CN102659693B - 3-methylquinoxaline-2-carboxylic acid artificial antigen and antibody obtained by the 3-methylquinoxaline-2-carboxylic acid artificial antigen - Google Patents
3-methylquinoxaline-2-carboxylic acid artificial antigen and antibody obtained by the 3-methylquinoxaline-2-carboxylic acid artificial antigen Download PDFInfo
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Abstract
The invention discloses a 3-methylquinoxaline-2-carboxylic acid artificial antigen and an antibody obtained by the 3-methylquinoxaline-2-carboxylic acid artificial antigen. The invention provides a compound shown in the formula (I). The compound shown in the formula (I) is prepared by structural modification of 3-methylquinoxaline-2-carboxylic acid, retains a feature structure of 3-methylquinoxaline-2-carboxylic acid as much as possible and has active groups which can couple with a carrier protein. The 3-methylquinoxaline-2-carboxylic acid artificial antigen provided by the invention is a conjugate prepared by coupling of the compound shown in the formula (I) and a carrier protein. The 3-methylquinoxaline-2-carboxylic acid artificial antigen is used for animal immunization so that high-specificity monoclonal and polyclonal antibodies are obtained. A preparation method of the high-specificity monoclonal and polyclonal antibodies is simple and practicable. The 3-methylquinoxaline-2-carboxylic acid artificial antigen can be used for detection of olaquindox metabolites. The antibodies obtained by the 3-methylquinoxaline-2-carboxylic acid artificial antigen can be used for detection of olaquindox metabolites.
Description
Technical field
The present invention relates to the antibody of a kind of 3-Jia based quinoxaline-2 carboxylic acid artificial antigen and preparation thereof.
Background technology
3-Jia based quinoxaline-2-carboxylic acid (MQCA) is the metabolite that olaquindox, mequindox and Quinocetone produce in animal body.Owing to can suppressing multiple gram-positive microorganism and negative bacterium, and it is very extensive in the application of China that poultry, fowl, flesh of fish class etc. are had to obvious growth promoting function ， quinoxaline medicine (comprising the medicines such as olaquindox, mequindox, Quinocetone).Research finds that ， quinoxaline original shape medicine and metabolite exist obvious safety issue, has the toxic side effect such as obvious teratogenesis, carcinogenic, mutagenesis, photosensitive and adrenal cortex infringement, serious harm the health of humans and animals.
European Union forbids adding olaquindox in feed in dispatch in 1998, and the pig being only approved for below 35kg in China is used.Mequindox approval be raw material and tablet, regulation is as the curative of swine dysentery and pig, ox bacterial enteritis.3-Jia based quinoxaline-2-carboxylic acid is mark and the monitored object that the foodstuff additive joint specialist council (JECFA) under the World Food Programme and the World Health Organization determines quinoxaline medicine residue.The maximum residue limit(MRL) of relevant quinoxaline medicine regulation is very strict in the world.European Union's regulation carbadox and olaquindox with and meta-bolites in animal food, must not detect and stipulate that these two kinds of veterinary drugs forbid selling.No. 235 regulation of China Ministry of Agriculture bulletin, olaquindox maximum residue limit(MRL) is olaquindox+3-Jia based quinoxaline-2-carboxylic acid: 4ug/kg (muscle), 50ug/kg (liver), 2mg/kg (feed).
When China carries out Residue Monitoring, often adopt immunochemical analyses to carry out primary dcreening operation, instrumental method is confirmed.Immunochemical analyses due to the advantage in uniqueness aspect the qualitative, quantitative of antigen-antibody and easy and simple to handle fast, cost is low, sensitivity is higher, analyzing samples amount is large advantage made up the deficiency of physico-chemical analysis.The basic factor that affects immunochemical analyses quality is specificity and the affinity of antibody, these character are decided by again the structure of immune hapten molecule, and therefore immune haptenic molecular designing is exactly the step that produces specific antibody and set up the most basic and most critical of small molecules residue of veterinary drug Fast Detection Technique with synthesizing.
Summary of the invention
The antibody that the object of this invention is to provide a kind of 3-Jia based quinoxaline-2 carboxylic acid artificial antigen and preparation thereof.
The invention provides a kind of compound, i.e. compound shown in formula (I).Compound shown in formula (I), for 3-Jia based quinoxaline-2 carboxylic acid is carried out to the compound that structure of modification obtains, had both at utmost retained the feature structure of 3-Jia based quinoxaline-2 carboxylic acids, 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 3-Jia based quinoxaline-2 carboxylic acid and γ-aminobutyric acid reaction, to obtain described compound.The mass ratio of described 3-Jia based quinoxaline-2 carboxylic acid and described γ-aminobutyric acid specifically can be 1: 2.Described reaction specifically can adopt pyridine as catalyzer.
The present invention also protects a kind of 3-Jia based quinoxaline-2 carboxylic acid 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 (11-8): 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 3-Jia based quinoxaline-2 carboxylic acid artificial antigen is suc as formula shown in (II).Described 3-Jia based quinoxaline-2 carboxylic acid artificial antigen can be used as immunogen and also can be used as coating antigen.By described 3-Jia based quinoxaline-2 carboxylic acid artificial antigen immune animal, can obtain monoclonal antibody and the polyclonal antibody of high specific, method is easy, easily capable.By described 3-first based quinoxaline-2 carboxylic acid artificial antigen coated elisa plate, also can be for the sero-fast detection of olaquindox metabolite (3-first based quinoxaline-2 carboxylic acid).The structural difference of coating antigen and immunogen can further improve sensitivity and the specificity of detection.
Carboxylic acid artificial antigen can be used for preparing 3-Jia based quinoxaline-2 carboxylic acid specific antibody in described 3-Jia based quinoxaline-2.Described antibody can be monoclonal antibody or polyclonal antibody.
The described 3-Jia based quinoxaline-2 carboxylic acid artificial antigen of take also belongs to protection scope of the present invention as the antibody that immunogen prepares.Described antibody can be monoclonal antibody or polyclonal antibody.
The present invention also protects a kind of hybridoma; the anti-olaquindox metabolite of called after monoclonal antibody hybridoma cell 5A2 (being called for short hybridoma 5A2); on February 15th, 2012, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center and (be called for short CGMCC; address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preserving number is CGMCC No.5778.
The monoclonal antibody of described hybridoma 5A2 secretion also belongs to protection scope of the present invention.
Described 3-Jia based quinoxaline-2 carboxylic acid artificial antigen can be used for detecting olaquindox metabolite (as carboxylic acid Huo quinoxaline-2,3-Jia based quinoxaline-2 carboxylic acid).
Described antibody (monoclonal antibody or polyclonal antibody) can be used for detecting olaquindox metabolite (as carboxylic acid Huo quinoxaline-2,3-Jia based quinoxaline-2 carboxylic acid).
The present invention has great value for the detection of olaquindox metabolite (3-Jia based quinoxaline-2 carboxylic acid).
Accompanying drawing explanation
Fig. 1 is the ultraviolet spectrogram of 3-Jia based quinoxaline-2 carboxylic acid artificial antigen.
The canonical plotting of Fig. 2 for adopting 3-Jia based quinoxaline-2 carboxylic acid 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.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.
3-Jia based quinoxaline-2-carboxylic acid is purchased from German Dr.Ehrenstorfer company, and catalog number is 81121, and formula (III) is shown in structural representation.
DMF (DMF) is suc as formula shown in (IV).
N-hydroxy-succinamide (NHS) is as shown in formula V.
Formula (V)
1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) is suc as formula shown in (VI).
Embodiment 1, preparation 3-Jia based quinoxaline-2-carboxylic acid haptens
One, the haptenic preparation of 3-Jia based quinoxaline-2-carboxylic acid
1, take 3-Jia based quinoxaline-2-carboxylic acid 10mg, be placed in 10mL reaction flask; Add proper amount of acetone and a small amount of DMF to make it dissolve (acetone dissolves 3-Jia based quinoxaline-2-carboxylic acid as reaction system, and the effect of DMF is to promote to dissolve) completely, add thionyl chloride 10 μ L (acting as activated carboxyl), heating reflux reaction 1h; Then add normal hexane 0.5ml and blow to constant volume with nitrogen, then add normal hexane 0.5ml and blow to constant volume with nitrogen, then add normal hexane 0.5ml and blow to constant volume with nitrogen, obtaining solution I.
2, take 20mg γ-aminobutyric acid, be dissolved in the KOH aqueous solution (dissolving γ-aminobutyric acid as reaction system) of 1ml 2mol/L, add 0.5ml pyridine (catalyzer reacting as 3-Jia based quinoxaline-2-carboxylic acid and γ-aminobutyric acid), obtain solution II.
3, in ice bath, solution I is dropwise joined in solution II, stirring reaction 1.5h, then with 6mol/L HCl, adjust pH to 6.0 left and right, with twice of dichloromethane extraction (each 5ml), merge the organic phase of twice extraction, wash with water 3 times, after organic phase is dry with anhydrous acid sodium, carry out concentrated by rotary evaporation, obtain 15mg product, be 3-Jia based quinoxaline-2-carboxylic acid haptens, hereinafter to be referred as MQCA-GABA.
Two, the haptenic sign of 3-Jia based quinoxaline-2-carboxylic acid
Product prepared by step 1 carries out ultimate analysis, and result is as follows:
C：61.51；H：5.52；N：15.40；O：17.57
Result shows, product prepared by step 1 is compound shown in formula (I).
The Preparation and characterization of embodiment 2,3-Jia based quinoxaline-2-carboxylic acid artificial antigen
One, the immunogenic synthetic and sign of 3-Jia based quinoxaline-2-carboxylic acid
1, the immunogenic preparation of 3-Jia based quinoxaline-2-carboxylic acid
(1) shown in formula 10mg embodiment 1 being prepared (I), 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 PBS damping fluid, fully dissolve, be solution IV.
(3) solution III is slowly dropped in solution IV, after slowly stirring 24h, enter dialysis tubing, 4 ℃ of dialysis 72h (water is changed 6 times in centre) in physiological saline, then under 4 ℃ of conditions, the centrifugal 30min of 8000rmp, gets supernatant, i.e. 3-Jia based quinoxaline-2-carboxylic acid immunogen solution, be sub-packed in ampere bottle-20 ℃ of preservations.3-Jia based quinoxaline-2-carboxylic acid immunogen is called for short MQCA-BSA, and 3-Jia based quinoxaline-2-carboxylic acid immunogen solution is called for short MQCA-BSA solution.
(4) after MQCA-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 MQCA-BSA concentration in former MQCA-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.MQCA-BSA concentration in MQCA-BSA solution is 6.2mg/ml.
2, the evaluation of 3-Jia based quinoxaline-2-carboxylic acid artificial antigen
By PBS damping fluid dilution (concentration that makes MQCA-BSA is 5mg/mL) for MQCA-BSA solution, as solution first; Using the PBS damping fluid containing 5mg/mL MQCA-GABA 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 comparing solution first with solution third, compound and BSA success coupling are described.
The maximum absorption wave long value of solution second is 297nm, and the maximum absorption wave long value of solution third is 280nm.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), calculate the optical extinction coefficient (K) of each compound.
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, with concentration value, divided by molecular weight, obtain the volumetric molar concentration of this compound, calculate coupling ratio, shown in formula (I), the coupling ratio of compound and BSA is 11: 1, and compound shown in 11 formulas (I) is in conjunction with 1 BSA.
Two, the Preparation and characterization of 3-Jia based quinoxaline-2-carboxylic acid coating antigen
1, the preparation of 3-Jia based quinoxaline-2-carboxylic acid coating antigen
With ovalbumin, replace bovine serum albumin, other is with 1 of step 1.
3-Jia based quinoxaline-2-carboxylic acid coating antigen is called for short MQCA-OVA, and 3-Jia based quinoxaline-2-carboxylic acid coating antigen solution is called for short MQCA-OVA solution.
MQCA-OVA concentration in MQCA-OVA solution is 3.2mg/ml.
2, the sign of 3-Jia based quinoxaline-2-carboxylic acid coating antigen
With MQCA-OVA, replace MQCA-BSA, with OVA, replace BSA, other is with 2 of step 1.
Shown in formula (I), the coupling ratio of compound and OVA is 8: 1, and compound shown in 8 formulas (I) is in conjunction with 1 OVA.
The preparation of embodiment 3,3-Jia based quinoxaline-2-carboxylic acid monoclonal antibody
Balb/c mouse: be purchased from Beijing Vital River Experimental Animals Technology Co., Ltd.;
SP2/0 myeloma cell: purchased from Siggma-Aldrich company, catalog number is 08060101.
One, animal immune
By the MQCA-BSA solution immunity Balb/c mouse of embodiment 2 preparations, every mouse single immunization 100 μ gMQCA-BSA, immunity is 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 secreting the hybridoma cell strain of 3-Jia based quinoxaline-2-carboxylic acid monoclonal antibody.By the anti-olaquindox metabolite of strain of hybridoma strain called after monoclonal antibody hybridoma cell 5A2 (being called for short hybridoma 5A2), on February 15th, 2012, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center and (be called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preserving number is CGMCC No.5778.
Three, cell cryopreservation and recovery
With frozen storing liquid, hybridoma 5A2 is made to 1 * 10
6the cell suspension of individual/ml is preserved for a long time in liquid nitrogen.During recovery, take out cryopreservation tube, put into immediately 37 ℃ 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 5A2 is placed in to cell culture medium, cultivates 2 days for 37 ℃, by sad-saturated ammonium sulphate method, the nutrient solution obtaining is carried out to purifying, obtain monoclonal antibody solution (20 ℃ of preservations).
Protein concn in monoclonal antibody (mg/ml)=1.45 * OD
280-0.74 * OD
260.
Adopting above formula to calculate the protein concn in monoclonal antibody, is 24.1mg/ml.
2, ascites preparation
Balb/c mouse peritoneal injection sterilizing paraffin oil (0.4mL/ only).7 days pneumoretroperitoneum injection hybridoma 5A2 (5 * 10
5individual/only).After 7 days, gather ascites, by sad-saturated ammonium sulphate method, carry out purifying, ℃ preservation of ascites-20 after purifying.
Five, the evaluation 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, utilize using non-competitive ELISA method to measure the avidity of monoclonal antibody
(1) use MQCA-OVA as coating antigen coated elisa plate
Adopt the MQCA-OVA solution (diluting with carbonate buffer solution) of embodiment 2 preparations to be coated with, 100 μ L/ holes; The coated concentration of following MQCA-OVA 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 ℃.
(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; Every kind of diluent arranges three multiple holes.
(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.
The natural logarithm value of the protein concn (mol/L) in monoclonal antibody of take is X-coordinate, take its corresponding absorbancy to make curve 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 antibody concentration corresponding to each curve 50%ODMAX.Adopt 1 μ g/mL to be coated with concentration, the antibody concentration that 50%ODMAX is corresponding is 4.2 * 10
-12mol/L.Adopt 0.5 μ g/mL to be coated with concentration, the antibody concentration that 50%ODMAX is corresponding is 19.4 * 10
-12mol/L.Adopt 0.25 μ g/mL to be coated with concentration, the antibody concentration that 50%ODMAX is corresponding is 185.7 * 10
-12mol/L.Adopt 0.125 μ g/mL to be coated with concentration, the antibody concentration that 50%ODMAX is corresponding is 415.8 * 10
-12mol/L.
By one group between two of 4 concentration, according to formula, calculate the affinity costant of monoclonal antibody
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 4.2 * 10
-12mol/L, 0.5 μ g/mL is coated with concentration, 50%OD
450corresponding antibody concentration is 19.4 * 10
-12mol/L, Ka=(2-1)/2 (2 * 19.4 * 10
-12-4.2 * 10
-12)=14.4 * 10
9m
-1.The like, obtain all the other 5 Ka values, be respectively 1.4 * 10
9m
-1, 0.7 * 10
9m
-1, 2.0 * 10
9m
-1, 0.9 * 10
9m
-1, 1.0 * 10
9m
-1, the affinity costant that calculates monoclonal antibody of averaging is 3.4 * 10
9m
-1.
3, the calculating of monoclonal antibody sensitivity
(1) adopt the MQCA-OVA solution (diluting with carbonate buffer solution) of embodiment 2 preparations to be coated with, 100 μ L/ holes; The coated concentration of MQCA-OVA is 1.0 μ g/mL.
Hatch 16 hours for (2) 4 ℃.
(3) seal and wash plate.
(4) every hole adds 50 μ L 3-Jia based quinoxaline-2-carboxylic acid standard solutions (3-Jia based quinoxaline-2-carboxylic acid and PBS damping fluid, to consist of; The concentration of 3-Jia based quinoxaline-2-carboxylic acid is respectively 0.5 μ g/L, 1.5 μ g/L, 4.5 μ g/L, 13.5 μ g/L, 40.5 μ g/L; By hole in contrast, the hole that only adds PBS damping fluid), 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 the standard solution that adopts each concentration is obtained (mean values in three multiple holes) is multiplied by 100 as ordinate zou again divided by the light absorption value of control wells, take the natural logarithm value of the 3-Jia based quinoxaline-2-carboxylic acid concentration (μ g/L) in each standard solution as X-coordinate curve plotting figure, see Fig. 2.
Contrast Fig. 2, when obtaining Y value and equaling 50%, corresponding 3-Jia based quinoxaline-2-carboxylic acid concentration is IC50 value.Monoclonal antibody detects the sensitivity (IC of 3-Jia based quinoxaline-2-carboxylic acid
50value) be 3.1ng/mL.
4, the calculating of cross reacting rate
(1) to (3) with (1) of step 3 to (3).
(4) every hole adds 50 μ L analog standard solutions (analog and PBS damping fluid, to consist of; The concentration of analog is respectively 0.5 μ g/L, 1.5 μ g/L, 4.5 μ g/L, 13.5 μ g/L, 40.5 μ g/L; By hole in contrast, the hole that only adds PBS damping fluid), each concentration arranges 3 multiple holes.
(5) to (10) with (5) of step 3 to (10).
The light absorption value that the analog that adopts each concentration is obtained (mean values in three multiple holes) is multiplied by 100 as ordinate zou again divided by the light absorption value of control wells, and the natural logarithm value of the similar substrate concentration (μ g/L) in each standard solution of take is 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.
Table 1 cross reacting rate
| Purchase approach | IC 50 | Cross reacting rate | |
| MQCA | Dr.Ehrenstorfer company, article No. is 81121 | 3.1 | 100％ |
| QCA | Huifeng, Wuhan reaches, article No. 87-96-52 | 7.2 | 43.2％ |
| Olaquindox | Dr.Ehrenstorfer GmbH company, article No. C 15716500 | Nothing | ＜0.1％ |
| Quinocetone | Dr.Ehrenstorfer GmbH company, article No. C 16709000 | Nothing | ＜0.1％ |
| Carbadox | Sigma company, article No. C6770 | Nothing | ＜0.1％ |
| Mequindox | Sigma-Aldrich company, article No. MO-189 | Nothing | ＜0.1％ |
Embodiment 4, the preparation of 3-Jia based quinoxaline-2-carboxylic acid polyclonal antibody and ELISA competition suppress experiment
New zealand white rabbit: be purchased from Beijing Vital River Experimental Animals Technology Co., Ltd..
New zealand white rabbit be take to the MQCA-BSA solution of embodiment 2 preparation and carry out immunity (immunization ways is the subcutaneous multi-point injection of nape portion).Every each immune 1mg of rabbit (in BSA amount), once, immunity is 7 times altogether in immunity in every two weeks, and immunity for the third time starts, after each immunity the 7th day, ear edge vein exploitating blood, gets serum, and polyclonal antibody, is put in-20 ℃ of environment and preserves.
Set up indirect ELISA method (see embodiment 3 step 53; Adopt 3-Jia based quinoxaline-2-carboxylic acid standard solution of 100ng/mL, by hole in contrast, the hole that only adds PBS damping fluid), with the serum of doubling dilution, replace monoclonal antibody solution.
Analytical results shows, compares with control wells, adds the light absorption value in each hole of 3-Jia based quinoxaline-2-carboxylic acid standard solution obviously to decline, and along with the increase of serum diluting multiple, light absorption value is the gradient of significantly successively decreasing.The rabbit anteserum that acquisition is described can be identified 3-Jia based quinoxaline-2-carboxylic acid specifically.
Claims (3)
1. anti-olaquindox metabolite monoclonal antibody hybridoma cell 5A2, its deposit number is CGMCC No.5778.
2. the monoclonal antibody that described in claim 1, hybridoma cell strain is secreted.
3. the application of monoclonal antibody in detecting olaquindox metabolite described in claim 2.
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| CN103146652B (en) * | 2013-02-19 | 2015-01-07 | 中国农业科学院兰州畜牧与兽药研究所 | Anti-olaquindox monoclonal antibody and its hybridoma cell line, preparation methods of antibody and cell line, and kit for detecting olaquindox in forage |
| CN103360328B (en) * | 2013-07-30 | 2015-12-02 | 中国农业大学 | A kind of desoxyquinocetone haptens and preparation method thereof and its application |
| CN103601662B (en) * | 2013-11-21 | 2016-04-06 | 深圳市药品检验所 | A kind of melatonin haptens, melatonin complete antigen and its preparation method and application |
| CN108426996B (en) * | 2017-02-15 | 2020-09-15 | 江苏美正生物科技有限公司 | Rapid detection kit for 3-methyl quinoxaline-2-carboxylic acid residues and preparation method and application thereof |
| CN109180519B (en) * | 2018-06-22 | 2021-08-03 | 华南农业大学 | Olaquindox metabolite antigen, antibody, enzyme-linked immunosorbent assay kit and detection method |
| CN109734675B (en) * | 2019-01-23 | 2021-02-26 | 北京市兽药监察所 | Method and product suitable for detecting olaquindox content in veterinary drug preparation |
| CN110927383A (en) * | 2019-10-08 | 2020-03-27 | 杭州佰昕科技有限公司 | Fluorescent microsphere immunochromatography test strip for detecting olaquindox residue and application thereof |
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| 冀宝庆.喹乙醇及其代谢残留的免疫检测技术研究.《中国优秀硕士学位论文全文数据库 工程科技I辑》.2009,(第4期),摘要、第23、25、28－39页. |
| 喹乙醇及其代谢残留的免疫检测技术研究;冀宝庆;《中国优秀硕士学位论文全文数据库 工程科技I辑》;20090415(第4期);摘要、第23、25、28－39页 * |
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