CN102659693A - 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 suppress multiple gram-positive microorganism and negative bacterium, and poultry, fowl, flesh of fish class etc. had tangible growth promoting function ， quinoxaline medicine (comprising medicines such as olaquindox, mequindox, Quinocetone) in the application of China very extensively.Discover that ， quinoxaline original shape medicine and metabolite exist tangible safety issue, have toxic side effect such as tangible teratogenesis, carcinogenic, mutagenesis, photosensitive and adrenal cortex infringement, serious harm the health of humans and animals.
European Union forbids in feed, adding olaquindox in dispatch in 1998, uses the pig that China only is approved for below the 35kg.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 confirms the De quinoxaline medicine residue.The 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.China Ministry of Agriculture announces regulation No. 235, and the olaquindox MRL is olaquindox+3-Jia based quinoxaline-2-carboxylic acid: 4ug/kg (muscle), 50ug/kg (liver), 2mg/kg (feed).
When China carries out residual monitoring, often adopt immunochemical analyses to carry out primary dcreening operation, instrumental method is proved conclusively.Immunochemical analyses since special advantages aspect the qualitative, quantitative of antigen-antibody and easy and simple to handle fast, cost is low, sensitivity is higher, the analyzing samples amount is big advantage remedied the deficiency of physico-chemical analysis.The basic factor that influences the immunochemical analyses quality is the specificity and the affinity of antibody; These character are decided by the structure of immune hapten molecule again, and therefore immune haptenic molecular designing is exactly the step that produces specific antibody and the most basic and most critical of setting up small molecules residue of veterinary drug Fast Detection Technique with synthesizing.
Summary of the invention
The antibody that the purpose 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 the formula (I).Compound shown in the formula (I) had both at utmost kept the feature structure of 3-Jia based quinoxaline-2 carboxylic acid for 3-Jia based quinoxaline-2 carboxylic acid is carried out the compound that structure of modification obtains, have again can with carrier proteins generation link coupled reactive group.
The present invention goes back the preparation method of compound shown in the protection (I), comprises the steps: 3-Jia based quinoxaline-2 carboxylic acid and γ-An Jidingsuan reaction are obtained said compound.The mass ratio of said 3-Jia based quinoxaline-2 carboxylic acid and said γ-An Jidingsuan specifically can be 1: 2.Said 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 the formula (I) and carrier protein couplet are obtained.Said carrier proteins is bovine serum albumin or oralbumin.The coupling ratio of compound and carrier proteins specifically can be (11-8) shown in the formula (I): 1.Said coupling ratio refers to mol ratio.Compound and said carrier proteins shown in the formula (I) specifically can pass through the active ester method coupling.Said 3-Jia based quinoxaline-2 carboxylic acid artificial antigen is suc as formula shown in (II).Said 3-Jia based quinoxaline-2 carboxylic acid artificial antigen can be used as immunogen and also can be used as coating antigen.With said 3-Jia based quinoxaline-2 carboxylic acid artificial antigen immune animal, can obtain the monoclonal antibody and the polyclonal antibody of high specific, easy, the easy row of method.With said 3-Jia based quinoxaline-2 carboxylic acid artificial antigen coated elisa plate, also can be used for the sero-fast detection of olaquindox metabolite (3-Jia based quinoxaline-2 carboxylic acid).The structural difference of coating antigen and immunogen can further improve the sensitivity and the specificity of detection.
Said 3-Jia based quinoxaline-2 carboxylic acid artificial antigen can be used for preparing 3-Jia based quinoxaline-2 carboxylic acid specific antibody.Said antibody can be monoclonal antibody or polyclonal antibody.
With said 3-Jia based quinoxaline-2 carboxylic acid artificial antigen is that the antibody that immunogen preparing obtains also belongs to protection scope of the present invention.Said 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); Be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on February 15th, 2012 and (be called for short CGMCC; The address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preserving number is CGMCC No.5778.
Said hybridoma 5A2 excretory monoclonal antibody also belongs to protection scope of the present invention.
Said 3-Jia based quinoxaline-2 carboxylic acid artificial antigen can be used for detecting olaquindox metabolite (like 3-Jia based quinoxaline-2 carboxylic acid Huo quinoxaline-2 carboxylic acid).
Said antibody (monoclonal antibody or polyclonal antibody) can be used for detecting olaquindox metabolite (like 3-Jia based quinoxaline-2 carboxylic acid Huo quinoxaline-2 carboxylic acid).
The present invention has great value for the detection of olaquindox metabolite (3-Jia based quinoxaline-2 carboxylic acid).
Description of drawings
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 among the following embodiment like no specified otherwise, is ordinary method.Used test materials among the following embodiment like no specified otherwise, is to buy from routine biochemistry reagent shop and obtains.Quantitative test in following examples all is provided with repeated experiments three times, results averaged.Used PBS damping fluid is the PBS damping fluid of pH7.4,0.01M among the embodiment.Used carbonate buffer solution is the sodium carbonate buffer of pH9.6,0.05mol/L among the embodiment.Bovine serum albumin is called for short BSA.Ovalbumin is called for short OVA.
3-Jia based quinoxaline-2-carboxylic acid is available from German Dr.Ehrenstorfer company, and catalog number is 81121, and formula (III) is seen in structural representation.
N, dinethylformamide (DMF) is suc as formula shown in (IV).
N-hydroxy-succinamide (NHS) is 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 haptin
One, 3-Jia based quinoxaline-haptenic preparation of 2-carboxylic acid
1, takes by weighing 3-Jia based quinoxaline-2-carboxylic acid 10mg, place the 10mL reaction flask; The adding proper amount of acetone makes it with a small amount of DMF dissolves fully (acetone is as reaction system dissolving 3-Jia based quinoxaline-2-carboxylic acid, and the effect of DMF is to promote to dissolve), adds THIONYL CHLORIDE 97 10 μ L (acting as activated carboxyl), heating reflux reaction 1h; Add normal hexane 0.5ml then and blow to constant volume, add normal hexane 0.5ml then and blow to constant volume, add normal hexane 0.5ml then and blow to constant volume, obtain solution I with nitrogen with nitrogen with nitrogen.
2, take by weighing the 20mg γ-An Jidingsuan; Be dissolved in the KOH aqueous solution (as reaction system dissolving γ-An Jidingsuan) of 1ml 2mol/L; Add 0.5ml pyridine (catalyzer that reacts as 3-Jia based quinoxaline-2-carboxylic acid and γ-An Jidingsuan), obtain solution II.
3, in ice bath, solution I is dropwise joined in the solution II, stirring reaction 1.5h transfers about pH to 6.0 with 6mol/L HCl then; With twice of dichloromethane extraction (each 5ml), merge the organic phase of twice extraction, with water washing 3 times; Organic phase is revolved inspissation after with anhydrous acid sodium drying to contract; Obtain the 15mg product, be 3-Jia based quinoxaline-2-carboxylic acid haptin, hereinafter to be referred as MQCA-GABA.
Two, 3-Jia based quinoxaline-haptenic sign of 2-carboxylic acid
Product to the step 1 preparation carries out ultimate analysis, and the result is following:
C：61.51；H：5.52；N：15.40；O：17.57
The result shows that the product of step 1 preparation is a compound shown in the formula (I).
Preparation and the sign 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
1,3-Jia based quinoxaline-immunogenic preparation of 2-carboxylic acid
(1) compound is dissolved in 2mL N shown in the formula that 10mg embodiment 1 is prepared (I); In N '-dimethylformamide; Add 10mg N-hydroxy-succinamide and 10mg 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, the room temperature lower magnetic force stirs 2h, obtains solution III.
(2) the 30mg bovine serum albumin is added in the 2mL PBS damping fluid, fully dissolving is solution IV.
(3) solution III is slowly dropped in the solution IV, go into dialysis tubing after slowly stirring 24h, 4 ℃ of dialysis 72h (water is changed 6 times in the centre) in saline water; 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 the 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) with MQCA-BSA solution with after the PBS damping fluid dilution; Measure the spectrophotometric value of 280nm and 260nm; By formula calculate the protein concentration in the diluent, the protein concentration that records is on duty to be the MQCA-BSA concentration in the former MQCA-BSA solution behind its extension rate.Protein concn (mg/ml)=1.45 * OD
280-0.74 * OD
260MQCA-BSA concentration in the MQCA-BSA solution is 6.2mg/ml.
2, the evaluation of 3-Jia based quinoxaline-2-carboxylic acid artificial antigen
MQCA-BSA solution is diluted (concentration that makes MQCA-BSA is 5mg/mL) with the PBS damping fluid, as the solution first; The PBS damping fluid that will contain 5mg/mL MQCA-GABA is as solution second; The PBS damping fluid that will contain 5mg/mL BSA is as solution third.Respectively solution first, solution second and solution third are carried out ultraviolet (200-380nm) spectral scan, the uv scan result sees Fig. 1.Considerable change has taken place in the uv-spectrogram of comparing the solution first with solution third, and compound and BSA success coupling is described.
The maximum absorption wave long value of solution second is 297nm, 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 the maximum absorption wave long value, and C is a strength of solution, and L is the thickness of liquid layer).
Adopt the maximum absorption wave long value of solution second and solution third that the solution first is carried out uv scan respectively; And according to the concentration of this compound of optical extinction coefficient backwards calculation in the solution first of this compound that has calculated; Obtain the volumetric molar concentration of this compound divided by molecular weight with concentration value; Calculate coupling ratio, the coupling ratio of compound and BSA is 11: 1 shown in the formula (I), and promptly compound shown in 11 formulas (I) combines 1 BSA.
Two, the preparation and the sign of 3-Jia based quinoxaline-2-carboxylic acid coating antigen
1, the preparation of 3-Jia based quinoxaline-2-carboxylic acid coating antigen
Replace bovine serum albumin with ovalbumin, 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 the MQCA-OVA solution is 3.2mg/ml.
2, the sign of 3-Jia based quinoxaline-2-carboxylic acid coating antigen
Replace MQCA-BSA with MQCA-OVA, replace BSA with OVA, other is with 2 of step 1.
The coupling ratio of compound and OVA is 8: 1 shown in the formula (I), and promptly compound shown in 8 formulas (I) combines 1 OVA.
Embodiment 3,3-Jia based quinoxaline-2-carboxylic acid MONOCLONAL ANTIBODIES SPECIFIC FOR
Balb/c mouse: purchase in Beijing Vital River Experimental Animals Technology Co., Ltd.;
SP2/0 myeloma cell: available from Siggma-Aldrich company, catalog number is 08060101.
One, animal immune
MQCA-BSA solution immunity Balb/c mouse with embodiment 2 preparations; Every mouse single immunization 100 μ gMQCA-BSA, immunity is 4 times altogether, each two weeks at interval; The immunization ways of first three time is the subcutaneous multi-point injection of nape portion, and back three times immunization ways is a 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 that cloning is carried out in positive hole, obtain to secrete the hybridoma cell strain of 3-Jia based quinoxaline-2-carboxylic acid monoclonal antibody.With the anti-olaquindox metabolite of strain of hybridoma strain called after monoclonal antibody hybridoma cell 5A2 (being called for short hybridoma 5A2); Be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on February 15th, 2012 and (be called for short CGMCC; The 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 processed 1 * 10
6The cell suspension of individual/ml is in the medium-term and long-term preservation of liquid nitrogen.During recovery, take out frozen pipe, put into 37 ℃ of water-bath middling speeds immediately and melt, move in the culturing bottle behind the centrifugal removal frozen storing liquid and cultivate.
Four, MONOCLONAL ANTIBODIES SPECIFIC FOR and purifying
1, increment culture method
The preparation method of cell culture medium (7.4): in the RPMI-1640 substratum, add calf serum and sodium hydrogencarbonate, the final concentration of calf serum is 20% (quality percentage composition), and the final concentration of sodium hydrogencarbonate is 0.2% (quality percentage composition).
5A2 places cell culture medium with hybridoma, cultivates 2 days for 37 ℃, with sad-saturated ammonium sulphate method the nutrient solution that obtains is carried out purifying, obtains monoclonal anti liquid solution (20 ℃ of preservations).
Protein concn in the monoclonal antibody (mg/ml)=1.45 * OD
280-0.74 * OD
260
Adopt above formula to calculate the protein concn in the monoclonal antibody, be 24.1mg/ml.
2, ascites preparation
Balb/c mouse peritoneal injection sterilization Yellow Protopet 2A (0.4mL/ only).7 days pneumoretroperitoneum injection hybridoma 5A2 (5 * 10
5Individual/only).Gather ascites after 7 days, carry out purifying, the ascites behind the purifying-20 ℃ preservation with sad-saturated ammonium sulphate method.
Five, the evaluation of monoclonal antibody
The monoclonal anti liquid solution that 1 of step 4 obtains is identified respectively as follows:
1, adopt ELISA monoclonal antibody hypotype detection kit (Sigma Company products, catalog number are 19285) to detect the hypotype of monoclonal antibody, the immunoglobulin subclass of monoclonal antibody is IgG1.
2, utilize noncompetitive ELISA method to measure the avidity of monoclonal antibody
(1) with MQCA-OVA as the coating antigen coated elisa plate
Adopt the MQCA-OVA solution (diluting) of embodiment 2 preparations to encapsulate 100 μ L/ holes with carbonate buffer solution; Following MQCA-OVA is set respectively encapsulates concentration: 1 μ g/mL, 0.5 μ g/mL, 0.25 μ g/mL, 0.125 μ g/mL.
Hatched 16 hours for (2) 4 ℃.
(3) seal and wash plate.
(4) every hole adds the diluent (diluting with the PBS damping fluid) of 100 μ L monoclonal anti liquid solutions; Protein concn in the 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 is provided with 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 colour developing liquid, lucifuge colour developing 15min.
(9) every hole adds 100 μ L 2mol/L sulfuric acid stopped reactions; Read OD
450Value.
Natural logarithm value with the protein concn in the monoclonal antibody (mol/L) is an X-coordinate, is that ordinate zou is made curve with its corresponding absorbancy.
Each antigen coated concentration obtains 1 S type curve, obtains 4 S type curves altogether.Find out the top of S curve, corresponding OD
450Value is set at ODMAX.Find out the corresponding AC of each bar curve 50%ODMAX respectively.Adopt 1 μ g/mL to encapsulate concentration, the AC that 50%ODMAX is corresponding is 4.2 * 10
-12Mol/L.Adopt 0.5 μ g/mL to encapsulate concentration, the AC that 50%ODMAX is corresponding is 19.4 * 10
-12Mol/L.Adopt 0.25 μ g/mL to encapsulate concentration, the AC that 50%ODMAX is corresponding is 185.7 * 10
-12Mol/L.Adopt 0.125 μ g/mL to encapsulate concentration, the AC that 50%ODMAX is corresponding is 415.8 * 10
-12Mol/L.
With one group in twos 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 the formula, two multiples that encapsulate concentration during n is every group, [Ab] t
1, [Ab] t
2Be respectively two ACs (mol/L) that 50%ODMAX is corresponding in every group.For example: 1 μ g/mL encapsulates concentration, 50%OD
450Corresponding AC is 4.2 * 10
-12Mol/L, 0.5 μ g/mL encapsulates concentration, 50%OD
450Corresponding AC is 19.4 * 10
-12Mol/L, Ka=(2-1)/2 (2 * 19.4 * 10
-12-4.2 * 10
-12)=14.4 * 10
9M
-1And 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, monoclonal antibody Sensitivity calculation
(1) adopt the MQCA-OVA solution (diluting) of embodiment 2 preparations to encapsulate 100 μ L/ holes with carbonate buffer solution; The concentration that encapsulates of MQCA-OVA is 1.0 μ g/mL.
Hatched 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 and (is made up of 3-Jia based quinoxaline-2-carboxylic acid and PBS damping fluid; 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; With the hole that only adds the PBS damping fluid as control wells), each concentration is provided with 3 multiple holes.
(5) every hole adds the monoclonal anti liquid solution that 1 of 50 μ L step 4 obtain.
(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 colour developing liquid, lucifuge colour developing 15min.
(10) every hole adds 100 μ L 2mol/L sulfuric acid stopped reactions; Read OD
450Value.
The light absorption value (MVs in three multiple holes) that the standard solution that adopts each concentration is obtained multiply by 100 as ordinate zou again divided by the light absorption value of control wells; Natural logarithm value with the 3-Jia based quinoxaline-2-carboxylic acid concentration (μ g/L) in each standard solution is X-coordinate curve plotting figure, sees Fig. 2.
Map 2 obtains Y value and equals 50% o'clock corresponding 3-Jia based quinoxaline-2-carboxylic acid concentration and be the 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 and (is made up of analog and PBS damping fluid; 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; With the hole that only adds the PBS damping fluid as control wells), each concentration is provided with 3 multiple holes.
(5) to (10) with (5) of step 3 to (10).
The light absorption value (MVs in three multiple holes) that the analog that adopts each concentration is obtained multiply by 100 as ordinate zou again divided by the light absorption value of control wells, is X-coordinate curve plotting figure with the natural logarithm value of the similar substrate concentration in each standard solution (μ g/L).Control curve figure obtains Y value and equals corresponding similar substrate concentration (μ g/L), the i.e. IC of analog at 50% o'clock
50Value.
Table 1 cross reacting rate
| ? | The purchase approach | IC 50 | Cross reacting rate |
| MQCA | Dr.Ehrenstorfer company, article No. is 81121 | 3.1 | 100％ |
| QCA | The Huifeng, Wuhan reaches, article No. 87-96-52 | 7.2 | 43.2％ |
| Olaquindox | Dr.Ehrenstorfer GmbH company, article No. C 15716500 | Do not have | ＜0.1％ |
| Quinocetone | Dr.Ehrenstorfer GmbH company, article No. C 16709000 | Do not have | ＜0.1％ |
| Carbadox | Sigma company, article No. C6770 | Do not have | ＜0.1％ |
| Mequindox | Sigma-Aldrich company, article No. MO-189 | Do not have | ＜0.1％ |
Embodiment 4,3-Jia based quinoxaline-2-carboxylic acid polyclonal antibody preparation and ELISA competition suppress experiment
New zealand white rabbit: purchase in Beijing Vital River Experimental Animals Technology Co., Ltd..
New zealand white rabbit is carried out immunity (immunization ways is the subcutaneous multi-point injection of nape portion) with the MQCA-BSA solution that embodiment 2 prepares.Every each immune 1mg of rabbit (in the BSA amount), per two all immunity once, immunity is 7 times altogether, immunity beginning for the third time, after each immunity the 7th day, ear edge vein exploitating blood is got serum, promptly polyclonal antibody is put in-20 ℃ of environment and preserves.
Set up indirect ELISA method (see embodiment 3 step 53; Adopt 3-first based quinoxaline-2-carboxylic acid standard solution of 100ng/mL, with the hole that only adds the PBS damping fluid as control wells), with the serum replacement monoclonal anti liquid solution of doubling dilution.
Analytical results shows, compares with control wells, and the light absorption value that adds each hole of 3-Jia based quinoxaline-2-carboxylic acid standard solution obviously descends, and along with the increase of serum diluting multiple, light absorption value is the gradient of significantly successively decreasing.Explain that the rabbit anteserum that obtains can discern 3-Jia based quinoxaline-2-carboxylic acid specifically.
Claims (10)
1. compound shown in the formula (I);
2. the preparation method of compound shown in the formula (I) comprises the steps: 3-Jia based quinoxaline-2-carboxylic acid and γ-An Jidingsuan reaction are obtained said compound.
3. the conjugate of compound and carrier proteins shown in the formula (I).
4. according to right 3 described conjugates, it is characterized in that: said carrier proteins is bovine serum albumin or oralbumin.
5. claim 3 or the 4 said conjugates application in preparation 3-Jia based quinoxaline-2-carboxylic acid specific antibody.
6. application as claimed in claim 5 is characterized in that: said antibody is monoclonal antibody or polyclonal antibody.
7. be the monoclonal antibody that immunogen preparing obtains with claim 3 or 4 said conjugates.
8. anti-olaquindox metabolite monoclonal antibody hybridoma cell 5A2, its deposit number is CGMCC No.5778.
9. the said hybridoma cell strain excretory of claim 8 monoclonal antibody.
10. claim 3 or 4 said conjugates, or, the application in detecting the olaquindox metabolite of claim 7 or 9 said monoclonal antibodies.
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| CN108426996A (en) * | 2017-02-15 | 2018-08-21 | 江苏美正生物科技有限公司 | A kind of quick detection kit and its preparation method and application that 3- Jia based quinoxalines -2- is carboxylic acid remained |
| CN109180519A (en) * | 2018-06-22 | 2019-01-11 | 华南农业大学 | A kind of olaquindox metabolite antigen, antibody and enzyme-linked immunologic detecting kit and detection method |
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| 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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| CN103146652A (en) * | 2013-02-19 | 2013-06-12 | 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 |
| 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 |
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| 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 |
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| 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 |
| CN108426996A (en) * | 2017-02-15 | 2018-08-21 | 江苏美正生物科技有限公司 | A kind of quick detection kit and its preparation method and application that 3- Jia based quinoxalines -2- is carboxylic acid remained |
| 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 |
| CN109180519A (en) * | 2018-06-22 | 2019-01-11 | 华南农业大学 | A kind of olaquindox metabolite antigen, antibody and enzyme-linked immunologic detecting kit and detection method |
| 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 |
| CN109734675A (en) * | 2019-01-23 | 2019-05-10 | 北京市兽药监察所 | A kind of method and product suitable for detecting Determination of olaquindox 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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