CN103360328A - Desoxyquinocetone hapten, and preparation method and application thereof - Google Patents
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Abstract
The invention discloses a desoxyquinocetone hapten, and a preparation method and an application thereof. A structure of the desoxyquinocetone hapten is shown as formula I. The desoxyquinocetone hapten immunizes an animal after being coupled with protein to obtain specific antibody for desoxyquinocetone. The antibody can be used for detecting residues of desoxyquinocetone in an animal product rapidly. The desoxyquinocetone hapten provided by the invention provides a novel material basis for establishing a fast, simple, cheap, sensitive and specific screening method for desoxyquinocetone.
Description
Technical field
The invention belongs to technical field of biochemical industry, be specifically related to a kind of desoxyquinocetone haptens and preparation method thereof and its application.
Background technology
Quinocetone (Quinocetone) molecular formula C
18H
14N
2O
3, the Shu quinoxaline medicine is that the livestock and poultry of Lanzhou Livestock and Animal Drug Inst., Chinese Academy of Agricultural Science development are antibiotic, antidiarrheal, growth promotion new drug, is the in the world pioneering new veterinary drug of a class of China.Such medicine has restraining effect to multiple pathogen enterobacteria (particularly Gram-negative bacteria), can obviously reduce the livestock and poultry diarrhea incidence, promotes to grow and improve food conversion ratio.The many countries and regions that comprise at present China all are widely used as animal feedstuff additive.Quinocetone is the widely used while in livestock industry, because the livestock industry producer is unfamiliar with physico-chemical property and the pharmacological toxicology characteristics of medicine, there is certain abuse condition, cause in the livestock product Quinocetone and the main metabolites desoxyquinocetone is residual exceeds standard, people healthy caused the serious potential danger side of body.The characteristics such as the while is difficult for storing, perishable owing to animal product has, in order to ensure the healthy of human consumer, in the urgent need to setting up a kind of quick, easy, inexpensive, sensitive, special Quinocetone and the screening method of main metabolites desoxyquinocetone thereof.
Summary of the invention
The purpose of this invention is to provide a kind of desoxyquinocetone haptens and preparation method thereof and its application.
Desoxyquinocetone haptens provided by the invention has the chemical structure shown in the formula I:
The haptenic preparation method of desoxyquinocetone provided by the invention comprises the steps (1) and (2):
(1) with mequindox and Sodium Hydrosulphite in 60 ℃ of reactions 2 hours, namely get 3-methyl-2-acetyl quinoxaline; The mol ratio of described mequindox and Sodium Hydrosulphite is 1:1.8;
(2) with 3-methyl-2-acetyl quinoxaline and p formyl benzoic acid under the alkaline catalysts effect, in 40 ℃ of reaction 12h, room temperature reaction spends the night again, namely gets the desoxyquinocetone haptens; The mol ratio of described 3-methyl-2-acetyl quinoxaline and p formyl benzoic acid is 1:1.2.
In the described step (1), need first mequindox to be dissolved in described solvent, add again Sodium Hydrosulphite.
The described heating for dissolving that is dissolved as; Be specially 30 ℃ of heating for dissolving.
In the described step (2), described room temperature is specially 25-27 ℃; Described reaction is spent the night and is specially reaction 12-14 hour.
In the described step (1), described reaction is carried out in solvent, and described solvent is the mixed solvent of tetrahydrofuran (THF), 100% ethanol and distilled water, and the volume ratio of described tetrahydrofuran (THF), 100% ethanol and distilled water is 2:10:1; In the described step (2), described reaction is carried out in solvent, and described solvent is the mixture of tetrahydrofuran (THF), second alcohol and water; In the described step (2), described alkaline catalysts is sodium hydroxide.
In the described step (1), also comprise concentration process and purge process after described reaction is complete.
Described concentration process is specially that rotary evaporation removes described solvent under 0.1MP, 50 ℃ of conditions.
Described purge process is specially with distilled water and chloroform extraction, and the dry organic phase that merges removes solvent and get final product.
In the described purge process, the volume ratio of described distilled water and trichloromethane is 4:9; Described extraction is specially 3 times; Described drying is specially uses anhydrous sodium sulfate drying.
In the described step (2), also comprise purge process after described reaction is complete.
Described purge process is specially filtration, product and cleans, is drying to obtain with hot ethanol and hot acetone successively.
In the described purge process, described hot ethanol and hot acetone refer to 35 ℃ ethanol and acetone; The volume number of described hot ethanol and hot acetone minute respectively is 50ml; Described cleaning is specially cleans 3 times; Described drying is specially uses anhydrous sodium sulfate drying.
Desoxyquinocetone haptens provided by the invention or the desoxyquinocetone haptens that directly obtained by described method are as haptenic purposes.
In the described purposes, describedly specifically refer to obtain the specific antibody for the Quinocetone meta-bolites with immune animal behind the described hapten conjugation albumen as haptenic purposes; Described Quinocetone meta-bolites specifically refers to desoxyquinocetone.
Desoxyquinocetone haptens provided by the invention or the purposes of desoxyquinocetone haptens in detecting desoxyquinocetone that is directly obtained by described method.
Desoxyquinocetone haptens provided by the invention or the purposes of desoxyquinocetone haptens in preparation detection desoxyquinocetone product that is directly obtained by described method.
Another object of the present invention provides a kind of antigen, the desoxyquinocetone haptens that directly obtains for described desoxyquinocetone haptens or by described method and the conjugate of bovine serum albumin.
Also purpose of the present invention provides a kind of polyclonal antibody, and described polyclonal antibody is that the conjugate of the desoxyquinocetone haptens that directly obtains with described desoxyquinocetone haptens or by described method and bovine serum albumin obtains after as antigen-immunized animal.
A further object of the present invention provides a kind of test kit of specific detection desoxyquinocetone, and described test kit comprises following 1)-9):
1) carbonate buffer solution:
Na
2CO
3 1.59g/L
NaHCO
3 2.93g/L
Solvent is distilled water;
2) the desoxyquinocetone haptens that directly obtains with the described desoxyquinocetone haptens of claim 1 or by any described method of claim 2-5 and the conjugate of ovalbumin are as envelope antigen;
3) confining liquid:
Calf serum 50ml/L
Sucrose 50g/L
Casein 2.5g/L
Na
2HPO
4·12H
2O 5.8g/L
NaH
2PO
4·2H
2O 0.593g/L
Proclin300 300μl/L
Solvent is distilled water;
4) phosphate buffered saline buffer:
Na
2HPO
4.12H
2O 3.12g/L
NaH
2PO
4.2H
2O 1.76g/L
Solvent is distilled water;
5) HRP-goat anti-rabbit igg;
6) substrate buffer solution:
Na
2HPO
4.12H
2O 36.8g/L
Citric acid 9.33g/L
Solvent is distilled water;
7) 3,3', 5,5'-tetramethyl biphenyl amine aqueous solution:
TMB 96.15mg/L
Ethylene glycol 38.46mL/L
Massfraction is 30% hydrogen peroxide 961 μ L/L
Na
2HPO
4.12H
2O 35.4g/L
Citric acid 8.97g/L
Solvent is distilled water;
8) H
2SO
4Solution:
Massfraction is 98% vitriol oil 2mol/L
Solvent is distilled water;
9) polyclonal antibody that the desoxyquinocetone haptens that directly obtains with the described desoxyquinocetone haptens of claim 1 or by any described method of claim 2-5 and the conjugate of bovine serum albumin obtain after as antigen-immunized animal.
Desoxyquinocetone synthesis of semiantigen of the present invention is simple, purity and productive rate are high, can be directly and behind the albumen coupling immune animal produce specific antibody for desoxyquinocetone, this antibody can be used for the residual of desoxyquinocetone in the rapid detection animal product.
Description of drawings
Fig. 1 is the haptenic chemical structural drawing of desoxyquinocetone.
Fig. 2 is the haptenic mass spectrum of desoxyquinocetone.
Fig. 3 is the haptenic nuclear magnetic spectrogram of desoxyquinocetone.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
Used solvent is other reagent of analytical pure level among the following embodiment.
Embodiment 1, desoxyquinocetone are haptenic synthetic
Fig. 1 seen in the haptenic chemical structural formula of desoxyquinocetone, and chemical name is the 2-(3-(4-carboxyphenyl) acryloyl)-3-Jia based quinoxaline, its building-up process is as described below.
(1) takes off the dioxy reaction
Mequindox generates 3-methyl-2-acetyl quinoxaline after taking off dioxy, and reaction formula is as follows:
The detailed process of taking off the dioxy reaction is:
Take by weighing the 10g mequindox, add the 40mL tetrahydrofuran (THF), the 200mL dehydrated alcohol, 20mL distilled water 30 ℃ of lower stirring heating dissolvings, adds the 9g Sodium Hydrosulphite, keeps solution temperature at 60 ℃, reacts to be cooled to room temperature (25-27 ℃) after 2 hours; 0.1MP about and rotate under 50 ℃ of conditions and is evaporated to about 20mL, add 40mL distilled water, with 90mL chloroform extraction three times, the merging organic phase; Obtain filtrate with suction filtration after the anhydrous sodium sulfate drying organic phase, rotary evaporated to dryness about 0.1MP and under 50 ℃ of conditions obtains 7.8g tawny 3-methyl-2-acetyl quinoxaline crude product, and is for subsequent use.
(2) formation of spacerarm reaction
The reaction formula of the formation reaction of spacerarm is as follows:
The haptenic synthetic detailed process of desoxyquinocetone is:
In the round-bottomed flask of a 500mL, add 3-methyl-2-acetyl quinoxaline 7.8g, p formylbenzoic acid (available from Li Deshi chemical company) 5.3g and sodium hydroxide 0.9g; in the 100ml dehydrated alcohol, stir, heat to 40 ℃; reaction 12h; then the lower reaction of room temperature (25-27 ℃) is spent the night, and namely reacts 12 hours.
Behind the reaction solution suction filtration, the mixture in the suction funnel respectively to be cleaned 3 times with 35 ℃ of dehydrated alcohols and 35 ℃ of acetone successively, every quantity of solvent all over using is 50ml; The product that will clean in suction funnel is transferred in the round-bottomed flask, again through anhydrous sodium sulphate (when dry anhydrous sodium sulphate do not mix with product or contact) drying after 6 hours, remove anhydrous sodium sulphate, with the liquid-solid mixture suction filtration that changes in the suction funnel, the yellow block product of gained is the desoxyquinocetone haptens in the suction funnel, yield 59% (calculation formula is as follows).Gained desoxyquinocetone haptens can be directly and behind the albumen coupling immune animal produce specific antibody for Quinocetone main metabolites desoxyquinocetone.
The calculation of yield formula:
(1) productive rate=haptenic actual mass/haptenic Theoretical Mass * 100%
(2) quality of haptenic Theoretical Mass=reactant mequindox * 318/219
Illustrate: 318 are haptenic molar mass; 219 is the molar mass of mequindox.
(3) the haptenic evaluation of desoxyquinocetone
1, Mass Spectrometric Identification
Mass Spectrometric Identification the results are shown in Figure 2.The peak of m/z319 is the quasi-molecular ion peak [M+H] of synthetic product among Fig. 2
+The fragmention that the m/z291 peak may obtain after losing carbonyl behind the molecular transposition for the quasi-molecular ion of synthetic product.The m/z273 peak may lose for synthetic product molecule-fragmention that COOH forms.The fragmention that the m/z143 peak produces for synthetic product molion side chain fracture.Therefore, Fig. 2 result shows that the synthetic compound of aforesaid method is compound shown in Figure 1.
2, nucleus magnetic resonance is identified
The mr qualification result is seen Fig. 3.Getting the haptenic nuclear magnetic data of desoxyquinocetone by Fig. 3 is:
1H NMR:d
6-DMSO δ: 2.87 (3H, s), 7.99 (8H, m), 8.21 (1H, d), 8.23 (1H, d), 13.16 (1H, s).Nuclear magnetic data shows that the synthetic compound of aforesaid method is compound shown in Figure 1.
Embodiment 2, the haptenic application of desoxyquinocetone
(1) preparation of desoxyquinocetone antigen
Synthesize the conjugate of desoxyquinocetone haptens and bovine serum albumin as antigen with the N-hydroxy-succinamide method.Its principle is: the reaction of the pendant carboxylic group on the above-mentioned haptens and N-hydroxy-succinamide generates the acid anhydrides intermediate, and activated carboxyl generates amido linkage and synthetic immunogen with amino reaction in the protein molecule.
Concrete steps are: take by weighing desoxyquinocetone haptens 15mg, use N, dinethylformamide 4mL dissolving then adds 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride 50mg and N-hydroxy-succinamide 50mg successively, and the room temperature lower magnetic force stirred 1 hour.Taking by weighing the 10mg bovine serum albumin is dissolved in the 1mL water.Bovine serum albumen solution is slowly added in the desoxyquinocetone haptens reaction mixture magnetic agitation 6 hours.Mixture is packed in the dialysis tubing, and dialysis is 6 days in phosphate buffered saline buffer (0.01mol/L, pH7.4), sooner or later changes dialyzate every day.With the dialysis after product at the 25-27 ℃ of centrifugal 5min of lower 3500r/min.Get supernatant, adopting ultraviolet spectrophotometer method to record its protein concentration is 35mg/mL, and packing places-20 ℃ of preservations.
Moiety and the content of used phosphate buffered saline buffer (pH7.4) of dialysing is:
Na
2HPO
4·12H
2O 2.9g
NaH
2PO
4·2H
2O 0.59g
NaCl 8.5g
KCl 0.2g
Distilled water 1000mL
(2) preparation of desoxyquinocetone polyclonal antibody
The new zealand white rabbit of choosing 37 ages in week is subjects.With the sodium chloride solution of 0.15mol/L the antigen diluent of above-mentioned preparation is become 1mg/mL(in carrier proteins).Get the antigen 1 mL after the dilution, add the equivalent Freund's complete adjuvant and make emulsifying agent, carry out head at the back of rabbit intracutaneous multi-point injection and exempt from.After 2 weeks, carry out the subcutaneous multi-point injection in back after the antigen of getting same concentration and volume adds Freund's incomplete adjuvant emulsification, per 2 all booster immunizations are (method is exempted from two) once, and immunity is 5 times altogether.Last immunity is the auricular vein injection.
(3) collection of desoxyquinocetone polyclonal antibody and bioactivity thereof
Replace bovine serum albumin with ovalbumin, employing prepares identical method with antigen and prepares envelope antigen.Mode with the posterior auricular vein blood sampling after the 5th immunity gathers White Rabbit blood, and centrifuging and taking serum carries out bioactivity.Determine the suitableeest working concentration of envelope antigen and polyclonal antibody with the square formation volumetry, antigen and antibody dilution when selecting the OD value to be 1.5 left and right sides are working concentration.
The step of the indirect elisa method of setting up is as follows:
1. coated: with carbonate buffer solution (pH9.6) the envelope antigen dilution is series concentration, each concentration is coated with delegation, 100 μ L/ holes, and 4 ℃ are spent the night;
Moiety and the content of used carbonate buffer solution (pH9.6) are:
Na
2CO
3 1.59g
NaHCO
3 2.93g
Distilled water 1000mL
2. washing and sealing: liquid in the hole of inclining, with phosphate buffered saline buffer (pH7.2) washing 3 times.Every hole adds 150 μ L confining liquids, and 37 ℃ of constant temperature sealed 1 hour, then washed 2 times;
The moiety and the content that wash used phosphate buffered saline buffer (pH7.2) are:
Na
2HPO
4.12H
2O 3.12g
NaH
2PO
4.2H
2O 1.76g
Distilled water 1000mL
The moiety of confining liquid and content are:
Calf serum 50ml
Sucrose 50g
Casein 2.5g
Na
2HPO
4·12H
2O 5.8g
NaH
2PO
4·2H
2O 0.593g
Proclin300 300μl
Distilled water 1000mL
3. application of sample: each is listed as the serum to be checked that the hole adds the doubling dilution that begins from 1:1000, every hole 100 μ L, 37 ℃ of reactions 1 hour; Washing is with 2.Every hole adds 100 μ L HRP-goat anti-rabbit iggs, and 37 ℃ were reacted 1 hour.Washing is with 2;
Moiety and the content of the substrate buffer solution (pH5.0) of dilution test serum are:
Na
2HPO
4.12H
2O 3.68g
Citric acid 0.933g
Adding distil water is to 100mL
4. chromogenic assay: every hole adds 3,3', 5,5'-tetramethyl benzidine (TMB) solution, 100 μ L, 37 ℃ of colour developing 20min, the then H that adds of every hole
2SO
4Solution (2mol/L) 50 μ L measure each hole in the OD at 450nm place value with microplate reader at last with termination reaction;
Used 3,3', moiety and the content of 5,5'-tetramethyl benzidine (TMB) solution are:
TMB 2.5mg
Ethylene glycol 1mL
Substrate buffer solution 25mL
Massfraction is 30% hydrogen peroxide 25 μ L
Substrate buffer solution is the damping fluid (pH5.0) of the described dilution test serum of step 3.
3,3', 5,5'-tetramethyl benzidine (TMB) solution faces fresh preparation of time spent.
H
2SO
4Moiety and the content of solution (2mol/L) are:
Massfraction is 98% the vitriol oil (18.4mol/L) 21.74mL
Distilled water 178.26mL
5. result of determination: tire greater than the ELISA of the highly diluted multiple of the serum of 2 times of negative control holes as serum take OD450nm.
The detected result that obtains is as follows: the suitableeest working concentration of envelope antigen is 1:1000; Obtain the best from certain antiserum(antisera) in the antiserum(antisera) after the 5th immunity of 3 White Rabbits and tire, i.e. 1:5000, this antiserum(antisera) is the desoxyquinocetone polyclonal antibody of acquisition.
(4) specific detection of IgG antibody
Adopt the square formation method to determine the suitableeest working concentration of coating antigen and antibody, adopt the indirect competitive ELISA method to detect the gained IgG antibody to the specificity of other quinoxaline medicine and meta-bolites (olaquindox, mequindox, carbadox, 3-Jia based quinoxaline-2-carboxylic acid are with Oxoquinoxaline-2-carboxylic acid), concrete operation step is with (three), the result shows, this antibody is only to the main metabolites desoxyquinocetone specific binding of Quinocetone, and is not obvious with other quinoxaline medicine and metabolite cross reaction.
Claims (10)
1. desoxyquinocetone haptens has the chemical structure shown in the formula I:
2. the described haptenic preparation method of claim 1 comprises the steps (1) and (2):
(1) with mequindox and Sodium Hydrosulphite in 60 ℃ of reactions 2 hours, namely get 3-methyl-2-acetyl quinoxaline; The mol ratio of described mequindox and Sodium Hydrosulphite is 1:1.8;
(2) with 3-methyl-2-acetyl quinoxaline and p formyl benzoic acid under the alkaline catalysts effect, in 40 ℃ of reaction 12h, room temperature reaction spends the night again, namely gets the desoxyquinocetone haptens; The mol ratio of described 3-methyl-2-acetyl quinoxaline and p formyl benzoic acid is 1:1.2.
3. method according to claim 2, it is characterized in that: in the described step (1), described reaction is carried out in solvent, and described solvent is the mixed solvent of tetrahydrofuran (THF), 100% ethanol and distilled water, and the volume ratio of described tetrahydrofuran (THF), 100% ethanol and distilled water is 2:10:1; In the described step (2), described reaction is carried out in solvent, and described solvent is the mixture of tetrahydrofuran (THF), second alcohol and water; In the described step (2), described alkaline catalysts is sodium hydroxide.
4. it is characterized in that: in the described step (1), also comprise concentration process and purge process after described reaction is complete according to claim 2 or 3 described methods.
5. it is characterized in that: in the described step (2), also comprise purge process after described reaction is complete according to claim 2 or 3 described methods.
6. the described desoxyquinocetone haptens of claim 1 or the desoxyquinocetone haptens that directly obtained by any described method of claim 2-5 are as haptenic purposes.
7. the described desoxyquinocetone haptens of claim 1 or the purposes of desoxyquinocetone haptens in detecting desoxyquinocetone that directly obtained by any described method of claim 2-5;
Or, the described desoxyquinocetone haptens of claim 1 or the purposes of desoxyquinocetone haptens in preparation detection desoxyquinocetone product that is directly obtained by any described method of claim 2-5.
8. antigen, the desoxyquinocetone haptens that directly obtains for the described desoxyquinocetone haptens of claim 1 or by any described method of claim 2-5 and the conjugate of bovine serum albumin.
9. polyclonal antibody, described polyclonal antibody are that the conjugate of the desoxyquinocetone haptens that directly obtains with the described desoxyquinocetone haptens of claim 1 or by any described method of claim 2-5 and bovine serum albumin obtains after as antigen-immunized animal.
10. the test kit of a specific detection desoxyquinocetone, described test kit comprises following 1)-9):
1) carbonate buffer solution:
Na
2CO
3 1.59g/L
NaHCO
3 2.93g/L
Solvent is distilled water;
2) the desoxyquinocetone haptens that directly obtains with the described desoxyquinocetone haptens of claim 1 or by any described method of claim 2-5 and the conjugate of ovalbumin are as envelope antigen;
3) confining liquid:
Calf serum 50ml/L
Sucrose 50g/L
Casein 2.5g/L
Na
2HPO
4·12H
2O 5.8g/L
NaH
2PO
4·2H
2O 0.593g/L
Proclin300 300μl/L
Solvent is distilled water;
4) phosphate buffered saline buffer:
Na
2HPO
4.12H
2O 3.12g/L
NaH
2PO
4.2H
2O 1.76g/L
Solvent is distilled water;
5) HRP-goat anti-rabbit igg;
6) substrate buffer solution:
Na
2HPO
4.12H
2O 36.8g/L
Citric acid 9.33g/L
Solvent is distilled water;
7) 3,3', 5,5'-tetramethyl biphenyl amine aqueous solution:
TMB 96.15mg/L
Ethylene glycol 38.46mL/L
Massfraction is 30% hydrogen peroxide 961 μ L/L
Na
2HPO
4.12H
2O 35.4g/L
Citric acid 8.97g/L
Solvent is distilled water;
8) H
2SO
4Solution:
Massfraction is 98% vitriol oil 2mol/L
Solvent is distilled water;
9) polyclonal antibody that the desoxyquinocetone haptens that directly obtains with the described desoxyquinocetone haptens of claim 1 or by any described method of claim 2-5 and the conjugate of bovine serum albumin obtain after as antigen-immunized animal.
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