2019-05-24 | 山西医科大学 | A kind of preparation method of N- methyl -4,4 '-bipyridyl iodide |
| CN110981791A (en) * | 2019-11-06 | 2020-04-10 | 苏州博源医疗科技有限公司 | Paraquat derivative, preparation method thereof and paraquat detection reagent |
| CN111205219A (en) * | 2020-01-09 | 2020-05-29 | 深圳大学 | Paraquat hapten, complete antigen, nano antibody, detection test paper, kit, preparation method and application |
| CN111925321A (en) * | 2020-09-24 | 2020-11-13 | 北京纳百生物科技有限公司 | Paraquat hapten, complete antigen, antibody, detection test paper and kit |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108251381A (en) * | 2018-04-04 | 2018-07-06 | 江南大学 | A kind of paraquat monoclonal antibody hybridoma cell strain and its application |
| CN108251381B (en) * | 2018-04-04 | 2020-09-04 | 江南大学 | Paraquat monoclonal antibody hybridoma cell strain and application thereof |
| CN109796396A (en) * | 2019-02-18 | 2019-05-24 | 山西医科大学 | A kind of preparation method of N- methyl -4,4 '-bipyridyl iodide |
| CN110981791A (en) * | 2019-11-06 | 2020-04-10 | 苏州博源医疗科技有限公司 | Paraquat derivative, preparation method thereof and paraquat detection reagent |
| CN111205219A (en) * | 2020-01-09 | 2020-05-29 | 深圳大学 | Paraquat hapten, complete antigen, nano antibody, detection test paper, kit, preparation method and application |
| CN111205219B (en) * | 2020-01-09 | 2022-11-25 | 深圳大学 | Paraquat hapten, complete antigen, nano antibody, detection test paper, kit, preparation method and application |
| CN111925321A (en) * | 2020-09-24 | 2020-11-13 | 北京纳百生物科技有限公司 | Paraquat hapten, complete antigen, antibody, detection test paper and kit |
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Application publication date: 20130605 |