CN106046083A - Avermectin artificial antigen and preparation method and application thereof - Google Patents
Avermectin artificial antigen and preparation method and application thereof Download PDFInfo
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- CN106046083A CN106046083A CN201610387441.0A CN201610387441A CN106046083A CN 106046083 A CN106046083 A CN 106046083A CN 201610387441 A CN201610387441 A CN 201610387441A CN 106046083 A CN106046083 A CN 106046083A
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- liquid
- avilamycin
- dichloromethane
- antigen
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
- C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
- C07H17/08—Hetero rings containing eight or more ring members, e.g. erythromycins
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
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- 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
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
-
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
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- Molecular Biology (AREA)
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- General Health & Medical Sciences (AREA)
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- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biotechnology (AREA)
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Abstract
The invention discloses an avermectin artificial antigen and a preparation method and application thereof. The avermectin artificial antigen is obtained by coupling an avermectin hapten shown in a formula I and a carrier protein. The avermectin artificial antigen is simple in synthesis method and high in purity and yield, and has great value for avermectin drug residue detection.
Description
Technical field
The invention belongs to biological medicine and chemical field, relate to a kind of avilamycin artificial antigen and preparation method thereof and answer
With.
Background technology
Avilamycin is the anti-parasite medicine of a kind of broad-spectrum high efficacy, ectoparasite in veterinary clinic is widely used in animal body
The preventing and treating of worm.But, Avermectins medicine mainly acts on neuromuscular junction, has potential neurotoxicity, and liposoluble
Property strong, residual in animal body was more than 35 days, and World Health Organization (WHO) is classified as high cytotoxic compound.Avilamycin enzyme linked immunological
The features such as test kit has easily and fast, sensitive, it is adaptable to batch samples detects.
At present, the method that detection of veterinary drugs in food is conventional has the physics and chemistry such as gas chromatogram, high performance liquid chromatography and gas chromatography mass spectrometry
Analysis method.These methods, while high specificity, highly sensitive, but sample pre-treatments complex operation step, relatively costly, also
It is not suitable for the selective mechanisms of batch samples.Immunochemical analyses is in view of unique excellent in terms of the qualitative, quantitative of antigen-antibody
Gesture and easy and simple to handle quickly, low cost, sensitivity is higher, analyze the big advantage of sample size compensate for the deficiency of physico-chemical analysis,
The residue detection of avilamycin plays the most important effect.
The basic factor affecting immunochemical analyses quality is specificity and the affinity of antibody, and these character are decided by again
The structure of immunity hapten molecule, the most immune haptenic MOLECULE DESIGN and synthesis are to produce specific antibody and set up little point
The step of the most basic and most critical of sub-residue of veterinary drug Fast Detection Technique.
Summary of the invention
It is an object of the invention to provide a kind of avilamycin artificial antigen and preparation method and application.
Avilamycin artificial antigen provided by the present invention is to build resisting of gained on the basis of avilamycin is haptenic
Former.
Described avilamycin hapten belongs to protection scope of the present invention, and its structure is shown in formula I.
Present invention also offers and prepare the haptenic method of described avilamycin.
Provided by the present invention prepare the haptenic method of described avilamycin, specifically can include the step of following A-C:
A. intermediate 1 is prepared according to the method comprising the steps (1)-(5):
(1) avilamycin is dissolved in dimethylformamide (DMF), is subsequently adding imidazoles, mixing, obtain I liquid；
Wherein, the proportioning of described avilamycin, described dimethylformamide (DMF) and described imidazoles is 1g:6ml:
0.47g；
(2) by tert-butyl chloro-silicane (t-BuMe2SiCl) it is dissolved in dimethylformamide (DMF), obtains II liquid；
Wherein, described tert-butyl chloro-silicane (t-BuMe2SiCl) with the proportioning of described dimethylformamide (DMF)
For 0.52g:2ml；
(3) described I liquid is mixed with described II liquid, 30 DEG C of stirring reaction 2h, obtain III liquid；
Wherein, the described avilamycin in described I liquid and the described tert-butyl chloro-silicane (t-in described II liquid
BuMe2SiCl) proportioning is 1g:0.52g；
Described I liquid is mixed particularly as follows: be added dropwise in described I liquid by described II liquid with described II liquid.
(4) use ethyl acetate (EtoAc) that described III liquid is extracted, separating ethyl acetate layer, MgSO4It is dried, subtracts
Pressure concentrates, and gained concentrate (slightly yellow dope) is dissolved in dichloromethane (CH2Cl2), obtain IV liquid；
(5) described IV liquid being carried out silica gel column chromatography, the silica gel granularity of employing is 200-300 mesh, and eluent is by volume ratio
Dichloromethane (CH for 95:52Cl2) and methanol (CH3OH) mix, use described eluent to collect symbol after carrying out eluting
The component of conjunction condition A, then carries out concentrating under reduced pressure, is vacuum dried (24h, lucifuge), obtains described intermediate 1；
The component of described eligible A is: at the mixed liquor of the dichloromethane using volume ratio as 95:5 and methanol as exhibition
Open agent, using granularity be 300-400 mesh silica gel as in the thin layer chromatography of fixing phase, Rf value is the component of Rf=0.3；
B. according to comprising the steps that the method for (6)-(9) is prepared intermediate 2 by described intermediate 1:
(6) described intermediate 1 is dissolved in dichloromethane (CH2Cl2After), be sequentially added into DMAP (DMAP),
Triethylamine and succinic anhydrides, obtain V liquid；
Wherein, described intermediate 1, described dichloromethane (CH2Cl2), described DMAP (DMAP), described three
The proportioning of ethamine and described succinic anhydrides is 0.5g:12ml:0.28g:0.45g:0.92g；
(7) described V liquid is refluxed 2.5h (solution is become brown, black from colourless) in 40 DEG C of water-baths, described in evaporated under reduced pressure
Dichloromethane (CH2Cl2), obtain VI liquid；
(8) use ether that described VI liquid is extracted, be filtered to remove insoluble matter, separate ether layer, first use mass fraction
It is HCl washing (2 times) of 3.6%, is washed with water and washs (2 times), then use MgSO4Being dried, concentrating under reduced pressure, by gained concentrate
(faint yellow dope) is dissolved in dichloromethane (CH2Cl2), obtain VII liquid；
(9) described VII liquid being carried out TLC separation, developing solvent is the dichloromethane of 95:5:5 by volume ratio
(CH2Cl2), oxolane (THF) and methanol (CH3OH) mix, fixing be mutually granularity be the silica gel of 300-400 mesh, collect
Rf value (Rf) is the zone of 0.5, with by the dichloromethane (CH that volume ratio is 1:12Cl2) and methanol (CH3OH) mix
Leacheate carries out drip washing, concentrating under reduced pressure, is vacuum dried (24h, lucifuge), obtains described intermediate 2；
C. according to comprising the steps that the method for (10)-(14) is prepared described compound by described intermediate 2:
(10) described intermediate 2 is dissolved in methanol (CH3OH), VIII liquid is obtained；
Wherein, described intermediate 2 and described methanol (CH3OH) proportioning is 0.3g:17ml；
(11) p-methyl benzenesulfonic acid is dissolved in methanol (CH3OH), IX liquid is obtained；
Wherein, described p-methyl benzenesulfonic acid and described methanol (CH3OH) proportioning is 0.26g:10ml；
(12) in 20-25 DEG C, being mixed with described IX liquid by described VIII liquid, stirring reaction 25min, 40 DEG C of concentrating under reduced pressure obtain
To X liquid；
Wherein, the described intermediate 2 in described VIII liquid with the proportioning of the described p-methyl benzenesulfonic acid in described IX liquid is
0.3g:0.26g；
Described VIII liquid is mixed with described IX liquid particularly as follows: described IX liquid is added dropwise to described VIII liquid under stirring
In.
(13) use ethyl acetate (EtoAc) that described X liquid is extracted, first with 2% (mass fraction) NaHCO3Solution
Wash 1 time, then wash 3 times with water, then use MgSO4It is dried, concentrating under reduced pressure, gained concentrate is dissolved in dichloromethane
(CH2Cl2), obtain XI liquid；
(14) described XI liquid being carried out TLC separation, developing solvent is the dichloromethane of 90:9.5:0.5 by volume ratio
(CH2Cl2), oxolane (THF) and acetic acid (HAc) mix, fixing be mutually granularity be the silica gel of 300-400 mesh, collect ratio
Shifting value (Rf) is the zone of 0.3, carries out drip washing, concentrating under reduced pressure by ethyl acetate (EtoAc), is vacuum dried (24h, lucifuge),
To described compound.
The avilamycin antigen building gained on the basis of avilamycin is haptenic falls within protection scope of the present invention.
Described avilamycin, for the antigen by described avilamycin hapten (Formulas I) Yu carrier protein couplet gained.At this
In one embodiment of invention, described carrier protein is specially bovine serum albumin (BSA) or ovalbumin (OVA).
Wherein, described avilamycin hapten (Formulas I) is 8.14:1 with the mol ratio of described carrier protein couplet.
The preparation method of described avilamycin antigen falls within protection scope of the present invention.
The preparation method of described avilamycin antigen, specifically can comprise the steps: described avilamycin hapten (formula
I) with carrier protein by amido link coupling, it is thus achieved that described avilamycin antigen.
In the present invention, described avilamycin antigen specifically prepares according to the method comprised the steps:
(a1) described avilamycin hapten (Formulas I) is dissolved in dimethylformamide (DMF), is subsequently adding 1-(3-
Dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) and N-hydroxy-succinamide (NHS), 20-25 DEG C of magnetic force stirs
Mix reaction 3h, obtain solution I；
Wherein, described avilamycin hapten (Formulas I), described dimethylformamide (DMF), described 1-(3-dimethylamino
Propyl group)-3-ethyl-carbodiimide hydrochloride (EDC), the proportioning of described N-hydroxy-succinamide (NHS) be 43.6mg:
1.5ml:26mg:25mg；
(a2) being placed in 0.1M carbonic acid buffer by described carrier protein, 4000rpm stirs 10mn, fully dissolves, obtains
Solution II；The pH of described 0.1M carbonic acid buffer is 9.6, and solvent is that water, solute and concentration are as follows: sodium carbonate 0.1mol/L, carbon
Acid hydrogen sodium 0.1mol/L；Described carrier protein is 50-67mg:3.5ml with the proportioning of described 0.1M carbonic acid buffer；
Wherein, if described carrier protein is bovine serum albumin (BSA), the most described bovine serum albumin (BSA) is with described
The proportioning of 0.1M carbonic acid buffer is 50mg:3.5ml；If described carrier protein is ovalbumin (OVA), the most described ovalbumin
(OVA) proportioning with described 0.1M carbonic acid buffer is 67mg:3.5ml；
(a3) described solution I and described solution II are mixed according to condition B, after mixing after stir in 20-25 DEG C of magnetic force
Mix reaction 24h, obtain solution III；
Described condition B is: described with described solution II of the described avilamycin hapten (Formulas I) in described solution I
The proportioning of 0.1M carbonic acid buffer is 43.6mg:3.5ml；
Wherein, described solution I and described solution II are mixed, is specially under the conditions of 4 DEG C, described solution I is dropwise added
Enter in described solution II, stirring while adding；
(a4) with phosphate buffer (0.01M PBS, pH=7.4), in 4 DEG C to described solution III stirring dialysis 3 days,
Obtain described avilamycin antigen；The solvent of described phosphate buffer is water, solute be potassium dihydrogen phosphate, disodium hydrogen phosphate,
Sodium chloride and potassium chloride；Described potassium dihydrogen phosphate concentration in described phosphate buffer is 0.27g/L, described phosphoric acid hydrogen two
Sodium concentration in described phosphate buffer is 1.42g/L, and described sodium chloride concentration in described phosphate buffer is
8g/L, described potassium chloride concentration in described phosphate buffer is 0.2g/L；The pH of described phosphate buffer is 7.4.
Described avilamycin hapten (Formulas I) or the application in following (a) or (b) of the described avilamycin antigen fall within
Protection scope of the present invention:
A () qualitatively or quantitatively detects avilamycin；
B () prepares avilamycin antibody.
The antibody utilizing described avilamycin antigen to prepare falls within protection scope of the present invention.
Described antibody can be polyclonal antibody, monoclonal antibody or antiserum.
Avilamycin hapten provided by the present invention, and described avilamycin antigen, synthetic method is simple, and purity is high
High with productivity, for the preparation of avilamycin antibody, and the detection of avilamycin drug residue has substantial worth.
Accompanying drawing explanation
" the Mass Spectrometer Method result of-o-succinoylAVM that Fig. 1 is avilamycin hapten 4.
Fig. 2 is the Mass Spectrometer Method result of BSA.
Fig. 3 is the Mass Spectrometer Method result of avilamycin antigen " avilamycin hapten+BSA ".
Detailed description of the invention
Experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
Avilamycin: lark prestige Science and Technology Ltd., its production code member is P-615N.
The haptenic preparation of embodiment 1, avilamycin and Structural Identification
One, the haptenic preparation of avilamycin
1, intermediate 5-o-t-BuMe2The synthesis of Si-R-4 〃-OH
(1) take 1.0g avilamycin (AVM), add 6mL dimethylformamide (DMF) and dissolve, add 0.47g imidazoles, mixing,
To I liquid.
(2) 0.52g tert-butyl chloro-silicane (t-BuMe is taken2SiCl) it is dissolved in 2mL DMF, obtains II liquid.
(3) mechanical agitation gained I liquid, is added dropwise over described II liquid, and 30 DEG C of stir about 2h obtain III liquid.
(4) in described III liquid add 100mL ethyl acetate (EtoAc), mixing, mixed liquor wash with water (3 times, every time
50mL), EtoAc layer, MgSO are separated4It is dried, concentrating under reduced pressure, gained concentrate (slightly yellow dope) is dissolved in appropriate dichloro
Methane (CH2Cl2), obtain IV liquid.
(5) described IV liquid being carried out silica gel column chromatography, the silica gel granularity of employing is 200-300 mesh, and eluent is by volume ratio
Dichloromethane (CH for 95:52Cl2) and methanol (CH3OH) mixing, flow velocity during eluting is 1～2/s, uses described
Eluent collects, after carrying out eluting, the component meeting following condition: in the dichloromethane with volume ratio as 95:5 and the mixing of methanol
Liquid as developing solvent, using granularity be 300-400 mesh silica gel as in the thin layer chromatography of fixing phase, Rf value is Rf=0.3's
Component.Then concentrating under reduced pressure, vacuum drying 24h (lucifuge), obtain intermediate 5-o-t-BuMe2Si-R-4〃-OH。
2, intermediate 5-o-t-BuM2Si-R-4 " the synthesis of-o-succinoyl
(1) the intermediate 5-o-t-BuM of step 1 synthesis is taken2Si-R-4 〃-OH 0.50g, adds 12mLCH2Cl2It is made to dissolve,
It is sequentially added into 0.28g dimethylamino naphthyridine (DMAP), 0.45g triethylamine and 0.92g succinic anhydrides, obtains V liquid.
(2) described V liquid is refluxed 2.5h in 40 DEG C of water-baths, solution from colourless become brown, black (with reaction raw materials without
Close), evaporated under reduced pressure CH2Cl2, obtain VI liquid.
(3) in described VI liquid, add 100mL ether, go in 250mL separatory funnel after being filtered to remove insoluble matter, separate
Ether layer, first washs 2 times with 100mL 3.6% (mass fraction) HCl, then washs 2 times with 100mL water, then use MgSO4Dry
Dry, concentrating under reduced pressure, by gained concentrate (faint yellow dope) with the addition of C H2Cl2Dissolve, obtain VII liquid.
(4) described VII liquid carrying out thin layer chromatography (TLC) separate, developing solvent is the dichloromethane of 95:5:5 by volume ratio
(CH2Cl2), oxolane (THF) and methanol (CH3OH) mix, fixing be mutually granularity be the silica gel of 300-400 mesh.TLC
Three zone occur, middle maximum zone (Rf value Rf is 0.5) is scraped, with being the dichloromethane of 1:1 by volume ratio
(CH2Cl2) and methanol (CH3OH) leacheate mixed carries out drip washing, concentrating under reduced pressure, vacuum drying 24h (lucifuge), obtains
Described intermediate 5-o-t-BuM2Si-R-4″-o-succinoyl。
3, the avilamycin hapten 4 " synthesis of-o-succinoylAVM
(1) by the intermediate 5-o-t-BuM of 2-in-1 for step one-tenth2"-o-succinoyl 0.30g joins 17mL first to Si-R-4
Alcohol makes it dissolve, and obtains VIII liquid.
(2) 0.26g p-methyl benzenesulfonic acid is dissolved in 10mL methanol, obtains IX liquid.
(3), under room temperature (20-25 DEG C) stirring, described IX liquid is added dropwise in described VIII liquid, continues stirring
25min, 40 DEG C are concentrated under reduced pressure to give X liquid.
(4) being washed by described X liquid with 80mL EtoAc, proceed in 250mL separatory funnel, first with 40mL 2%, (quality is divided
Number) NaHCO3Solution washs 1 time, then washs 3 times with 80mL water, then uses MgSO4It is dried, concentrating under reduced pressure, gained concentrate is used
The addition of C H2Cl2Dissolve, obtain XI liquid.
(5) described XI liquid carrying out TLC isolated and purified, developing solvent is the dichloromethane of 90:9.5:0.5 by volume ratio
(CH2Cl2), oxolane (THF) and acetic acid (HAc) mix, fixing be mutually granularity be the silica gel of 300-400 mesh, TLC shows
Show three zone, be that 0.3 middle zone scrapes by Rf value Rf, EtoAc drip washing, concentrating under reduced pressure final vacuum is dried (lucifuge) 24h,
Obtain described avilamycin hapten 4 "-o-succinoylAVM.
Reaction equation is as follows:
Two, the haptenic Structural Identification of avilamycin
Result is as it is shown in figure 1, "-o-succinoylAVM carries out Mass Spectrometer Method, M+:M+Na=to step one gained 4
995.5, M-:M-H=971.5.
Result shows its chemical structural formula shown in formula I, is avilamycin hapten.
Embodiment 2, the preparation of avilamycin artificial antigen and Structural Identification
One, the preparation of avilamycin artificial antigen
1, immunogenic synthesis
(1) hapten (Formulas I) 43.6mg embodiment 1 prepared is dissolved in 1.5mL dimethylformamide (DMF)
In, after being completely dissolved, it is sequentially added into 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) 26mg, N-hydroxyl
Butanimide (NHS) 25mg, room temperature (20-25 DEG C) magnetic agitation reaction 3h, obtain solution I.
(2) weighing 50mg bovine serum albumin (BSA), be dissolved in 3.5mL 0.1M carbonic acid buffer, 400rpm stirs
10min, fully dissolves, and obtains solution II.
Wherein, the pH of described 0.1M carbonic acid buffer is 9.6, and solvent is that water, solute and concentration thereof are as follows:
Na2CO31.59g/L, NaHCO3 2.94g/L。
(3) take above-mentioned solution I, be added dropwise in above-mentioned solution II under ice-water bath (4 DEG C) environment, stirring while adding,
Room temperature (20-25 DEG C) magnetic agitation reaction 24h, obtains solution III.
(4) described solution III is loaded 1 the clean bag filter of distilled water flushing (15cm), 1L 0.01M PBS (pH7.4)
(formula: potassium dihydrogen phosphate 0.27g, disodium hydrogen phosphate 1.42g, sodium chloride 8g, potassium chloride 0.2g add deionized water about 800mL and fill
Dividing stirring and dissolving, be subsequently adding concentrated hydrochloric acid and adjust pH to 7.4, last constant volume, to 1L, is 0.01M pH7.4PBS) dialyse 3 days, 4
DEG C stirring dialysis, change dialysis solution every day 3 times, dialysis product 4500rpm be centrifuged 6min, 0.5ml/ pipe subpackage, by antigen compile
Number ,-20 DEG C save backup.
2, the synthesis of coating antigen
(1) hapten (Formulas I) 43.6mg embodiment 1 prepared is dissolved in 1.5mL dimethylformamide (DMF)
In, after being completely dissolved, it is sequentially added into 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) 26mg, N-hydroxyl
Butanimide (NHS) 25mg, room temperature (20-25 DEG C) magnetic agitation reaction 3h, obtain solution I.
(2) weighing 67.0mg ovalbumin (OVA), (formula is same to be dissolved in 3.5mL 0.1M carbonic acid buffer pH=9.6
On) in, 400rpm stirs 10min, fully dissolves, obtains solution II.
(3) take above-mentioned solution I, be added dropwise in above-mentioned solution II under ice-water bath (4 DEG C) environment, stirring while adding,
Room temperature (20-25 DEG C) magnetic agitation reaction 24h, obtains solution III.
(4) described solution III is loaded 1 the clean bag filter of distilled water flushing (15cm), 1L 0.01M PBS (pH7.4)
(formula is ibid) dialyses 3 days, 4 DEG C of stirring dialysis, changes dialysis solution every day 3 times, and dialysis product 4500rpm is centrifuged 6min,
0.5ml/ pipe subpackage, numbers antigen, and-20 DEG C save backup.
Two, the qualification of avilamycin artificial antigen
As shown in Figure 2 and Figure 3, immunogen MALDI-TOF-MS qualification result display coupling ratio is: R=(74205.876-
66291.676)/972.5=8.14.I.e. in immunogen, described avilamycin hapten (Formulas I) and bovine serum albumin (BSA)
The mol ratio of coupling is 8.14:1.
Embodiment 3, avilamycin artificial antigen immune animal prepare antiserum
One, animal immune
With the avilamycin artificial antigen " avilamycin-BSA " of step embodiment 2 acquisition as immunogen immune New Zealand
White Rabbit, each immunizing dose is 100～200 μ g, and immunization ways is double shoulder and the subcutaneous multi-point injection of rear thigh, each region
With about the immunogen of 1/4.By immunogen normal saline dilution when head exempts from, then carry out 1:1 (body with incomplete Freund's adjuvant
Long-pending than) be mixed and made into emulsifying agent, at interval of within 2 weeks, taking after same dose immunogen adds equal-volume incomplete Freund's adjuvant mixing and emulsifying
Booster immunization once, after using this mode to add altogether and exempt from 3 times, is spaced and within 3～4 weeks, takes same dose immunogen Jia Fushi again and not exclusively help
Agent carries out final immunization, and arteria auricularis takes blood examination and surveys antibody titer.Final immunization uses carotid artery blood-letting, every rabbit after 7-10 days
Can obtain blood about 100-120ml, the blood taken is placed 5～6 hours at 4 DEG C of refrigerators, is then centrifuged 10min with 5000rpm, separates
Serum.
Two, antiserum titre measures
Use the antibody titer of indirect elisa method determination step one gained serum, specific as follows:
1) it is coated: in 96 hole ELISA Plate, add " avilamycin-OVA " solution that 100 μ L concentration are 2 μ g/mL (with being coated
Buffer is diluted), the comparison of not envelope antigen is set simultaneously, 4 DEG C are coated overnight, wash 3 times with PBS.
It is coated buffer: (solvent is water, solute and dense for the sodium carbonate-bicarbonate buffer of pH9.6,0.05mol/L
Spend as follows: Na2CO31.59g/L and NaHCO3 2.93g/L)。
2) close: add the confining liquid in 150 μ L/ holes, hatch 2h at 37 DEG C, abandon confining liquid, wash 3 times, pat dry.It is placed in 4
DEG C Refrigerator store is standby.
Confining liquid: delay containing 0.5% (volumn concentration) calf serum, 3% (3g/100ml) caseic phosphate
Rush liquid, pH7.4.
3) add testing sample: draw different dilution test serum 100 μ l, add in corresponding ELISA Plate, incubate for 37 DEG C
Educate 30min, wash plate 4 times, pat dry.
The comparison of non-immunized rabbit anteserum is set simultaneously；The comparison (negative control hole) of detected sample is replaced with PBS.
4) ELIAS secondary antibody is added: take HRP mark goat anti-rabbit igg antibody (Jackson ImmunoResearch company, article No. 111-
035-003), by volume 1:5000 times dilute after, 100 μ l/ holes, hatch 20 to 30min for 37 DEG C, wash 4 times, pat dry.
5) colour developing: 20 × TMB is diluted to 1 × TMB, is added by 100 μ l/ holes, 37 DEG C of colour developing 15-30min.
6) terminate: add stop buffer (2M H2SO4) 50 μ l/ holes.
7) reading: measure each hole OD value with 450nm Single wavelength, (to replace the right of testing sample with PBS with negative control hole
According to) ratio (P/N) of OD value is limited, as the critical point being judged as serum titer more than 2.1.
ELISA result decision method: with P/N > 2.1 serum maximum dilution multiple represent.
Result shows that the antibody titer in serum is 1:16000.
Embodiment 4, avilamycin enzyme linked immunological kit detection avilamycin
One, the assembling of avilamycin enzyme linked immunological kit
1, the composition of avilamycin enzyme linked immunological kit includes the following:
(1) avilamycin standard substance working solution: 6 bottles, 1.5mL/ bottle, concentration is 0ng/ml, 0.5ng/ml, 1.5ng/ml,
4.5ng/ml、13.5ng/ml、40.5ng/ml；
(2) avilamycin ELISA Plate: 1 piece (8 hole × 12), for be coated that embodiment 2 prepares " avilamycin-
OVA " ELISA Plate.
(3) enzyme marker diluent: 1 bottle (10mL), for PBS.
(4) enzyme marker working solution: 1 bottle (11 ×, 1mL), wherein enzyme marker is specially horseradish peroxidase labelling
The antibody of anti-avilamycin, described antibody is the antiserum that embodiment 3 prepares.
(5) sample diluting liquid: 1 bottle (50ml), for the PBS of 0.1M pH7.4.
(6) cleaning mixture: 1 bottle (20 ×, 25mL), for the PBST solution of 0.01mol/L pH7.4.
(7) substrate A liquid, substrate B liquid each 1 bottle (7mL).Wherein, substrate A is 2% urea peroxide aqueous solution.Substrate B is 1%
Tetramethyl biphenyl amine aqueous solution.
(8) stop buffer: 1 bottle (7mL), for 2mol/L H2SO4Solution.
(9) cover plate film；
(10) valve bag.
2, the equipment needed and do not provide and material
(1) equipment
Microplate reader (detection wavelength 450nm, reference wavelength 630nm), vortex oscillator, centrifuge (4000g), Nitrogen evaporator,
Micropipettor, timer.
(2) reagent
25% methanol buffer: accurately measure 5mL methanol and 15mL sample diluting liquid, mix standby.
Acetonitrile (analytical pure), normal hexane (analytical pure).
3, storage
This test kit is stored in 2-8 DEG C, is sure not freezing, 1 year effect duration.
The ELIAS strip being finished is not made to seal, 2-8 DEG C of preservation.
4, test kit Cleaning Principle
The antigen-specific sexual competition enzyme marker that avilamycin in sample is fixing with in ELISA Plate, passes through substrate for enzymatic activity
Colour developing, carrys out the content of avilamycin in judgement sample according to the depth of colour developing.Content is few, and colour developing is deep；Content is many, develops the color shallow.
Two, the using method of avilamycin enzyme linked immunological kit
1, sample pre-treatments
(1) raw milk method one (coefficient of dilution: 2)
A) take the fresh raw milk of 1mL in 10mL centrifuge tube, add 3mL methanol, high speed whirling motion 1min；B) 4000g is centrifuged
10min；C) taking 1mL supernatant in 4mL centrifuge tube, in 50 DEG C of water-baths, nitrogen dries up；D) 0.5mL 25% methanol buffer is added
(see step one 2), abundant whirling motion 1min；E) take 50 μ L to detect.
(2) raw milk method two (coefficient of dilution: 10)
A) take the fresh raw milk of 1mL in 10mL centrifuge tube, add 3mL methanol, abundant whirling motion 1min；B) 4000g is centrifuged
10min；C) take 300 μ L of supernatant liquid in new centrifuge tube, add 600 μ L sample diluents, abundant whirling motion 1min；D) 50 μ L are taken
Detect.
(3) Carnis Gallus domesticus, Carnis Sus domestica, Hepar Gallus domesticus (coefficient of dilution: 4)
A) tissue sample after 2 ± 0.01g homogenizing is accurately weighed in 50mL centrifuge tube；B) be sequentially added into 3mL normal hexane,
6mL acetonitrile, after whirlpool dissipates the most one by one, high speed whirling motion 1min；C) 4000g is centrifuged 10min, discards upper strata normal hexane；D) 2.7mL is taken
Supernatant, in 4mL centrifuge tube, is previously added 0.4g scarvenger A and 0.5g scarvenger B, high speed whirling motion immediately in centrifuge tube
1min；E) 4000g is centrifuged 5min；F) taking 1mL supernatant in 4mL centrifuge tube, in 60 DEG C of water-baths, nitrogen dries up；G) Carnis Sus domestica, chicken
Meat sample: be initially charged 250 μ L methanol, adds 750 μ L sample diluents after whirling motion 5s；Hepar Gallus domesticus sample: be initially charged 150 μ L methanol,
850 μ L sample diluents are added after whirling motion 5s；H) high speed whirling motion 1min；I) take 50 μ L to detect.
2, detecting step
(1) lath is inserted on ELISA Plate frame, and record the position of each standard substance and sample, it is proposed that all do diplopore and put down
OK, after untapped lath valve bag seals, it is stored in immediately in 2-8 DEG C of environment；
(2) avilamycin standard substance working solution (or testing sample solution) of the 50 each concentration of μ L is separately added into the mark of correspondence
In quasi-product (or testing sample hole)；
(3) in every hole, add 80 μ L enzyme marker working solution (see step 1)；
(4) building cover plate film, vibration ELISA Plate 10s, fully mixes gently, and under room temperature (25 ± 2 DEG C), lucifuge is reacted
40min；
(5) cover plate film is opened；
(6) outwelling liquid in plate hole, add 260 μ L wash operating solutions in every hole, fully washing 4 times, soaks 15-every time
30s；
(7) outwell liquid in plate hole, ELISA Plate is inverted in absorbent paper, pats dry；
(8) (substrate A liquid, substrate B liquid are by body to add the mixed liquor of 100 μ L substrate A liquid and substrate B liquid immediately in every hole
Long-pending 1:1 mixing, it is necessary to fully mixing, mixed liquor uses in 5min, it is to avoid use metal to contain, stir reagent)；
(9) building cover plate film, vibration ELISA Plate 10s, fully mixes gently, under room temperature (25 ± 2 DEG C), and lucifuge reaction 10-
15min；
(10) opening cover plate film, add 50 μ L stop buffers in every hole, vibration ELISA Plate 10s, fully mixes gently；
(11) after terminating, the interior microplate reader of 5min reads ELISA Plate absorbance under dual wavelength 450nm, 630nm.
3, result calculates or judges
(1) mean absorbance values of each standard substance (or testing sample), divided by zero standard (concentration is the standard substance of 0ng/ml)
Absorbance, is multiplied by 100, can obtain the percentage ratio of absorbance corresponding to each standard substance, i.e. percentage absorbance:
(2) with the percentage absorbance of each standard substance as vertical coordinate, draw with corresponding avilamycin concentration for abscissa
Standard curve.
(3) the percentage absorbance of testing sample is substituted into standard curve equation, the concentration that testing sample is corresponding can be drawn,
Being multiplied by the extension rate of respective sample again, side obtains the actual content of avilamycin in raw sample to be measured.
Three, avilamycin enzyme linked immunological kit detection avilamycin
1, specific detection
The specificity of avilamycin enzyme linked immunological kit is next really by carrying out cross reaction test with corresponding material
Fixed.Cross reaction is the least, and specificity is the best.
Avilamycin and other analog (ivermectin, angstrom general rhzomorph, doractin) are done serial dilution respectively, respectively
Operate according to as above step 22, substitute " avilamycin therein with the serial dilutions of avilamycin and other analog
Standard substance working solution ", make standard curve, and on curve, find out respective 50% inhibition concentration (IC50), concrete grammar is as follows:
Obtain the Y value avilamycin concentration (ng/mL) equal to 50% correspondence, i.e. IC50Value.With following formula calculate test kit to Ah
Dimension rhzomorph and the cross reacting rate of each analog.
Result is as shown in table 1, from table 1 it follows that the friendship that avilamycin enzyme linked immunological kit is to various analogs
Fork response rate is respectively less than 1%.This explanation avilamycin enzyme linked immunological kit has high specificity to avilamycin, can have
The interference getting rid of other analog of effect, can be specifically designed to the detection of avilamycin.
The specificity of table 1 avilamycin enzyme linked immunological kit
| Medicine name | Cross reacting rate (%) |
| Avilamycin | 100 |
| Ivermectin | ＜ 1 |
| Angstrom general rhzomorph | ＜ 1 |
| Doractin | ＜ 1 |
2, the lowest detectable limit of different samples measures
Measure respectively and use avilamycin enzyme linked immunological kit that avilamycin in raw milk, Hepar Gallus domesticus, Carnis Sus domestica and Carnis Gallus domesticus is entered
Lowest detectable limit during row detection.Concrete grammar such as step 2.
Result shows, uses the sample-pretreating method in above step 22 (1) to process raw milk, the lowest detection recorded
Limit up to 2ng/ml (in i.e. every ml raw milk, the avilamycin containing 2ng can be detected, lower same)；Use above step 2 (2)
In sample-pretreating method process raw milk, the lowest detectable limit recorded is up to 5ng/ml；Use the sample in above step 2 (3)
Product pre-treating method processes Hepar Gallus domesticus, Carnis Sus domestica and Carnis Gallus domesticus, and the lowest detectable limit recorded is up to 2ng/g.
3, the mensuration of error between avilamycin enzyme linked immunological kit plate inner panel
Measure error between error and plate in the plate of avilamycin enzyme linked immunological kit respectively.Concrete grammar such as step 2.
Result shows, in the plate of test kit absorbance, error is less than 5%, and between plate, error is less than 10%.
4, the determination of recovery rates of avilamycin enzyme linked immunological kit detection avilamycin
Measure the response rate using avilamycin enzyme linked immunological kit detection avilamycin.Concrete grammar such as step 2.
Result shows, the response rate scope using avilamycin enzyme linked immunological kit detection avilamycin is 90% ±
30%.
5, the sensitivity determination of avilamycin enzyme linked immunological kit
Measure the sensitivity of avilamycin enzyme linked immunological kit.Concrete grammar such as step 2.
Result shows, the sensitivity of avilamycin enzyme linked immunological kit is 0.5ppb, standard curve range 0.5ppb-
40.5ppb (note: ppb=μ g/kg).
Claims (9)
1. a compound, its structure shown in formula I:
2. the method for compound described in preparation claim 1, including the step of following A-C:
A. intermediate 1 is prepared according to the method comprising the steps (1)-(5):
(1) avilamycin is dissolved in dimethylformamide, is subsequently adding imidazoles, mixing, obtain I liquid；
Wherein, the proportioning of described avilamycin, described dimethylformamide and described imidazoles is 1g:6ml:0.47g；
(2) tert-butyl chloro-silicane is dissolved in dimethylformamide, obtains II liquid；
Wherein, described tert-butyl chloro-silicane is 0.52g:2ml with the proportioning of described dimethylformamide；
(3) described I liquid is mixed with described II liquid, 30 DEG C of reaction 2h, obtain III liquid；
Wherein, the proportioning of the described avilamycin in described I liquid and the described tert-butyl chloro-silicane in described II liquid is
1g:0.52g；
(4) use ethyl acetate that described III liquid is extracted, separating ethyl acetate layer, MgSO4Being dried, concentrating under reduced pressure, by institute
Obtain concentrate and be dissolved in dichloromethane, obtain IV liquid；
(5) described IV liquid being carried out silica gel column chromatography, the silica gel granularity of employing is 200-300, and eluent is 95:5 by volume ratio
Dichloromethane and methanol mixed form, use described eluent after carrying out eluting, to collect the component of eligible A, then carry out
Concentrating under reduced pressure, vacuum drying, obtain described intermediate 1；
The component of described eligible A is: at the mixed liquor of the dichloromethane using volume ratio as 95:5 and methanol as developing solvent,
Using granularity be 300-400 mesh silica gel as in the thin layer chromatography of fixing phase, Rf value is the component of Rf=0.3；
B. according to comprising the steps that the method for (6)-(9) is prepared intermediate 2 by described intermediate 1:
(6), after described intermediate 1 being dissolved in dichloromethane, it is sequentially added into DMAP, triethylamine and succinic anhydrides,
Obtain V liquid；
Wherein, described intermediate 1, described dichloromethane, described DMAP, described triethylamine and described succinic anhydrides
Proportioning be 0.5g:12ml:0.28g:0.45g:0.92g；
(7) described V liquid is refluxed 2.5h in 40 DEG C of water-baths, dichloromethane described in evaporated under reduced pressure, obtain VI liquid；
(8) use ether that described VI liquid is extracted, separate ether layer, first with the HCl washing that mass fraction is 3.6%, then
Wash with water, then use MgSO4It is dried, concentrating under reduced pressure, gained concentrate is dissolved in dichloromethane, obtains VII liquid；
(9) described VII liquid being carried out TLC separation, developing solvent is the dichloromethane of 95:5:5, oxolane by volume ratio
Form with methanol mixed, fixing be mutually granularity be the silica gel of 300-400 mesh, collecting Rf value is the zone of 0.5, with by volume ratio
Carry out drip washing, concentrating under reduced pressure, vacuum drying for the dichloromethane of 1:1 and the leacheate of methanol mixed, obtain described centre
Body 2；
C. according to comprising the steps that the method for (10)-(14) is prepared described compound by described intermediate 2:
(10) described intermediate 2 is dissolved in methanol, obtains VIII liquid；
Wherein, described intermediate 2 is 0.3g:17ml with the proportioning of described methanol；
(11) p-methyl benzenesulfonic acid is dissolved in methanol, obtains IX liquid；
Wherein, described p-methyl benzenesulfonic acid is 0.26g:10ml with the proportioning of described methanol；
(12) in 20-25 DEG C, being mixed with described IX liquid by described VIII liquid, react 25min, 40 DEG C are concentrated under reduced pressure to give X liquid
Wherein, the described intermediate 2 in described VIII liquid is 0.3g with the proportioning of the described p-methyl benzenesulfonic acid in described IX liquid:
0.26g；
(13) using ethyl acetate to extract described X liquid, first is the NaHCO of 2% with mass fraction3Solution washs, then with water
Washing, then uses MgSO4It is dried, concentrating under reduced pressure, gained concentrate is dissolved in dichloromethane, obtains XI liquid；
(14) described XI liquid being carried out TLC separation, developing solvent is the dichloromethane of 90:9.5:0.5, tetrahydrochysene by volume ratio
Furan and acetic acid mix, fixing be mutually granularity be the silica gel of 300-400 mesh, collecting Rf value is the zone of 0.3, uses acetic acid
Ethyl ester carries out drip washing, concentrating under reduced pressure, vacuum drying, obtains described compound.
3. avilamycin antigen, for the antigen by compound described in claim 1 Yu carrier protein couplet gained.
Avilamycin antigen the most according to claim 3, it is characterised in that: described carrier protein be bovine serum albumin or
Ovalbumin.
5. according to the avilamycin antigen described in claim 3 or 4, it is characterised in that: compound described in claim 1 is with described
The mol ratio of carrier protein couplet is 8.14:1.
6. the preparation method of arbitrary described avilamycin antigen in claim 3-5, comprises the steps: claim 1
Described compound and carrier protein are by amido link coupling, it is thus achieved that described avilamycin antigen.
Method the most according to claim 6, it is characterised in that: described method comprises the steps:
(a1) by compound dissolution described in claim 1 in dimethylformamide, be subsequently adding 1-(3-dimethylamino-propyl)-
3-ethyl-carbodiimide hydrochloride and N-hydroxy-succinamide, 20-25 DEG C of reaction 3h, obtain solution I；
Described compound, described dimethylformamide, described 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride,
The proportioning of described N-hydroxy-succinamide is 43.6mg:1.5ml:26mg:25mg；
(a2) described carrier protein is dissolved in 0.1M carbonic acid buffer, obtains solution II；
Described carrier protein is 50-67mg:3.5ml with the proportioning of described 0.1M carbonic acid buffer；
(a3) described solution I and described solution II are mixed according to condition B, after mixing after in 20-25 DEG C react 24h,
To solution III；
Described condition B is: the described compound in described solution I is joined with the described 0.1M carbonic acid buffer in described solution II
Ratio is 43.6mg:3.5ml；
(a4) with phosphate buffer, in 4 DEG C, described solution III is dialysed 3 days, obtain described avilamycin antigen.
8. in compound described in claim 1 or claim 3-5 arbitrary described avilamycin antigen at following (a) or (b)
In application:
A () qualitatively or quantitatively detects avilamycin；
B () prepares avilamycin antibody.
9. utilize the antibody that in claim 3-5 prepared by arbitrary described avilamycin antigen.
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| CN106771154A (en) * | 2016-11-29 | 2017-05-31 | 百奥森（江苏）食品安全科技有限公司 | The detection card and its detection method of AVM in a kind of detection tableware |
| CN108794507A (en) * | 2017-04-26 | 2018-11-13 | 北京维德维康生物技术有限公司 | A kind of rifaximin haptens, artificial antigen and the preparation method and application thereof |
| CN109180760A (en) * | 2018-08-30 | 2019-01-11 | 华中农业大学 | The monoclonal antibody and its application of a kind of ivermectin derivative and anti-Avermectins medicine |
| CN110618269A (en) * | 2019-09-04 | 2019-12-27 | 北京勤邦生物技术有限公司 | Preparation and application of emamectin benzoate monoclonal antibody |
| CN110646607A (en) * | 2019-07-12 | 2020-01-03 | 广东工业大学 | Mesoporous silica-coated positive charge nanogold coupled antibody and preparation method and application thereof |
| CN113817008A (en) * | 2021-07-15 | 2021-12-21 | 湖州师范学院 | Preparation method and application of novel succinyl sixteen-membered macrolide |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106771154A (en) * | 2016-11-29 | 2017-05-31 | 百奥森（江苏）食品安全科技有限公司 | The detection card and its detection method of AVM in a kind of detection tableware |
| CN108794507A (en) * | 2017-04-26 | 2018-11-13 | 北京维德维康生物技术有限公司 | A kind of rifaximin haptens, artificial antigen and the preparation method and application thereof |
| CN109180760A (en) * | 2018-08-30 | 2019-01-11 | 华中农业大学 | The monoclonal antibody and its application of a kind of ivermectin derivative and anti-Avermectins medicine |
| CN109180760B (en) * | 2018-08-30 | 2021-09-17 | 华中农业大学 | Ivermectin derivative, monoclonal antibody of anti-avermectin drug and application of monoclonal antibody |
| CN110646607A (en) * | 2019-07-12 | 2020-01-03 | 广东工业大学 | Mesoporous silica-coated positive charge nanogold coupled antibody and preparation method and application thereof |
| CN110618269A (en) * | 2019-09-04 | 2019-12-27 | 北京勤邦生物技术有限公司 | Preparation and application of emamectin benzoate monoclonal antibody |
| CN113817008A (en) * | 2021-07-15 | 2021-12-21 | 湖州师范学院 | Preparation method and application of novel succinyl sixteen-membered macrolide |
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