CN102617493B - Mequindox artificial antigens and antibodies prepared by same - Google Patents
Mequindox artificial antigens and antibodies prepared by same Download PDFInfo
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Abstract
The invention discloses mequindox artificial antigens and antibodies that are prepared by the mequindox artificial antigens, and provides a compound that is shown in the formula (I). The compound that is shown in the formula (I) is obtained by structurally modifying mequindox, so that feature structures of mequindox can remain to the maximum extent, and the compound also has reactive groups that can be coupled with carrier protein. The invention also protects the mequindox artificial antigens that are conjugates obtained when the compound shown in the formula (I) is coupled with carrier protein. The mequindox artificial antigens are utilized to immunize animals, high-specificity monoclonal antibodies and polyclonal antibodies can be obtained, and the method is simple, convenient and feasible. The artificial antigens can be used for detecting quinoxalines compounds. The antibodies can be used for detecting the quinoxalines compounds.
Description
Technical field
The present invention relates to the antibody of a kind of mequindox artificial antigen and preparation thereof.
Background technology
Quinoxaline compound is equal You quinoxaline-1 in molecule, the antimicrobial drug of 4-dioxide mother nucleus structure synthetic, Major Members has olaquindox (olaquindox, OLA), carbadox (cabadox, CBX), mequindox (mequindox, and Quinocetone (quinocetone, QCT) etc. MEQ).Owing to can suppressing multiple gram-positive microorganism and negative bacterium, and poultry, fowl, fish etc. are had to obvious growth promoting function ， quinoxaline compound extensively applied as fodder additives.
Along with going deep into of research, find that quinoxaline compound has obvious toxic side effect, carbadox has genetoxic and carcinogenic suspicion, olaquindox has strong cumulative toxicity and mutagenicity, therefore in the world about the maximum residue limit(MRL) of quinoxaline compound also has very strict regulation, China has put into effect the standardize use of quinoxaline compound of corresponding regulation.China forbids using carbadox as fodder additives, olaquindox can only be used for piglet medicated feed additive, forbid for avian production.In addition, the research of Chinese scholars and production practice show that mequindox toxicity is bigger than normal, are also unsuitable for as fowl medicated feed additive, although Quinocetone diseases prevention do growth result good, have no side effect, a kind of gene toxic agent and reproduction mutagenic compound, have mutagenesis, teratogenesis and carinogenicity.
At present, less for the residual detection method research of feed Zhong quinoxaline compound.Therefore set up one sensitive, quick, simple to operate, be applicable to great amount of samples examination quinoxaline compound test method be to there is important economic implications and social effect.
Summary of the invention
The object of this invention is to provide the antibody of a kind of mequindox artificial antigen and preparation thereof.
The invention provides a kind of compound, i.e. compound shown in formula (I).Shown in formula (I), compound is that mequindox is carried out to the compound that structure of modification obtains, and has both at utmost retained the feature structure of mequindox, have again can with the active group of carrier proteins generation coupling.
The present invention goes back the preparation method of compound shown in protection (I), comprises the steps:, by mequindox and carboxymethyl azanol reaction, to obtain described compound.Described reaction specifically can be carried out in the mixed solvent of anhydrous propanone and DMF (volume ratio of anhydrous propanone and DMF specifically can be 1: 1).The mol ratio of described mequindox and described carboxymethyl azanol specifically can be 1: 1.5.
The present invention also protects a kind of mequindox artificial antigen, is the conjugate that compound shown in formula (I) and carrier protein couplet are obtained.Described carrier proteins is bovine serum albumin or oralbumin.Shown in formula (I), the coupling ratio of compound and carrier proteins specifically can be (8-10): 1.Described coupling ratio refers to mol ratio.Compound and described carrier proteins shown in formula (I) specifically can pass through active ester method coupling.Described mequindox artificial antigen is suc as formula shown in (II).Described mequindox artificial antigen can be used as immunogen and also can be used as coating antigen.By described mequindox artificial antigen immune animal, can obtain monoclonal antibody and the polyclonal antibody of high specific, method is easy, Yi Hang.By described mequindox artificial antigen coated elisa plate, also can be for the sero-fast detection of quinoxaline compound.The structural difference of coating antigen and immunogen can further improve sensitivity and the specificity of detection.
Formula (I).
Formula (II).
Described mequindox artificial antigen can be used for preparing quinoxaline compound specificity antibody; Described quinoxaline compound is mequindox, carbadox, olaquindox or Quinocetone.Described antibody can be monoclonal antibody or polyclonal antibody.
The antibody preparing taking described mequindox artificial antigen as immunogen also belongs to protection scope of the present invention.Described antibody can be monoclonal antibody or polyclonal antibody.Described monoclonal antibody specifically can be the monoclonal antibody of anti-mequindox monoclonal antibody hybridoma cell 1A10 secretion.
The present invention also protects a kind of hybridoma; the anti-mequindox monoclonal antibody hybridoma cell of called after 1A10; be called for short hybridoma 1A10; be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on February 15th, 2012 and (be called for short CGMCC; address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preserving number is CGMCC No.5780.
The monoclonal antibody of described hybridoma 1A10 secretion also belongs to protection scope of the present invention.
Described mequindox artificial antigen can be used for detecting quinoxaline compound.
Described antibody (monoclonal antibody or polyclonal antibody) can be used for detecting quinoxaline compound.
Arbitrary described quinoxaline compound is to have quinoxaline-1 in molecule above, the compound of 4-dioxide mother nucleus structure.Arbitrary described quinoxaline compound is mequindox, carbadox, olaquindox or Quinocetone above.
The present invention has great value for the detection of quinoxaline compound.
Brief description of the drawings
Fig. 1 is the artificial antigen ultraviolet spectrogram of mequindox.
Fig. 2 is the canonical plotting that adopts mequindox to make.
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.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples, all arranges and repeats experiment, results averaged for three times.Mequindox is purchased from Sigma-Aldrich company, and catalog number is MO-189.In embodiment, PBS damping fluid used is the PBS damping fluid of pH7.4,0.01M.In embodiment, carbonate buffer solution used is the sodium carbonate buffer of pH9.6,0.05mol/L.Bovine serum albumin is called for short BSA.Ovalbumin is called for short OVA.
Mequindox is suc as formula shown in (III), and molecular weight is 218.21.
Formula (III)
Carboxymethyl azanol is suc as formula shown in (IV), and molecular weight is 77.04.
Formula (IV)
DMF (DMF) is as shown in formula V.
Formula (V)
N-hydroxy-succinamide (NHS) is suc as formula shown in (VI).
Formula (VI)
1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) is suc as formula shown in (VII).
Formula (VII)
Embodiment 1, prepare mequindox haptens
One, the haptenic preparation of mequindox
Mequindox 216mg (1mmol) is dissolved in the mixed solvent (volume ratio of anhydrous propanone and DMF is 1: 1) of 3ml anhydrous propanone and DMF, add in brown reaction flask, process lucifuge, take carboxymethyl azanol 116mg (1.5mmol) and add reaction flask, 40 DEG C are reacted 16 hours, add a small amount of shrend reaction of going out, adjust pH to 10 left and right with the NaOH aqueous solution of 0.1mol/L, use CH
2cl
2extraction, water intaking is adjusted pH to 3 left and right with the HCl aqueous solution of 0.1mol/L mutually, is extracted with ethyl acetate, and gets oil phase and carries out vacuum-drying, obtains 56mg product.
Two, the haptenic sign of mequindox
Product prepared by step 1 carries out ultimate analysis, and result is as follows:
C：52.06；H：4.32；N：15.10；O：28.38。
Result shows, product prepared by step 1 is compound shown in formula (I).
Formula (I).
The Preparation and characterization of embodiment 2, mequindox artificial antigen
One, the immunogenic synthetic and sign of mequindox
1, mequindox is immunogenic synthetic
(1) shown in the formula (I) 10mg embodiment 1 being prepared, compound is dissolved in 2mL N, in N '-dimethylformamide, add 10mg N-hydroxy-succinamide and 10mg 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, room temperature lower magnetic force stirs 2h, obtains solution III.
(2) 30mg bovine serum albumin is added in 2mL deionized water, fully dissolve, be solution IV.
(3) solution IV is added in solution III, after slowly stirring 6h, enter dialysis tubing, 4 DEG C of dialysis 72h (water is changed 6 times in centre) in physiological saline, then under 4 DEG C of conditions, the centrifugal 30min of 8000rmp, get supernatant, be mequindox immunogen solution, be sub-packed in ampere bottle-20 DEG C of preservations, mequindox immunogen is called for short MEQ-BSA, and mequindox immunogen solution is called for short MEQ-BSA solution.
(4) after MEQ-BSA solution is diluted with PBS damping fluid, measure the spectrophotometric value of 280nm and 260nm, press formula and calculate the protein concentration in diluent, be the MEQ-BSA concentration in former MEQ-BSA solution after the protein concentration value recording is multiplied by its extension rate.Protein concn (mg/ml)=1.45 × OD
280-0.74 × OD
260.MEQ-BSA concentration in MEQ-BSA solution is 5.3mg/ml.
2, the immunogenic sign of mequindox
By PBS damping fluid dilution (concentration that makes MEQ-BSA is 5mg/mL) for MEQ-BSA solution, as solution first; Using the PBS damping fluid containing 5mg/mL mequindox as solution second; Using the PBS damping fluid containing 5mg/mL BSA as solution third.Respectively solution first, solution second and solution third are carried out to ultraviolet (200-380nm) spectral scan, uv scan the results are shown in Figure 1.There is considerable change in the uv-spectrogram of solution first compared with solution third, and compound and BSA success coupling are described.
The maximum absorption wave long value of solution second is 372nm, and the maximum absorption wave long value of solution third is 280nm.Calculate the optical extinction coefficient (K) of each compound according to formula K=A/CL (A is the absorbancy under maximum absorption wave long value, and C is strength of solution, the thickness that L is liquid layer).
Adopt respectively the maximum absorption wave long value of solution second and solution third to carry out uv scan to solution first, and according to this compound of optical extinction coefficient backwards calculation of this compound having calculated the concentration in solution first, obtain the volumetric molar concentration of this compound divided by molecular weight with concentration value, calculate coupling ratio, shown in formula (I), the coupling ratio of compound and BSA is 8: 1, and compound shown in 8 formulas (I) is in conjunction with 1 BSA.
Two, the Preparation and characterization of mequindox coating antigen
1, the preparation of mequindox coating antigen
Replace bovine serum albumin with ovalbumin, other is with 1 of step 1.
Mequindox coating antigen is called for short MEQ-OVA, and mequindox coating antigen solution is called for short MEQ-OVA solution.
MEQ-OVA concentration in MEQ-OVA solution is 3.0mg/ml.
2, the sign of mequindox coating antigen
Replace MEQ-BSA with MEQ-OVA, replace BSA with OVA, other is with 2 of step 1.
Shown in formula (I), the coupling ratio of compound and OVA is 10: 1, and compound shown in 10 formulas (I) is in conjunction with 1 OVA.
The preparation of embodiment 3, quinoxaline compound monoclonal antibodies
Balb/c mouse: be purchased from Beijing Vital River Experimental Animals Technology Co., Ltd.;
SP2/0 myeloma cell: purchased from Sigma-Aldrich company, catalog number is 08060101.
One, animal immune
MEQ-BSA solution immunity Balb/c mouse prepared by embodiment 2, every mouse single immunization 100 μ gMEQ-BSA, immunity 4 times altogether, every minor tick two weeks, the immunization ways of first three time is the subcutaneous multi-point injection of nape portion, and the immunization ways of latter three times is peritoneal injection.
Two, cytogamy and cloning
1, the 4th immunity be after 3 days, and extracting spleen cell merges in 5: 1 (quantitative proportion) ratios and SP2/0 myeloma cell, adopts indirect competitive ELISA to measure cell conditioned medium liquid, screens positive hole.
2, utilize limiting dilution assay to carry out cloning to positive hole, obtain a strain and can secrete the hybridoma of mequindox monoclonal antibody, the anti-mequindox monoclonal antibody hybridoma cell of called after 1A10 (being called for short hybridoma 1A10).Hybridoma 1A10 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on February 15th, 2012 and (is called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preserving number is CGMCC No.5780.
Three, cell cryopreservation and recovery
Hybridoma 1A10 is made to 1 × 10 with frozen storing liquid
6the cell suspension of individual/ml is preserved for a long time in liquid nitrogen.When recovery, take out cryopreservation tube, put into immediately 37 DEG C of water-bath middling speeds and melt, after centrifugal removal frozen storing liquid, move in culturing bottle and cultivate.
Four, the preparation and purification of monoclonal antibody
1, increment culture method
The preparation method of cell culture medium (7.4): add calf serum and sodium bicarbonate in RPMI-1640 substratum, the final concentration of calf serum is 20% (quality percentage composition), and the final concentration of sodium bicarbonate is 0.2% (quality percentage composition).
Hybridoma 1A10 is placed in to cell culture medium, cultivates 2 days for 37 DEG C, by sad-saturated ammonium sulphate method, the nutrient solution obtaining is carried out to purifying, obtain monoclonal antibody solution (20 DEG C of preservations).
Protein concn (mg/ml)=1.45 × OD in monoclonal antibody
280-0.74 × OD
260.
Adopting above formula to calculate the protein concn in monoclonal antibody, is 21.8mg/ml.
2, ascites preparation
Balb/c mouse peritoneal injection sterilizing paraffin oil (0.4mL/ only).7 days pneumoretroperitoneum injection hybridoma 1A10 (5 × 10
5individual/only).After 7 days, gather ascites, carry out purifying, DEG C preservation of ascites-20 after purifying by sad-saturated ammonium sulphate method.
Five, the qualification of monoclonal antibody
1 of the step 4 monoclonal antibody solution obtaining is identified respectively as follows:
1, adopt ELISA monoclonal antibody hypotype detection kit (Sigma company product, catalog number is 19285) to detect the hypotype of monoclonal antibody, the immunoglobulin subclass of monoclonal antibody is IgG1.
2, the mensuration of antibody titer
(1) adopt MEQ-OVA solution (adopting carbonate buffer solution to regulate concentration) prepared by embodiment 2 to be coated with 100 μ L/ holes; The coated concentration of MEQ-OVA is 1.0 μ g/mL.
Hatch 16 hours for (2) 4 DEG C.
(3) seal and wash plate.
(4) every hole adds 1 monoclonal antibody solution obtaining or its diluent (adopting PBS damping fluid to carry out gradient dilution) of 100 μ L step 4.
(5) incubated at room 2h, washes plate.
(6) every hole adds the sheep anti-mouse igg of 100 μ L horseradish peroxidase-labeled, incubated at room 2h.
(7) wash plate.
(8) add TMB nitrite ion, lucifuge colour developing 15min.
(9) every hole adds 100 μ L 2mol/L sulfuric acid stopped reactions; Read OD
450value.
While reaching 1.0 left and right with OD value, be judged to be the positive.Antibody titer is 1: 320000.
3, the calculating of monoclonal antibody sensitivity
(1) to (3) with (1) of step 2 to (3).
(4) every hole adds 50 μ L mequindox standard solutions (to be made up of mequindox and PBS damping fluid; The concentration of mequindox is respectively 0.3 μ g/L, 0.9 μ g/L, 2.7 μ g/L, 8.1 μ g/L, 24.3 μ g/L; The hole in contrast, hole of PBS damping fluid will only be added); Each concentration arranges 3 multiple holes.
(5) every hole adds 1 monoclonal antibody solution obtaining of 50 μ L step 4.
(6) incubated at room 2h, washes plate.
(7) every hole adds the sheep anti-mouse igg of 100 μ L horseradish peroxidase-labeled, incubated at room 2h.
(8) wash plate.
(9) add TMB nitrite ion, lucifuge colour developing 15min.
(10) every hole adds 100 μ L 2mol/L sulfuric acid stopped reactions; Read OD
450value.
The light absorption value that adopts the standard solution of each concentration to obtain (mean values in three multiple holes) is multiplied by 100 as ordinate zou again divided by the light absorption value of control wells, taking the natural logarithm value of the mequindox concentration in each standard solution (μ g/L) as X-coordinate curve plotting figure, see Fig. 2.
Contrast Fig. 2, obtains the mequindox concentration (μ g/L) that Y value equals 50% correspondence, is IC
50value.Monoclonal antibody detects the sensitivity (IC of mequindox
50value) be 1.2 μ g/L.
4, the calculating of cross reacting rate
(1) to (3) with (1) of step 2 to (3).
(4) every hole adds 50 μ L analog standard solutions (to be made up of analog and PBS damping fluid; The concentration of analog is respectively 0.3 μ g/L, 0.9 μ g/L, 2.7 μ g/L, 8.1 μ g/L, 24.3 μ g/L; To only add the hole in contrast, hole of PBS damping fluid), each concentration arranges 3 multiple holes.
Analog is: olaquindox, Quinocetone, carbadox, quinoline match many, 3-Jia based quinoxaline-2-carboxylic acid (MQCA) Oxoquinoxaline-2-carboxylic acid (QCA), Streptomycin sulphate, tsiklomitsin, sulfaquinoxaline, penicillin, monensin, clenbuterol or Sulphadiazine Sodium.
Olaquindox: Dr.Ehrenstorfer GmbH company product, article No. C 15716500.Quinocetone: Dr.Ehrenstorfer GmbH company product, article No. C 16709000.Carbadox: sigma company product, article No. C6770.Streptomycin sulphate: sigma company product, article No. 85886.Tsiklomitsin: sigma company product, article No. 31741.Sulfaquinoxaline: sigma company product, article No. 45662.Penicillin: sigma company product, article No. 46609.Monensin: sigma company product, article No. M5273.Clenbuterol: sigma company product, article No. C5423.Sulphadiazine Sodium: sigma company product, article No. S8626.
(5) to (10) with (5) of step 3 to (10).
The light absorption value that adopts the analog of each concentration to obtain (mean values in three multiple holes) is multiplied by 100 as ordinate zou again divided by the light absorption value of control wells, taking the natural logarithm value of the similar substrate concentration in each standard solution (μ g/L) as X-coordinate curve plotting figure.Control curve figure, corresponding similar substrate concentration (μ g/L) when obtaining Y value and equaling 50%, the i.e. IC of analog
50value.
Cross reacting rate by following formula calculating monoclonal antibody to other analog.
The results are shown in Table 1.
Table 1 monoclonal antibody specificity
| Analog | Sensitivity (IC 50Value) | Cross reacting rate | Analog | Sensitivity (IC 50Value) | Cross reacting rate |
| Mequindox | 1.20μg/ |
100％ | Olaquindox | 1.36μg/L | 88％ |
| Quinocetone | 0.68μg/L | 176％ | Carbadox | 1.87μg/L | 64％ |
| Quinoline match is many | 3.24μg/L | 37％ | MQCA | 6.67μg/L | 18％ |
| QCA | 20.3μg/L | 6％ | Streptomycin sulphate | Nothing | ＜1％ |
| Tsiklomitsin | Nothing | ＜1％ | Sulfaquinoxaline | Nothing | ＜1％ |
| Penicillin | Nothing | ＜1％ | Monensin | Nothing | ＜1％ |
| Clenbuterol | Nothing | ＜1％ | Sulphadiazine Sodium | Nothing | ＜1％ |
Monoclonal antibody is Yu carbadox, olaquindox and Quinocetone in quinoxaline compound have cross reaction, there is respectively 18% and 6% cross reaction with the residual mark 3-of olaquindox Jia based quinoxaline-2-carboxylic acid (MQCA) and the residual mark Oxoquinoxaline-2-carboxylic acid of carbadox (QCA), with other analog, catalyzer, derivative reagent no cross reaction.
5, stability
By 1 of step 4 monoclonal antibody solution-20 that obtain DEG C placement, different time sampling, with detecting and tire after the dilution of PBS damping fluid, concrete steps are as follows:
(1) to (3) with (1) of step 2 to (3).
(4) every hole adds 50 μ L monoclonal antibody solutions or its diluent (adopting PBS damping fluid to carry out gradient dilution).
(5) to (9) with (6) of step 3 to (10).
OD
450value is in table 2.Result shows, constant tiring of 120 days monoclonal antibodies of-20 DEG C of preservations.
The stability of table 2 monoclonal antibody
6, affinity costant is measured
(1) use MEQ-OVA as coating antigen coated elisa plate
Adopt MEQ-OVA solution (adopting carbonate buffer solution to regulate concentration) prepared by embodiment 2 to be coated with 100 μ L/ holes; The coated concentration of following OVA-SAL is set respectively: 1 μ g/mL, 0.5 μ g/mL, 0.25 μ g/mL, 0.125 μ g/mL.
Hatch 16 hours for (2) 4 DEG C.
(3) seal and wash plate.
(4) every hole adds the diluent (diluting with PBS damping fluid) of 100 μ L monoclonal antibody solutions; Protein concn in diluent is respectively 1.25,0.625,0.3125,1.5625 × 10
-1, 7.8 × 10
-2, 3.9 × 10
-2, 1.95 × 10
-2, 9.75 × 10
-3, 4.88 × 10
-3, 2.44 × 10
-3, 1.22 × 10
-3, 6.1 × 10
-4mg/L;
(5) incubated at room 2h, washes plate.
(6) every hole adds the sheep anti-mouse igg of 100 μ L horseradish peroxidase-labeled, incubated at room 2h.
(7) wash plate.
(8) add TMB nitrite ion, lucifuge colour developing 15min.
(9) every hole adds 100 μ L 2mol/L sulfuric acid stopped reactions; Read OD
450value.
Taking the natural logarithm value of the protein concn in monoclonal antibody (mol/L) as X-coordinate, make curve taking its corresponding absorbancy as ordinate zou.
Each antigen coated concentration obtains 1 S type curve, obtains altogether 4 S type curves.Find out the top of S curve, corresponding OD
450value is set as ODMAX.Find out respectively each the antibody concentration that curve 50%ODMAX is corresponding.Adopt 1 μ g/mL to be coated with concentration, the antibody concentration that 50%ODMAX is corresponding is 8.9 × 10
-12mol/L.Adopt 0.5 μ g/mL to be coated with concentration, the antibody concentration that 50%ODMAX is corresponding is 27.4 × 10
-12mol/L.Adopt 0.25 μ g/mL to be coated with concentration, the antibody concentration that 50%ODMAX is corresponding is 180.9 × 10
-12mol/L.Adopt 0.125 μ g/mL to be coated with concentration, the antibody concentration that 50%ODMAX is corresponding is 374.6 × 10
-12mol/L.
By one group between two of 4 concentration, calculate the affinity costant of monoclonal antibody according to formula
Ka＝(n-1)/2(n[Ab]t
1-[Ab]t
2)
In formula, n be every group in the multiple of two coated concentration, [Ab] t
1, [Ab] t
2be respectively two antibody concentration (mol/L) that 50%ODMAX is corresponding in every group.For example: 1 μ g/mL is coated with concentration 50% OD
450corresponding antibody concentration is 8.9 × 10
-12mol/L, coated concentration 50% OD of coated 0.5 μ g/mL
450corresponding antibody concentration is 27.4 × 10
-12mol/L, Ka=(2-1)/2 (2 × 27.4 × 10
-12-8.9 × 10
-12)=10.8 × 10
9m
-1.The like, obtain all the other 5 Ka values, be respectively 1.49 × 10
9m
-1, 0.91 × 10
9m
-1, 2.1 × 10
9m
-1, 1.0 × 10
9m
-1, 1.17 × 10
9m
-1, the affinity costant that calculates monoclonal antibody of averaging is 2.91 × 10
9m
-1.
Claims (9)
1. compound shown in formula I;
2. the preparation method of compound described in claim 1, comprises the steps:, by mequindox and carboxymethyl azanol reaction, to obtain described compound.
3. the conjugate of compound and carrier proteins described in claim 1.
4. conjugate as claimed in claim 3, is characterized in that: described carrier proteins is bovine serum albumin or oralbumin.
Described in claim 3 or 4 conjugate in the application of preparing in quinoxaline compound specificity antibody.
6. application as claimed in claim 5, is characterized in that: described antibody is monoclonal antibody or polyclonal antibody.
7. anti-mequindox monoclonal antibody hybridoma cell 1A10, its deposit number is CGMCC No.5780.
8. the monoclonal antibody of hybridoma secretion described in claim 7.
9. conjugate described in claim 3 or 4, or monoclonal antibody is in the application detecting in quinoxaline compound described in claim 8;
Described quinoxaline compound is carbadox, olaquindox, Quinocetone, 3-Jia based quinoxaline-2-carboxylic acid and/or Oxoquinoxaline-2-carboxylic acid.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2083817A (en) * | 1980-09-12 | 1982-03-31 | Egyt Gyogyszervegyeszeti Gyar | Process for the preparation of 2-methylene-quinoxaline-1,4-dioxide derivatives |
| CN101046474A (en) * | 2006-03-30 | 2007-10-03 | 中国农业科学院上海家畜寄生虫病研究所 | Enzyme-linked immunological kit for detecting quinoxaline medicine residue |
| CN101074214A (en) * | 2007-06-21 | 2007-11-21 | 广东新南都饲料科技有限公司 | Synthesis of antibiotics N,N-oxazoline dioxide-2-methanal hydrazone compound |
| CN101433824A (en) * | 2008-12-03 | 2009-05-20 | 中国农业大学 | Method for extracting sulfabenzpyrazine from animal sample and special immune affinity sorbent thereof |
-
2012
- 2012-02-22 CN CN201210042215.0A patent/CN102617493B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2083817A (en) * | 1980-09-12 | 1982-03-31 | Egyt Gyogyszervegyeszeti Gyar | Process for the preparation of 2-methylene-quinoxaline-1,4-dioxide derivatives |
| CN101046474A (en) * | 2006-03-30 | 2007-10-03 | 中国农业科学院上海家畜寄生虫病研究所 | Enzyme-linked immunological kit for detecting quinoxaline medicine residue |
| CN101074214A (en) * | 2007-06-21 | 2007-11-21 | 广东新南都饲料科技有限公司 | Synthesis of antibiotics N,N-oxazoline dioxide-2-methanal hydrazone compound |
| CN101433824A (en) * | 2008-12-03 | 2009-05-20 | 中国农业大学 | Method for extracting sulfabenzpyrazine from animal sample and special immune affinity sorbent thereof |
Non-Patent Citations (2)
| Title |
|---|
| 喹乙醇单克隆抗体的制备及其免疫学特性的鉴定;宋春美等;《核农学报》;20101231;第24卷(第4期);777-783 * |
| 宋春美等.喹乙醇单克隆抗体的制备及其免疫学特性的鉴定.《核农学报》.2010,第24卷(第4期),777-783. |
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