CN102621322B - Kit for detecting 3-methyl-quinoxaline-2-carboxylic acid - Google Patents
Kit for detecting 3-methyl-quinoxaline-2-carboxylic acid Download PDFInfo
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Abstract
The invention discloses a kit for detecting 3-methyl-quinoxaline-2-carboxylic acid. The enzyme linked immunosorbent assay (ELISA) kit for detecting 3-methyl-quinoxaline-2-carboxylic acid comprises a monoclonal antibody which has an anti-preservation number of CGMCC No.5778 and is excreted by an olaquindox metabolite monoclonal antibody hybrid tumor 5A2. The kit comprises the monoclonal antibody, and a conjugate of a compound and a barrier protein, which is shown as a formula (I). The kit has the advantages of high sensitivity, high accuracy, high precision, low cost, simplicity for operation, short detection time, suitability for use by various units, simplicity for storage and long shelf life, can be used for quickly detecting a large number of samples at the same time, and can realize on-site high-flux quick detection. The ELISA kit is suitable for determining the residual quantity of olaquindox metabolites in feeds, animal tissues or excrements. The antibody, the kit and a detection method can play a major role in detection of the olaquindox metabolites.
Description
Technical field
The present invention relates to a kind of kit of the 3-of detection Jia based quinoxaline-2-carboxylic acid.
Background technology
3-Jia based quinoxaline-2-carboxylic acid (MQCA) is the metabolin that olaquindox, mequindox and quinocetone produce in animal body.By research, find that quinoxaline original shape medicine and metabolin exist obvious safety issue, there is the toxic and side effects such as obvious teratogenesis, carcinogenic, mutagenesis, photosensitive and adrenal cortex infringement, serious harm the health of humans and animals.In the world about the maximum residue limit(MRL) regulation of quinoxaline medicine is very strict.European Union's regulation carbadox and olaquindox with and metabolic product in animal food, must not detect and stipulate these two kinds of veterinary drugs forbid sell.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).
Research mainly contains chromatography (TLC), high performance liquid chromatography (HPLC) and immuno analytical method etc. both at home and abroad at present.The specificity of TLC method is poor, sensitivity is also relatively poor, required standard items concentration is higher, potential contaminative is higher, the required instrument and equipment investment of HPLC method is large, operative technique requires high, decontaminating column consumes more, testing cost is higher, immune affinity column specificity is better, but need could quantitatively detect in conjunction with HPLC instrument and fluorophotometer, detection technique requirement and cost are higher, and ELISA method Comparatively speaking, easy and simple to handle, fast, less pollution, sensitivity is also higher, be applicable to general inspection and the screening of batch sample, the kit of particularly succeeding in developing, to testing staff, bring great convenience, simultaneously, also provide cost savings, meet the current developing direction of detection field in the world.Immune colloidal gold chromatography technology does not need any instrument and equipment and reagent, is particularly suitable for detection and large area generaI investigation that grass-roots unit is pressed for time in enormous quantities, and therefore enzyme immunoassay is widely used in feed safety analysis.
Summary of the invention
The object of this invention is to provide a kind of kit of the 3-of detection Jia based quinoxaline-2-carboxylic acid.
The enzyme linked immunological kit of detection 3-Jia based quinoxaline-2-carboxylic acid provided by the invention, comprises the monoclonal antibody that anti-olaquindox metabolin monoclonal antibody hybridoma cell 5A2 secretes.Described anti-olaquindox metabolin monoclonal antibody hybridoma cell 5A2 (being called for short hybridoma 5A2) 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.5778.
Described kit can be following 1) to 4) in any one:
1) comprise the kit of compound shown in formula (I) and the conjugate of carrier protein, described monoclonal antibody and enzyme labeling antiantibody; Wherein, described conjugate is as coating antigen;
2) comprise the kit of the enzyme labeling thing of compound shown in formula (I), described monoclonal antibody and antiantibody; Wherein, described antiantibody is as coating antigen;
3) comprise the enzyme labeling thing of compound shown in formula (I) and the kit of described monoclonal antibody; Wherein, described monoclonal antibody is as coating antigen;
4) comprise the kit of compound shown in formula (I) and the conjugate of carrier protein and the enzyme labeling thing of described monoclonal antibody; Wherein, described conjugate is as coating antigen.
Described carrier protein can be ovalbumin (OVA) or bovine serum albumin(BSA) (BSA).
In described conjugate, shown in formula (I), the coupling ratio of compound and carrier protein specifically can be (11-8): 1.Described coupling ratio refers to mol ratio.Compound and described carrier protein shown in formula (I) specifically can pass through active ester method coupling.Described conjugate specifically can be suc as formula shown in (II).
Formula (II).
Described kit also comprises at least one in cleansing solution, sample concentration liquid, substrate nitrite ion and stop buffer.
Every 1 liter of cleansing solution can be prepared and obtain as follows: 10ml polysorbas20,5g sodium azide and 990ml phosphate buffer are mixed, obtain cleansing solution.Described phosphate buffer can be the phosphate buffer of pH7.2-7.6,0.005M-0.015M, and the concentration specifically can be can be pH7.4,0.01M) sodium phosphate buffer.
Described sample concentration liquid can be the phosphate buffer that concentration is 0.03mol/L-0.05mol/L, is preferably the PBS damping fluid of pH7.4,0.04mol/L.
Described substrate nitrite ion comprises nitrite ion A and nitrite ion B, can be nitrite ion A and the nitrite ion B of independent packaging, also can directly nitrite ion A and nitrite ion B equal-volume be mixed to get.Described nitrite ion can be superoxol or urea peroxide solution; Described nitrite ion B can be o-phenylenediamine (OPD) solution or tetramethyl benzidine (TMB) solution.Described nitrite ion A specifically can be 2% (g/100ml) urea peroxide aqueous solution.Described nitrite ion B specifically can be 1% (g/100ml) tetramethyl biphenyl amine aqueous solution.
Described stop buffer specifically can be 0.2M aqueous sulfuric acid.
Arbitrary described kit all can be used for detecting olaquindox metabolin (as carboxylic acid Huo quinoxaline-2,3-Jia based quinoxaline-2 carboxylic acid) above.
Arbitrary described kit all can be used for detecting in sample to be tested, whether to contain olaquindox metabolin (as carboxylic acid Huo quinoxaline-2,3-Jia based quinoxaline-2 carboxylic acid) above.
The present invention adopts 3-Jia based quinoxaline-2-carboxylic acid monoclonal antibody of high specific, and the method for inspection is convenient and easy, has that specificity is high, highly sensitive, degree of accuracy is high, accuracy high.Kit of the present invention and detection method require low to the pre-treatment of sample, sample pretreatment process is simple, simultaneously fast detecting batch samples; That kit of the present invention has advantages of is highly sensitive, accuracy is high, precision is high, cost is low, simple to operate, detection time is short, be applicable to various units uses, stores simple, long shelf-life; Fast detecting batch samples, can realize on-the-spot high flux fast detecting simultaneously.Enzyme linked immunological kit provided by the invention, is applicable to measure the residual quantity of olaquindox metabolin in feed, animal tissue or excreta.Antibody of the present invention, kit and detection method will be brought into play significant role in the detection of olaquindox metabolin.
Accompanying drawing explanation
Fig. 1 is the ultraviolet spectrogram of 3-Jia based quinoxaline-2-carboxylic acid artificial antigen.
Fig. 2 is the canonical plotting that adopts 3-Jia based quinoxaline-2-carboxylic acid to make.
Fig. 3 is the canonical plotting of kit.
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 conventional method.Test material 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.PBS damping fluid used in embodiment, if no special instructions, 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(BSA) 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 structural representation is shown in formula (III).
Formula (III).
DMF (DMF) is suc as formula shown in (IV).
Formula (IV)
N-hydroxy-succinamide (NHS) is suc as formula shown in (V).
Formula (V)
1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) is suc as formula shown in (VI).
Formula (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 bulb; Add proper amount of acetone and a small amount of DMF to make it dissolve that (acetone dissolves 3-Jia based quinoxaline-2-carboxylic acid as reaction system completely, the effect of DMF is to promote to dissolve), 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).
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 the formula (I) 10mg embodiment 1 being prepared, compound is dissolved in 2mL N, in N '-dimethylformamide, add 10mg N-hydroxy-succinamide and 10mg1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, room temperature lower magnetic force stirs 2h, obtains solution III.
(2) 30mg bovine serum albumin(BSA) 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 bag filter, 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 immunogene 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 dilution, be the MQCA-BSA concentration in former MQCA-BSA solution after the protein concentration value recording is multiplied by its extension rate.Protein concentration (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 significant 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 297nm, and the maximum absorption wave long value of solution third is 280nm.According to formula K=A/CL (A is the absorbance under maximum absorption wave long value, and C is solution concentration, the thickness that L is liquid layer), calculate the 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 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
Replace bovine serum albumin(BSA) 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 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.
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 Sigma-Aldrich company, catalog number is 08060101.
One, animal immune
MQCA-BSA solution immunity Balb/c mouse prepared by embodiment 2, every mouse single immunization 100 μ gMQCA-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 intraperitoneal injection.
Two, Fusion of Cells 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 metabolin 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
Hybridoma 5A2 is made to 1 × 10 with cryopreserving liquid
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 cryopreserving liquid, move in culture flask and cultivate.
Four, the preparation and purification of monoclonal antibody
1, increment cultivation
The preparation method of cell culture medium (7.4): add calf serum and sodium bicarbonate in RPMI-1640 nutrient culture media, 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 sulfate method, the nutrient solution obtaining is carried out to purifying, obtain monoclonal antibody solution (20 ℃ of preservations).
Protein concentration (mg/ml)=1.45 × OD in monoclonal antibody
280-0.74 × OD
260.
Adopting above formula to calculate the protein concentration 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, carry out purifying, ℃ preservation of ascites-20 after purifying by sad-saturated ammonium sulfate method.
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 affinity of monoclonal antibody
(1) use MQCA-OVA as coating antigen coated elisa plate
Adopt the MQCA-OVA solution prepared of embodiment 2 (diluting with carbonate buffer solution) 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 dilution (diluting with PBS damping fluid) of 100 μ L monoclonal antibody solutions; Protein concentration in dilution 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 dilution 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.
Take the natural logarithm value of the protein concentration in monoclonal antibody (mol/L) as horizontal ordinate, take its corresponding absorbance as ordinate, make curve.
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 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
9w
-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 prepared of embodiment 2 (diluting with carbonate buffer solution) 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; To only add the hole in contrast, hole of 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 adopts the standard solution of each concentration to obtain (mean values in three multiple holes) is multiplied by 100 as ordinate 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 horizontal ordinate 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.The sensitivity (IC50 value) that monoclonal antibody detects 3-Jia based quinoxaline-2-carboxylic acid is 3.1ng/mL.
The preparation of embodiment 4,3-Jia based quinoxaline-2-carboxylic acid polyclonal antibody
New zealand white rabbit: be purchased from Beijing Vital River Experimental Animals Technology Co., Ltd..
New zealand white rabbit is carried out to immunity (immunization ways is as the subcutaneous multi-point injection of nape portion) take 3-Jia based quinoxaline-2-carboxylic acid immunogene (MQCA-BSA) of preparation in embodiment 2.Every each immune 1.5mg of rabbit (in BSA amount), immunity in every three weeks once, when immunity is mixed and made into emulsifying agent by 3-Jia based quinoxaline-2-carboxylic acid immunogene and Freund's complete adjuvant and carries out immunity for the first time, six immunity of for the second time to the are mixed and made into emulsifying agent by 3-Jia based quinoxaline-2-carboxylic acid immunogene and incomplete Freund's adjuvant and carry out immunity, the 7th immunity only carried out immunity by 3-Jia based quinoxaline-2-carboxylic acid immunogene, the 7th immunity be heart blood sampling after 10 days, obtains the polyclonal antibody of purifying with ammonium sulfate precipitation.
The preparation of the enzyme linked immunological kit of embodiment 5, detection 3-Jia based quinoxaline-2-carboxylic acid
One, the composition of enzyme linked immunological kit
Enzyme linked immunological kit is comprised of following substances:
1, cleansing solution: every 1 liter of cleansing solution is prepared and obtained as follows: 10ml polysorbas20,5g sodium azide and 990ml PBS damping fluid damping fluid are mixed, obtain cleansing solution.
2, the ELISA Plate of coated MQCA-OVA
The MQCA-OVA solution dilution (concentration that makes MQCA-OVA is 0.5 μ g/mL) of with carbonate buffer solution being prepared by embodiment 2, is coating buffer; Coating buffer is added to 96 hole polystyrene ELISA Plate (48 holes also can), every hole 100 μ L, 37 ℃ of incubation 2h; The coating buffer that inclines, with cleansing solution washing 3 times, each 30s, pats dry; Then in every hole, add 200 μ L confining liquids, 37 ℃ of incubation 2h, liquid in the hole of inclining, dry rear with aluminium film vacuum seal preservation.
Every 1 liter of described confining liquid is prepared as follows: by 5ml horse serum, 1g sodium azide and 30g for casein phosphate buffer (pH7.2,0.02M) dissolve and be settled to 1000ml, obtain confining liquid.
3, sample concentration liquid: PBS damping fluid (pH7.4,0.04M).
Sample concentration liquid is diluted with water to 20 times of volumes, is sample diluting liquid.
4, antibody working fluid: 1 of the step 4 of embodiment 3 monoclonal antibody solution obtaining is diluted with sample diluting liquid, and making protein concentration is 6.5ng/mL.
5, ELIAS secondary antibody working fluid
The sheep anti-mouse antibody of horseradish peroxidase (HRP) mark is purchased from U.S. Sigma-Aldrich company, and catalog number is A7058, by specification preparation ELIAS secondary antibody working fluid.
6, standard solution
Standard items are 3-Jia based quinoxaline-2-carboxylic acid.
With sample diluting liquid dilution, dissolve 3-Jia based quinoxaline-2-carboxylic acid, obtain each standard solution.In each standard solution, 3-Jia based quinoxaline-2-carboxylic acid concentration 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.
Sample diluting liquid is the standard solution (0 standard) of 0 μ g/L as 3-Jia based quinoxaline-2-carboxylic acid concentration.
7, substrate nitrite ion
By A liquid and B liquid equal-volume, mixed, A liquid is 2% (g/100ml) urea peroxide aqueous solution, and B liquid is 1% (g/100ml) tetramethyl biphenyl amine aqueous solution.
8, stop buffer: 0.2M aqueous sulfuric acid.
Two, kit test method
The kit that adopts step 1 to prepare detects as follows:
(1) sample pre-treatments
Detect sample while being pork or pork liver: accurately take tissue sample after homogeneous in centrifuge tube, add sample extracting solution (containing the sample diluting liquid of 0.2% polysorbas20), fully whirling motion disperses to tissue, and the above centrifugal 10min of 4000g, gets 50 μ L supernatants as sample to be tested solution.
When detection sample is urine sample: the centrifugal 10min of 3000g, draw supernatant and sample diluting liquid by 1: 1 volume mixture, get 50 μ L mixed liquors as sample to be tested solution.
(2) kit test method
1, the making of typical curve
In the ELISA Plate of coated MQCA-OVA, add standard solution (50 μ L/ holes; Each standard solution arranges three multiple holes), add again antibody working fluid (50 μ L/ hole), with cover plate film shrouding, in 37 ℃ of constant temperature ovens, react 60min, pour out liquid in hole, wash 4 times (step of each washing is: every hole adds 250 μ L cleansing solutions, pours out liquid in hole after 30s), pat dry with thieving paper.Add ELIAS secondary antibody working fluid (100 μ L/ hole), in 37 ℃ of constant temperature ovens, react 30min, pour out liquid in hole, wash 4 times (step is the same), every hole adds substrate nitrite ion 100 μ L, vibration mixes gently, 37 ℃ of constant temperature oven lucifuge colour developing 15min, and every hole adds stop buffer 50 μ L, vibration mixes gently, measure every hole absorbance (OD630 and OD450 dual wavelength, the 450th, predominant wavelength, the 630th, reference wavelength) by microplate reader.
Divided by the absorbance (B0) of first standard solution (0 standard), be multiplied by again 100% with the absorbance mean value (B) of the standard solution of each concentration, obtain percentage absorbance.Use Originpro 7.0 softwares to analyze data result, take the natural logarithm value of standard items concentration (μ g/L) as X-axis, percentage absorbance is that Y-axis simulates typical curve.Typical curve is shown in Fig. 3.
2,3-Jia based quinoxaline-2-carboxylic acid concentration's mensuration in sample
In the ELISA Plate of coated MQCA-OVA, add sample to be tested solution or its dilution (50 μ L/ holes; Three multiple holes are set), then add antibody working fluid (50 μ L/ hole), with cover plate film shrouding, in 37 ℃ of constant temperature ovens, react 60min, pour out liquid in hole, wash 4 times (step is the same), pat dry with thieving paper.Add ELIAS secondary antibody working fluid (100 μ L/ hole), in 37 ℃ of constant temperature ovens, react 30min, wash 4 times (step is the same), every hole adds substrate nitrite ion 100 μ L, and vibration mixes gently, 37 ℃ of constant temperature oven lucifuge colour developing 15min, every hole adds stop buffer 50 μ L, and vibration mixes gently, measures every hole absorbance (OD630 and OD450 dual wavelength by microplate reader, the 450th, predominant wavelength, the 630th, reference wavelength).
Result judgement: be multiplied by again 100% divided by the absorbance (B0) of first standard solution (0 standard) with the absorbance mean value (B) of each sample to be tested solution, obtain percentage absorbance.The percentage absorbance of corresponding each sample to be tested solution, can read from typical curve the concentration value of 3-Jia based quinoxaline-2-carboxylic acid, then is multiplied by the extension rate of respective sample, converses the content of 3-Jia based quinoxaline-2-carboxylic acid in sample to be tested solution.
Three, kit detects effect assessment
(1) accuracy and precision test
To not adding 3-Jia based quinoxaline-2-carboxylic acid standard items containing in pork, pork liver and the pig urine samples of 3-Jia based quinoxaline-2-carboxylic acid, make the final concentration of 3-Jia based quinoxaline-2-carboxylic acid standard items in sample be respectively 4 μ g/kg, 10 μ g/kg, 4 μ g/kg; Sample after adding is carried out to pre-treatment according to method described in () of step 2 respectively, obtain test sample solution.
From the kit of three different batches, 3 kits of each extraction detect, detection method is described in (two) of step 2, each experiment repeats 5 times, the content that detects 3-Jia based quinoxaline-2-carboxylic acid in sample according to the cubage of 3-Jia based quinoxaline-2-carboxylic acid in sample to be tested solution, the results are shown in Table 1.
Each kit of table 1 application detects the content (μ g/kg) of 3-Jia based quinoxaline-2-carboxylic acid in the sample drawing
| Kit 1 | Kit 2 | Kit 3 | |
| The final concentration of 3-Jia based quinoxaline-2-carboxylic acid in pork is 4 μ g/kg | 3.34 | 3.63 | 3.48 |
| The final concentration of 3-Jia based quinoxaline-2-carboxylic acid in pork liver is 10 μ g/kg | 9.31 | 7.93 | 8.52 |
| The final concentration of 3-Jia based quinoxaline-2-carboxylic acid in pig urine is 4 μ g/kg | 3.53 | 3.72 | 3.35 |
Calculate recovery rate and the coefficient of variation, the results are shown in Table 2 respectively.
Content × 100% of the actual 3-Jia based quinoxaline-2-carboxylic acid adding in the content ÷ milk of 3-Jia based quinoxaline-2-carboxylic acid that the recovery=application kit detection computations goes out.
The coefficient of variation (the CV)=standard deviation of measurement result and the number percent of its mean value.
The computing method of plate within variance coefficient: the coefficient of variation of certain sample (being generally medium level) replication number of times acquired results in plate within variance coefficient=same same plate of once measuring.
The computing method of variation within batch coefficient: the coefficient of variation of each parallel samples in variation within batch coefficient=same once mensuration.
The computing method of interassay coefficient of variation: interassay coefficient of variation=same sample, in the coefficient of variation of different batches measurement result, is got its mean value.
Table 2 accuracy and Precision test result
Result shows: the interpolation recovery of all samples is 79.3%～93.1%, and variation within batch coefficient is 4.3%～8.3%, and interassay coefficient of variation is 12.3%～14.7%.
(2) kit storage life
Kit preservation condition is 2-8 ℃, and through the mensuration of 12 months, the maximum absorbance value (zero standard) of kit, 50% inhibition concentration, 3-Jia based quinoxaline-2-carboxylic acid added practical measurement value all within normal range.Consider in transportation and use procedure, have improper preservation condition and occur, kit is placed 8 days under the condition of 37 ℃ of preservations, carry out accelerated deterioration experiment, result shows that the indices of this kit meets the requirements completely.Consider that the freezing situation of kit occurs, kit is put into-20 ℃ of refrigerator freezings 8 days, measurement result also shows that kit indices is completely normal.From above result, can show that kit can at least can preserve more than 12 months at 2-8 ℃.
(3) cross reacting rate test
In the ELISA Plate of coated MQCA-OVA, add analogue standard solution (by analogue and PBS damping fluid, to be formed; The concentration of analogue 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; The hole in contrast, hole of PBS damping fluid will only be added; 50 μ L/ holes; Each concentration arranges 3 multiple holes), then add antibody working fluid (50 μ L/ hole), with cover plate film shrouding, in 37 ℃ of constant temperature ovens, react 60min, pour out liquid in hole, wash 4 times (step is the same), pat dry with thieving paper.Add ELIAS secondary antibody working fluid (100 μ L/ hole), in 37 ℃ of constant temperature ovens, react 30min, wash 4 times (step is the same), every hole adds substrate nitrite ion 100 μ L, and vibration mixes gently, 37 ℃ of constant temperature oven lucifuge colour developing 15min, every hole adds stop buffer 50 μ L, and vibration mixes gently, measures every hole absorbance (OD630 and OD450 dual wavelength by microplate reader, the 450th, predominant wavelength, the 630th, reference wavelength).
The light absorption value that adopts the analogue of each concentration to obtain (mean values in three multiple holes) is multiplied by 100 as ordinate again divided by the light absorption value of control wells, take the natural logarithm value of the analogue concentration in each standard solution (μ g/L) as horizontal ordinate curve plotting figure.Control curve figure, corresponding analogue concentration (μ g/L) when obtaining Y value and equaling 50%, the i.e. IC of analogue
50value.
Cross reacting rate with following formula calculating kit to other analogue.The results are shown in Table 3.
The specificity of table 3 kit
| Purchase approach | Cross reacting rate | |
| MQCA | Dr.Ehrenstorfer company, catalog number is 81121 | 100％ |
| QCA | Huifeng, Wuhan reaches, article No. 879652 | 43.2％ |
| Olaquindox | Dr.Ehrenstorfer GmbH company, article No. C 15716500 | ＜0.1％ |
| Quinocetone | Dr.Ehrenstorfer GmbH company, article No. C 16709000 | ＜0.1％ |
| Mequindox | Sigma Aldrich company, catalog number is M0189 | ＜0.1％ |
Claims (6)
1. detect an enzyme linked immunological kit for 3-first based quinoxaline-2-carboxylic acid, comprise the monoclonal antibody of anti-olaquindox metabolin monoclonal antibody hybridoma cell 5A2 secretion; The preserving number of described anti-olaquindox metabolin monoclonal antibody hybridoma cell 5A2 is CGMCC No.5778.
2. kit as claimed in claim 1, is characterized in that: described kit is following 1) to 4) in any one:
1) comprise the kit of compound shown in formula (I) and the conjugate of carrier protein, described monoclonal antibody and enzyme labeling antiantibody; Wherein, described conjugate is as coating antigen;
2) comprise the kit of the enzyme labeling thing of compound shown in formula (I), described monoclonal antibody and antiantibody; Wherein, described antiantibody is as coating antigen;
3) comprise the enzyme labeling thing of compound shown in formula (I) and the kit of described monoclonal antibody; Wherein, described monoclonal antibody is as coating antigen;
4) comprise the kit of compound shown in formula (I) and the conjugate of carrier protein and the enzyme labeling thing of described monoclonal antibody; Wherein, described conjugate is as coating antigen;
3. kit as claimed in claim 2, is characterized in that: described carrier protein is ovalbumin or bovine serum albumin(BSA).
4. as the kit as described in arbitrary in claims 1 to 3, it is characterized in that: described kit also comprises at least one in cleansing solution, sample concentration liquid, substrate nitrite ion and stop buffer.
5. the application of arbitrary described kit in detection 3-Jia based quinoxaline-2-carboxylic acid in claim 1 to 4.
6. in claim 1 to 4, whether arbitrary described kit contains the application in 3-Jia based quinoxaline-2-carboxylic acid at detection sample to be tested.
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| CN201210089054.0A CN102621322B (en) | 2012-03-29 | 2012-03-29 | Kit for detecting 3-methyl-quinoxaline-2-carboxylic acid |
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| CN103342683B (en) * | 2013-07-30 | 2015-07-15 | 中国农业大学 | Quinocetone hapten as well as preparation method and application thereof |
| 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 |
| CN110927376B (en) * | 2019-10-08 | 2024-04-30 | 浙江工商大学 | Magnetic immunochemistry luminescence detection kit of olaquindox and application thereof |
| CN110927377A (en) * | 2019-10-08 | 2020-03-27 | 杭州佰昕科技有限公司 | Magnetic immunochemiluminescence detection kit for olaquindox and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN201852838U (en) * | 2010-10-27 | 2011-06-01 | 北京勤邦生物技术有限公司 | Sulfaquinoxaline ELISA (Enzyme Linked Immunosorbent Assay) detecting reagent kit |
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| US7081346B2 (en) * | 2002-05-14 | 2006-07-25 | Marquette University | Method of screening binding of a compound to a receptor |
| CN1971279B (en) * | 2006-12-06 | 2011-05-18 | 华中农业大学 | Oxoquinoxaline-2-carboxylic acid remained enzyme linked immunity detecting method and reagent kits |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN201852838U (en) * | 2010-10-27 | 2011-06-01 | 北京勤邦生物技术有限公司 | Sulfaquinoxaline ELISA (Enzyme Linked Immunosorbent Assay) detecting reagent kit |
Non-Patent Citations (2)
| Title |
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| 冀宝庆等.喹乙醇代谢物3-甲基-喹啉-2-羧酸的合成与鉴定.《中国饲料》.2008,(第12期),33-35. |
| 喹乙醇代谢物3-甲基-喹啉-2-羧酸的合成与鉴定;冀宝庆等;《中国饲料》;20080620(第12期);33-35 * |
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