CN102608316B - Kit or test strip for detecting quinoxaline compound - Google Patents
Kit or test strip for detecting quinoxaline compound Download PDFInfo
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- CN102608316B CN102608316B CN201210042221.6A CN201210042221A CN102608316B CN 102608316 B CN102608316 B CN 102608316B CN 201210042221 A CN201210042221 A CN 201210042221A CN 102608316 B CN102608316 B CN 102608316B
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Abstract
The invention discloses a kit or a test strip for detecting a quinoxaline compound. The ELISA (Enzyme Linked Immunosorbent Assay) kit or test strip for detecting the quinoxaline compound, provided by the invention, comprises a monoclonal antibody secreted by an anti-mequindox monoclonal antibody hybridoma 1A10; and the preservation number of the anti-mequindox monoclonal antibody hybridoma 1A10 is CGMCC No.5780. The kit and the test strip disclosed by the invention have the advantages of high sensitivity, high accuracy, high precision, low cost, simplicity in operation, short detection time, suitable for use in various units, simpleness for storage and long quality guarantee period, and meanwhile, can be used for rapidly detecting a large batch of samples to realize high-throughput rapid detection on site. The ELISA kit or test strip provided by the invention is suitable for determining residues of the quinoxaline compound in animal feed and animal tissues. The antibody, the kit, the test strip and a detection method, disclosed by the invention, are about to play a great role in detecting the quinoxaline compound.
Description
Technical field
The present invention relates to detect kit or the test strips of quinoxaline compound.
Background technology
Quinoxaline compound is equal You quinoxaline-1 in molecule, the artificial synthetic antimicrobial of 4-dioxide mother nucleus structure, Major Members has olaquindox (olaquindox, OLA), carbadox (cabadox, CBX), mequindox (mequindox, MEQ) and quinocetone (quinocetone, QCT) etc.Quinoxaline compound can antibacterial growth promotion, therefore by widely as feed addictive.Along with going deep into of research, find that quinoxaline compound has obvious toxic and side effect, long-term unreasonable use can cause animal body itself poisoning, produces carcinogenic, teratogenesis, mutagenicity, can cause the medicament residue of animal derived food simultaneously.
The detection of Dui quinoxaline compound adopts instrumental method more both at home and abroad at present.(HPLC) is highly sensitive for high performance liquid chromatography, but must special messenger operate, and is not suitable for the screening of batch sample.Sensitive in the urgent need to setting up, quick, simple to operate, the method for applicable great amount of samples examination.Based on immunoenzymatic ELISA method have highly sensitive, be easy to use, the advantage such as equipment is simple, quick, immune colloidal gold chromatography technology does not need any instrument and equipment and reagent, be particularly suitable for detection and large area generaI investigation that grass-roots unit is pressed for time in enormous quantities, in feed safety analysis, be widely used.
Summary of the invention
The object of this invention is to provide detect quinoxaline compound kit or test strips.
The enzyme linked immunological kit of detection quinoxaline compound provided by the invention, comprises the monoclonal antibody that anti-mequindox monoclonal antibody hybridoma cell 1A10 secretes.
Anti-mequindox monoclonal antibody hybridoma cell 1A10, be called for short hybridoma 1A10, 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.5780.
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.
Formula (I).
The conjugate of compound and carrier protein shown in formula (I) is specifically suc as formula shown in (II).
Formula (II).
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 (8-10): 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 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 quinoxaline compound above.
Arbitrary described kit all can be used for detecting in sample to be tested, whether to contain quinoxaline compound above.
The present invention also protects a kind of colloidal gold strip that detects quinoxaline compound, absorption of sample pad, collaurum pad, reaction film and adsorptive pads, consists of; Along test strips axially, described absorption of sample pad, described collaurum pad, described reaction film and described adsorptive pads are linked in sequence successively, the end of absorption of sample pad is connected with the top of collaurum pad, the end of collaurum pad is connected with the top of reaction film, and the end of reaction film is connected with the top of adsorptive pads;
On described collaurum pad, be coated with the monoclonal antibody of colloid gold label; Described monoclonal antibody is the monoclonal antibody of anti-mequindox monoclonal antibody hybridoma cell 1A10 secretion;
On described reaction film, have detection zone and Quality Control district, detection zone (T line) is and the axial vertical ribbon of described test paper with Quality Control district (C line); Detection zone is positioned at the side near collaurum pad end; Quality Control district is positioned at the side away from collaurum pad end; Detection zone is coated with the conjugate (coating antigen) of compound shown in formula (I) and carrier protein, and the coated sheep anti mouse two in Quality Control district is anti-.
Described absorption of sample pad is cellulose filter membrane.Described collaurum pad is the glass fibre membrane of the coated described monoclonal antibody by colloid gold label.Described reaction film is nitrocellulose filter (NC film).Described adsorptive pads is thieving paper.
Described sample well is positioned in sample absorbent the one end away from collaurum pad end.
Described colloidal gold strip can be used for detecting quinoxaline compound.
Described colloidal gold strip is for detection of whether containing quinoxaline compound in sample to be tested.
Arbitrary described quinoxaline compound can be and in molecule, has quinoxaline-l, the compound of 4-dioxide mother nucleus structure above.Arbitrary described quinoxaline compound specifically can be olaquindox (olaquindox, OLA), carbadox (cabadox, CBX), mequindox (mequindox, MEQ) or quinocetone (quinocetone, QCT) above.
The present invention adopts high specific quinoxaline compound monoclonal antibody, 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 and test strips (claiming again colloidal gold test paper card) have 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 or colloidal gold strip, be applicable to measure the residual quantity of animal feed and the Zhong of animal tissue quinoxaline compound.In the detection of antibody of the present invention, kit, colloid gold test paper and detection method quinoxaline compound, will bring into play significant role.
Accompanying drawing explanation
Fig. 1 is the artificial antigen ultraviolet spectrogram of mequindox.
Fig. 2 is the canonical plotting of mequindox.
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.Mequindox is purchased from Sigma-Aldrich company, and catalog number is MO-189.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.
Mequindox is suc as formula shown in (III), and molecular weight is 218.21.
Formula (III)
Ethyloic azanol is suc as formula shown in (IV), and molecular weight is 77.04.
Formula (IV)
DMF (DMF) is suc as formula shown in (V).
Formula (V)
N-hydroxy-succinamide (NHS) is suc as formula shown in (VI).
Formula (VI)
1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) is suc as formula shown in (VII).
Formula (VII)
Embodiment 1, prepare mequindox haptens
One, the haptenic preparation of mequindox
Mequindox 216mg (1mmol) is dissolved in the mixed solvent (volume ratio of anhydrous propanone and DMF is 1: 1) of 3ml anhydrous propanone and DMF, add in brown reaction bulb, process lucifuge, take ethyloic azanol 116mg (1.5mmol) and add reaction bulb, 40 ℃ are reacted 16 hours, add a small amount of shrend reaction of going out, by the NaOH aqueous solution of 0.1mol/L, adjust pH to 10 left and right, use CH
2cl
2extraction, water intaking is adjusted pH to 3 left and right by the HCl aqueous solution of 0.1mol/L mutually, is extracted with ethyl acetate, and gets oil phase and carries out vacuum drying, obtains 56mg product.
Two, the haptenic sign of mequindox
Product prepared by step 1 carries out ultimate analysis, and result is as follows:
C：52.06；H：4.32；N：15.10；O：28.38。
Result shows, product prepared by step 1 is compound shown in formula (I).
Formula (I).
The Preparation and characterization of embodiment 2, mequindox artificial antigen
One, the immunogenic synthetic and sign of mequindox
1, mequindox is immunogenic synthetic
(1) shown in 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(BSA) is added in 2mL deionized water, fully dissolve, be solution IV.
(3) solution IV is added in solution III, after slowly stirring 6h, enter bag filter, 4 ℃ of dialysis 72 h (water is changed 6 times in centre) in physiological saline, then under 4 ℃ of conditions, the centrifugal 30min of 8000rmp, get supernatant, be mequindox immunogen solution, be sub-packed in ampere bottle-20 ℃ of preservations, mequindox immunogene is called for short MEQ-BSA, and mequindox immunogen solution is called for short MEQ-BSA solution.
(4) after MEQ-BSA solution is diluted with PBS damping fluid, measure the spectrophotometric value of 280nm and 260nm, press formula and calculate the protein concentration in dilution, be the MEQ-BSA concentration in former MEQ-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.MEQ-BSA concentration in MEQ-BSA solution is 5.3mg/ml.
2, the immunogenic sign of mequindox
By PBS damping fluid dilution (concentration that makes MEQ-BSA is 5mg/mL) for MEQ-BSA solution, as solution first; Using the PBS damping fluid containing 5mg/mL mequindox as solution second; Using the PBS damping fluid containing 5mg/mL BSA as solution third.Respectively solution first, solution second and solution third are carried out to ultraviolet (200-380nm) spectral scan, uv scan the results are shown in Figure 1.There is significant 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 372nm, 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 8: 1, and compound shown in 8 formulas (I) is in conjunction with 1 BSA.
Two, the Preparation and characterization of mequindox coating antigen
1, the preparation of mequindox coating antigen
With ovalbumin, replace bovine serum albumin(BSA), other is with 1 of step 1.
Mequindox coating antigen is called for short MEQ-OVA, and mequindox coating antigen solution is called for short MEQ-OVA solution.
MEQ-OVA concentration in MEQ-OVA solution is 3.0mg/ml.
2, the sign of mequindox coating antigen
With MEQ-OVA, replace MEQ-BSA, with OVA, replace BSA, other is with 2 of step 1.
Shown in formula (I), the coupling ratio of compound and OVA is 10: 1, and compound shown in 10 formulas (I) is in conjunction with 1 OVA.
The preparation of embodiment 3, quinoxaline compound monoclonal antibodies
Balb/c mouse: be purchased from Beijing Vital River Experimental Animals Technology Co., Ltd.;
SP2/0 myeloma cell: purchased from Sigma-Aldrich company, catalog number is 08060101.
One, animal immune
By the MEQ-BSA solution immunity Balb/c mouse of embodiment 2 preparations, every mouse single immunization 100 μ gMEQ-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 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 the hybridoma that a strain can be secreted mequindox monoclonal antibody, the anti-mequindox monoclonal antibody hybridoma cell of called after 1A10 (being called for short hybridoma 1A10).Hybridoma 1A10 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on February 15th, 2012, and (be called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preserving number is CGMCC No.5780.
Three, cell cryopreservation and recovery
With cryopreserving liquid, hybridoma 1A10 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 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 1A10 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 in monoclonal antibody (mg/ml)=1.45 * OD
280-0.74 * OD
260.
Adopting above formula to calculate the protein concentration in monoclonal antibody, is 21.8mg/ml.
2, ascites preparation
Balb/c mouse peritoneal injection sterilizing paraffin oil (0.4mL/ only).7 days pneumoretroperitoneum injection hybridoma 1A10 (5 * 10
5individual/only).After 7 days, gather ascites, by sad-saturated ammonium sulfate 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, the mensuration of antibody titer
(1) adopt the MEQ-OVA solution (adopting carbonate buffer solution to regulate concentration) of embodiment 2 preparations to be coated with 100 μ L/ holes; The coated concentration of MEQ-OVA is 1.0 μ g/mL.
Hatch 16 hours for (2) 4 ℃.
(3) seal and wash plate.
(4) every hole adds 1 monoclonal antibody solution obtaining or its dilution (adopting PBS damping fluid to carry out gradient dilution) of 100 μ L step 4.
(5) incubated at room 2h, washes plate.
(6) every hole adds the sheep anti-mouse igg of 100 μ L horseradish peroxidase-labeled, incubated at room 2h.
(7) wash plate.
(8) add TMB nitrite ion, lucifuge colour developing 15min.
(9) every hole adds 100 μ L 2mol/L sulfuric acid stopped reactions; Read OD
450value.
While reaching 1.0 left and right with OD value, be judged to be the positive.Antibody titer is 1: 320000.
3, the calculating of monoclonal antibody sensitivity
(1) to (3) with (1) of step 2 to (3).
(4) every hole adds 50 μ L mequindox standard solutions (mequindox and PBS damping fluid, to consist of; The concentration of mequindox is respectively 0.3 μ g/L, 0.9 μ g/L, 2.7 μ g/L, 8.1 μ g/L, 24.3 μ g/L; 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 again divided by the light absorption value of control wells, take the natural logarithm value of the mequindox concentration (μ g/L) in each standard solution as horizontal ordinate curve plotting figure, see Fig. 2.
Contrast Fig. 2, obtains the mequindox concentration (μ g/L) that Y value equals 50% correspondence, is IC
50value.Monoclonal antibody detects the sensitivity (IC of mequindox
50value) be 1.2 μ g/L.
4, stability
By 1 of step 4 monoclonal antibody solution-20 that obtain ℃ placement, different time sampling, with detecting and tire after the dilution of PBS damping fluid, concrete steps are as follows:
(1) to (3) with (1) of step 2 to (3).
(4) every hole adds 50 μ L monoclonal antibody solutions or its dilution (adopting PBS damping fluid to carry out gradient dilution).
(5) to (9) with (6) of step 3 to (10).
OD
450value is in Table 1.Result shows, constant tiring of 120 days monoclonal antibodies of-20 ℃ of preservations.
The stability of table 1 monoclonal antibody
5, affinity costant is measured
(1) use MEQ-OVA as coating antigen coated elisa plate
Adopt the MEQ-OVA solution (adopting carbonate buffer solution to regulate concentration) of embodiment 2 preparations to be coated with 100 μ L/ holes; The coated concentration of following OVA-SAL is set respectively: 1 μ g/mL, 0.5 μ g/mL, 0.25 μ g/mL, 0.125 μ g/mL.
Hatch 16 hours for (2) 4 ℃.
(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;
(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 concentration (mol/L) in monoclonal antibody of take is horizontal ordinate, take its corresponding absorbance to make curve as ordinate.
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 8.9 * 10
-12mol/L.Adopt 0.5 μ g/mL to be coated with concentration, the antibody concentration that 50%ODMAX is corresponding is 27.4 * 10
-12mol/L.Adopt 0.25 μ g/mL to be coated with concentration, the antibody concentration that 50%ODMAX is corresponding is 180.9 * 10
-12mol/L.Adopt 0.125 μ g/mL to be coated with concentration, the antibody concentration that 50%ODMAX is corresponding is 374.6 * 10
-12mol/L.
By one group between two of 4 concentration, 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 8.9 * 10
-12mol/L, coated concentration 50% OD of coated 0.5 μ g/mL
450corresponding antibody concentration is 27.4 * 10
-12mol/L, Ka=(2-1)/2 (2 * 27.4 * 10
-12-8.9 * 10
-12)=10.8 * 10
9m
-1.The like, obtain all the other 5 Ka values, be respectively 1.49 * 10
9m
-1, 0.91 * 10
9m
-1, 2.1 * 10
9m
-1, 1.0 * 10
9m
-1, 1.17 * 10
9m
-1, the affinity costant that calculates monoclonal antibody of averaging is 2.91 * 10
9m
-1.
The preparation of embodiment 4, quinoxaline compound polyclonal antibodies
New zealand white rabbit: be purchased from Beijing Vital River Experimental Animals Technology Co., Ltd..
New zealand white rabbit be take to the mequindox immunogene (MEQ-BSA) of preparation in embodiment 2 and carry out immunity (immunization ways is the subcutaneous multi-point injection of nape portion).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 mequindox 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 mequindox immunogene and incomplete Freund's adjuvant and carry out immunity, the 7th immunity only carried out immunity by mequindox 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 quinoxaline compound
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 phosphate buffer (pH7.4,0.01M) are mixed, obtain cleansing solution.
2, the ELISA Plate of coated MEQ-OVA
With carbonate buffer solution, by the MEQ-OVA solution dilution (concentration that makes MEQ-OVA is 0.5 μ g/mL) of embodiment 2 preparations, be 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 that olaquindox is (purchased from U.S. Sigma-Aldrich company; Catalog number is G1397).
With sample diluting liquid dilution, dissolve olaquindox, obtain each standard solution.In each standard solution, olaquindox concentration is respectively 0.3 μ g/L, 0.9 μ g/L, 2.7 μ g/L, 8.1 μ g/L, 24.3 μ g/L.
Sample diluting liquid is the standard solution (0 standard) of 0 μ g/L as olaquindox 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
When detection sample is feed, carry out as follows pre-treatment: take the equal pledge of 1 ± 0.05g to 50mL polystyrene centrifuge tube, add the quick whirling motion 20s of 10mL deionized water, put into water-bath, 65 ℃ of standing 30min, the centrifugal 15min of 4000r/min; Get sample diluting liquid to the 4 times volume for supernatant after centrifugal; Get 50 μ L as sample to be tested solution.
When detection sample is animal tissue, carry out as follows pre-treatment: the animal tissue taking after 1 ± 0.01g homogeneous is placed in 10mL centrifuge tube, add 5mL ethyl acetate-acetonitrile mixed solvent (volume ratio 1: 1), vibration 20min; 20 ℃-25 ℃, the centrifugal 20min of 4000r/min; Get upper solution in another centrifuge tube, add 1mL 0.2mol/LNaOH aqueous solution, vibration 10min, standing 10min; Sucking-off organic phase, in another centrifuge tube, dries up with nitrogen 50-60 ℃ of water-bath; Add 2mL normal hexane, abundant whirling motion 10s, then add 1mL sample diluting liquid, low speed whirling motion 1min, the centrifugal 5min of 4000g; Discard upper strata normal hexane and interstitial impurity completely; Get 50 μ L as sample to be tested solution.
(2) kit test method
1, the making of typical curve
In the ELISA Plate of coated MEQ-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 30min, pour out liquid in hole, wash 5 times (step of each washing is: every hole adds 250 μ L cleansing solutions, pours out liquid in hole after 30s), with thieving paper, pat dry.Add ELIAS secondary antibody working fluid (100 μ L/ hole), in 37 ℃ of constant temperature ovens, react 30min, pour out liquid in hole, wash 5 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, by microplate reader, measure every hole absorbance (OD630 and OD450 dual wavelength, the 450th, predominant wavelength, the 630th, reference wavelength).
With the absorbance mean value (B) of the standard solution of each concentration, divided by the absorbance (B0) of first standard solution (0 standard), be multiplied by again 100%, obtain percentage absorbance.Use Originpro 7.0 softwares to analyze data result, the natural logarithm value of standard items concentration (μ g/L) of take is X-axis, and percentage absorbance is that Y-axis simulates typical curve.Typical curve is shown in Fig. 3.
2, the mensuration of sample Zhong quinoxaline compound concentration
In the ELISA Plate of coated MEQ-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 30min, pour out liquid in hole, wash 5 times (step is the same), with thieving paper, pat dry.Add ELIAS secondary antibody working fluid (100 μ L/ hole), in 37 ℃ of constant temperature ovens, react 30min, wash 5 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, by microplate reader, measures every hole absorbance (OD630 and OD450 dual wavelength, the 450th, predominant wavelength, the 630th, reference wavelength).
Result judgement: be multiplied by again 100% with the absorbance mean value (B) of each sample to be tested solution divided by the absorbance (B0) of first standard solution (0 standard), obtain percentage absorbance.The percentage absorbance of corresponding each sample to be tested solution, can read from typical curve the concentration value of quinoxaline compound, then is multiplied by the extension rate of respective sample, converses the content of sample to be tested solution Zhong quinoxaline compound.
Three, kit detects effect assessment
(1) accuracy and precision test
In the Feed Sample of Han quinoxaline compound not, add olaquindox (standard items), make the final concentration of olaquindox in sample be respectively 5 μ g/kg, 10 μ g/kg, 20 μ g/kg; Sample after adding is carried out to pre-treatment according to method described in () of step 2 respectively, obtain sample to be tested solution.
From the kit of three different batches, respectively extracting 3 kits detects, detection method is described in (two) of step 2, each experiment repeats 5 times, detects the content of olaquindox in sample according to the cubage of olaquindox in sample to be tested solution, the results are shown in Table 2.
Each kit of table 2 application detects the content (μ g/kg) of olaquindox in the Feed Sample drawing
| Kit 1 | Kit 2 | Kit 3 | |
| The final concentration of olaquindox in sample is 5 μ g/kg | 4.23 | 9.36 | 17.86 |
| The final concentration of olaquindox in sample is 10 μ g/kg | 4.26 | 8.52 | 15.66 |
| The final concentration of olaquindox in sample is 20 μ g/kg | 4.28 | 7.53 | 18.14 |
Calculate recovery rate and the coefficient of variation, the results are shown in Table 3 respectively.
Content * 100% of the actual olaquindox adding in the content ÷ feed of the olaquindox 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 3 recovery and coefficient of variation result
Result shows: the recovery of all samples is 75.3%～93.6%, and variation within batch coefficient is 5.7%～8.1%, and interassay coefficient of variation is 9.2%～11.6%.
(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, olaquindox 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 MEQ-OVA, add analogue standard solution (by analogue and PBS damping fluid, to be formed; The concentration of analogue is respectively 0.3 μ g/L, 0.9 μ g/L, 2.7 μ g/L, 8.1 μ g/L, 24.3 μ g/L; By hole in contrast, the hole that only adds PBS damping fluid; 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 30min, pour out liquid in hole, wash 5 times (step is the same), with thieving paper, pat dry.Add ELIAS secondary antibody working fluid (100 μ L/ hole), in 37 ℃ of constant temperature ovens, react 30min, wash 5 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, by microplate reader, measures every hole absorbance (OD630 and OD450 dual wavelength, the 450th, predominant wavelength, the 630th, reference wavelength).
Analogue is: mequindox, olaquindox, quinocetone, carbadox, quinoline match many, 3-Jia based quinoxaline-2-carboxylic acid (MQCA) Oxoquinoxaline-2-carboxylic acid (QCA), tetracycline, sulfaquinoxaline, coban or sulphadiazine.
Olaquindox: Dr.Ehrenstorfer GmbH company product, article No. C 15716500.Quinocetone: Dr.Ehrenstorfer GmbH company product, article No. C 16709000.Carbadox: sigma company product, article No. C6770.Tetracycline: sigma company product, article No. 31741.Sulfaquinoxaline: sigma company product, article No. 45662.Coban: sigma company product, article No. M5273.Sulphadiazine: sigma company product, article No. S8626.
With following formula, calculate the cross reacting rate of kit to other analogue.
The results are shown in Table 4.
The specificity of table 4 kit
| Medicine name | Cross reacting rate (%) |
| Olaquindox | 100.0 |
| Mequindox | 134.0 |
| Quinocetone | 200.0 |
| Carbadox | 72.7 |
| Quinoline match is many | 20.4 |
| MQCA | 6.8 |
| QCA | ＜1％ |
| Tetracycline | ＜1％ |
| Coban | ＜1％ |
| Sulphadiazine | ＜1％ |
Experiment shows, kit of the present invention can specific detection quinoxaline compound.
Test strips and the preparation and application thereof of embodiment 6, detection quinoxaline compound
One, the structure of test strips
Described test strips is comprised of absorption of sample pad, collaurum pad, reaction film and adsorptive pads;
Along test strips axially, absorption of sample pad, collaurum pad, reaction film and adsorptive pads are linked in sequence successively, the end of absorption of sample pad is connected with the top of collaurum pad, and the end of collaurum pad is connected with the top of reaction film, and the end of reaction film is connected with the top of adsorptive pads;
On described collaurum pad, be coated with 1 monoclonal antibody obtaining of step 4 of the embodiment 3 of colloid gold label;
On described reaction film, have detection zone and Quality Control district, detection zone (T line) is and the axial vertical ribbon of test strips with Quality Control district (C line); Detection zone is positioned at the side near collaurum pad end; Quality Control district is positioned at the side away from collaurum pad end; The MEQ-OVA of coated embodiment 2 preparations in detection zone, the coated sheep anti mouse two in Quality Control district is anti-.
Sample well is positioned in sample absorbent the one end away from collaurum pad end.
Two, the preparation of test strips
(1) colloidal gold labeled monoclonal antibody
1. the preparation of colloidal gold solution
Get 0.01% aqueous solution of chloraurate 100mL and be heated to boiling with thermostatic electromagnetic stirrer, in the situation that continues to stir, add 1% trisodium citrate aqueous solution 2.5mL, continue agitating heating 20min, solution is bright redness.Room temperature is cooling, with deionized water, returns to original volume, 2-8 ℃ of preservation.
2. the preparation of golden labeling antibody solution
Use 0.1mol/L K
2cO
3aqueous solution regulates the pH to 8.2 of colloidal gold solution, then getting 10mL adds in 50mL beaker, magnetic stirrer 250r/min stirs, 1 monoclonal antibody solution obtaining (containing 0.35mg protein) that dropwise adds the step 4 of embodiment 3, dropwise add 3mL 5g/100mL BSA aqueous solution, continue to stir 10min.
3. by the centrifugal 20min of golden labeling antibody solution normal temperature low speed (1500r/min), discard the precipitation being formed by the gold grain condensing, get red supernatant solution.
4. by 4 ℃ of step solution 3., the centrifugal 40min of 11000r/min, solution is divided into three layers (gold grain layers of black densification on transparent supernatant, the flowable kermesinus precipitation in the pipe end and pipe diapire), flowable kermesinus precipitation is transferred in another one centrifuge tube, with the 0.01mol/L phosphate buffer containing 1g/100mL BSA, be suspended to the volume of former golden labeling antibody solution, spend the night, 4 ℃, the centrifugal 40min of 11000r/min, collecting precipitation.
5. with the 0.01mol/L phosphate buffer containing 1g/100mL BSA and 0.02g/100mL NaN3 step precipitation is 4. suspended to former golden labeling antibody solution volume 1/40,2-8 ℃ of preservation.
(2) metal spraying: the suspension that step (1) is obtained is sprayed onto on glass fibre membrane, makes collaurum pad.
(3) spray film: the T line position on reaction film is sprayed MEQ-OVA solution, the C line position of embodiment 2 preparations and sprayed sheep anti-mouse antibody.
(4) assembling: absorption of sample pad (cellulose filter membrane), collaurum pad, nitrocellulose filter (NC film), adsorptive pads (thieving paper) are assembled according to a conventional method, then slitting, test strips is packed in plastics fabrication, form test card.
Three, with test card, detect
(1) sample pre-treatments and detection
Detection sample is feed; Sample treatment is with (one) of the step 2 of embodiment 5.
Take out test card, behind Kaifeng, lie against desktop, draw sample to be tested solution and dropwise add 4 in sample well; 5-10min judged result, the judged result after 15min is invalid.
Result criterion:
Negative: the colour developing of C line, T line naked eyes are visible, and no matter shade is all judged to feminine gender;
Positive: the colour developing of C line, T line does not develop the color, and is judged to the positive;
Invalid: C line does not develop the color, no matter whether T line develops the color, and it is invalid that this test card is all judged to.
Four, the effect of test card
(1) false positive rate and false negative rate
The negative feed (Han quinoxaline compound L EssT.LTssT.LT 100.0 μ g/kg of the confirmation of learning from else's experience) 50 parts, the positive milk (Han quinoxaline compound >=100.0 μ g/kg of the confirmation of learning from else's experience) 50 parts.Sample is detected with test card respectively, calculate false positive rate and false positive rate.
Result: in 50 parts of negative Feed Samples are measured, test card detects totally 1 part of positive, and false positive rate is 2%.In 50 parts of positive Feed Samples are measured, test card detects 0 part of negative sample, and false negative rate is 0%.
(2) test card storage life
Stability test result shows, this test card can be preserved 1 year at shady and cool dry place under 2-8 ℃ or room temperature condition.
Claims (9)
1. detect an enzyme linked immunological kit for quinoxaline compound, comprise the monoclonal antibody of anti-mequindox monoclonal antibody hybridoma cell 1A10 secretion; The deposit number of described anti-mequindox monoclonal antibody hybridoma cell 1A10 is CGMCC No.5780.
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. detect a colloidal gold strip for quinoxaline compound, by absorption of sample pad, collaurum pad, reaction film and adsorptive pads, formed;
Along test strips axially, described absorption of sample pad, described collaurum pad, described reaction film and described adsorptive pads are linked in sequence successively, the end of absorption of sample pad is connected with the top of collaurum pad, the end of collaurum pad is connected with the top of reaction film, and the end of reaction film is connected with the top of adsorptive pads;
On described collaurum pad, be coated with the monoclonal antibody of colloid gold label; Described monoclonal antibody is the monoclonal antibody of anti-mequindox monoclonal antibody hybridoma cell 1A10 secretion; The deposit number of described anti-mequindox monoclonal antibody hybridoma cell 1A10 is CGMCC No.5780;
On described reaction film, have detection zone and Quality Control district, detection zone is and the axial vertical ribbon of described test paper with Quality Control district; Detection zone is positioned at the side near collaurum pad end; Quality Control district is positioned at the side away from collaurum pad end; Detection zone is coated with the conjugate of compound shown in formula (I) and carrier protein, and the coated sheep anti mouse two in Quality Control district is anti-;
6. the application of colloidal gold strip in detecting quinoxaline compound described in arbitrary described kit or claim 5 in claim 1 to 4.
7. in claim 1 to 4, described in arbitrary described kit or claim 5, whether colloidal gold strip contains the application in quinoxaline compound at detection sample to be tested.
8. as the kit as described in arbitrary in claims 1 to 3, or colloidal gold strip as claimed in claim 5, or the application as described in claim 6 or 7, it is characterized in that: described quinoxaline compound is to have quinoxaline-l in molecule the compound of 4-dioxide mother nucleus structure.
9. as the kit as described in arbitrary in claims 1 to 3, or colloidal gold strip as claimed in claim 5, or the application as described in claim 6 or 7, it is characterized in that: described quinoxaline compound is mequindox, olaquindox, carbadox or quinocetone.
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| CN103792355A (en) * | 2012-11-04 | 2014-05-14 | 江苏维赛科技生物发展有限公司 | Olaquindox metabolite residue rapid detection test strip |
| CN103353524A (en) * | 2012-12-12 | 2013-10-16 | 河南省农业科学院 | Test strip for rapidly detecting microscale olaquindox |
| 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 |
| CN103499690B (en) * | 2013-10-14 | 2016-04-27 | 河南科技学院 | Olaquindox metabolite immunochromatographytest test paper card and preparation method thereof |
| CN103524435B (en) * | 2013-10-22 | 2015-11-04 | 华南农业大学 | A kind of mequindox remains the haptens and complete antigen and preparation method thereof that marker takes off dioxy mequindox |
| CN106645462A (en) * | 2016-11-17 | 2017-05-10 | 石家庄学院 | Detection method for metabolite residues of carbadox and olaquindox in animal tissues |
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