CN1971279B - Oxoquinoxaline-2-carboxylic acid remained enzyme linked immunity detecting method and reagent kits - Google Patents
Oxoquinoxaline-2-carboxylic acid remained enzyme linked immunity detecting method and reagent kits Download PDFInfo
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Abstract
The invention belongs to fields of immunochemistry analysis. It discloses an enzyme-uniting immune method and reagent box used in detection of quinoxaline-2-carboxyl acid residues, the method mainly contain medicine reconstruction, the preparation of immunogen, peridium antigen and antibody and pretreatment of sample and building of ELISA detecting method. The reagent box comprises quinoxaline-2-carboxyl acid (QCA) specificity antibody, QCA standard substance, enzyme target-object peridiumed with coupling substance of egg albumin and reaction production of QCA and gamma-aminobutyric acid. The sample is hydrolysed by metaphosphoric acid to discharge QCA, and cleaned by MAX column, derivatized by butyl amine, indirect competition ELISA is adopted in detection. The method and reagent box in the invention has advantages of simpleness, speediness, sensitivity and accuracy, large-lot samples can be detected fast and simultaneously, lowest detecting limit is 0.6 mug/kg.
Description
Technical field
The invention belongs to the food fields of immunochemistry analysis.Be specifically related to a kind of enzyme-linked immunoassay method and kit that is used for Oxoquinoxaline-2-carboxylic acid (QCA) residue detection, be applicable to the residual quantity of the residual marker Oxoquinoxaline-2-carboxylic acid of carbadox (QCA) in the edible tissue of measuring food animal such as muscle, the liver.
Background technology
Carbadox is a quinoxaline medicine, is the broad spectrum antibiotic of synthetic, and the Ceng Zuowei growth accelerator is widely used in poultry, the fowl breed.Toxicologic study finds that carbadox has carcinogenicity and mutagenicity.For health that ensures the consumer and the trade contacts that enlarge animal food, residual detection is the task of top priority to carbadox in the animal food in reinforcement.
Carbadox is metabolized to Oxoquinoxaline-2-carboxylic acid (QCA) in animal body, the QCA that combines with histone in vivo the hold-up time long, be regarded as the residual marker of carbadox, so analyze residual its metabolic product of common analysis of carbadox QCA.Detect the residual method of QCA at present and high performance liquid chromatography (HPLC) is arranged, LC-MS (LC-MS), gas chromatography mass spectrometry (GC-MS).
The required instrument of instrumental method (HPLC, LC-MS, GC-MS) commonly used costs an arm and a leg, and higher to operating personnel's technical requirement, analysis time is longer, can not carry out the screening of high flux sample, is not suitable for vast basic unit and detects the unit application, is difficult to promote.Immunochemical analyses method particularly enzyme-linked immunosorbent analytical technique (ELISA) has advantages such as quick, highly sensitive, simple to operate, that selectivity is good, is fit to high-throughout sample screening.Yet there are no the ELISA detection method of the residual marker Oxoquinoxaline-2-carboxylic acid of carbadox (QCA) and the report of kit at present both at home and abroad.
Enzyme-linked immunoassay method provided by the present invention and kit are pressed the competitive ELISA method between adopting, the sensing range broad, false positive rate is low, and testing result is reliable, accurate, be applicable to that QCA detects in the edible tissue of food animal such as muscle, the liver, have very important economic implications and social effect.
Summary of the invention
The 1st purpose of the present invention provides the residual enzyme-linked immune detection method of a kind of Oxoquinoxaline-2-carboxylic acid (QCA).
The 2nd purpose of the present invention provides the residual enzyme-linked immunologic detecting kit of a kind of Oxoquinoxaline-2-carboxylic acid (QCA).
Method of the present invention and kit are exclusively used in the residual enzyme linked immunosorbent detection of edible animal tissue Zhong Oxoquinoxaline-2-carboxylic acid (QCA), compare with existing method, outstanding advantages such as method of the present invention and kit have highly sensitive, detect fast, and easy to use and detection cost is lower.
Technical scheme of the present invention is:
A kind of Oxoquinoxaline-2-carboxylic acid remained detection enzyme-linked immunoassay method, its step comprise the preparation of medicine transformation, immunogene and coating antigen, the preparation and the sample pre-treatments of antibody, it is characterized in that according to following steps:
(1) obtains product QCA-ABA Jiang Oxoquinoxaline-2-carboxylic acid (QCA) and γ-An Jidingsuan (ABA) reaction;
(2) with the described product QCA-ABA of step (1) and ovalbumin mutually coupling obtain coating antigen;
(3) just the described product of step (1) and bovine serum albumin(BSA) mutually coupling obtain immunogene;
(4) the immunogen immune rabbit with step (2) obtains the specificity rabbit polyclonal antibody;
(5) use the coating antigen bag of step (1) by solid phase carrier;
(6) with described sample earlier after acidolysis is extracted after the MAX column purification, add derivative reagent at last and catalyzer is handled, obtain product to be measured;
(7) product to be measured with step (6) carries out enzyme linked immunosorbent detection.
Wherein:
Described solid phase carrier is an ELISA Plate.For example can adopt the ELISA Plate in 48 or 96 holes to make solid phase carrier.
Described derivative reagent is a butylamine.
Described catalyzer is the itrile group diethyl phosphate.
A kind of enzyme-linked immunologic detecting kit based on said method comprises box body, is located at the hole enzyme mark utmost point in the box body and is located at the interior reagent of box body, in every hole of ELISA Plate, is coated with described coating antigen; Described reagent comprises anti-Oxoquinoxaline-2-carboxylic acid specificity rabbit polyclonal antibody, horseradish peroxidase mark goat anti-rabbit antibody, concentrated cleaning solution, concentrating sample dilution ， Oxoquinoxaline-2-carboxylic acid standard solution, substrate colour developing A liquid, substrate colour developing B liquid, stop buffer, derivative reagent and catalyzer.
Wherein:
Described substrate colour developing A liquid is tetramethyl benzidine or o-phenylenediamine.
Described substrate colour developing B liquid is hydrogen peroxide or urea peroxide.
Described stop buffer is sulfuric acid solution or hydrochloric acid solution.
Described concentrated cleaning solution is the phosphate buffer that contains 0.05～0.1% Tween-20.
Described concentrating sample dilute solution is the phosphate buffer of pH7.4.
Enzyme-linked immunoassay method of the present invention and kit mainly adopt in the qualitative or detection by quantitative edible animal tissue of indirect competitive ELISA method such as liver, the muscle QCA residual.The QCA polyclonal antibody that it adopts high specific has characteristics such as high specific, high sensitivity, pinpoint accuracy, pin-point accuracy, organizes lowest detection to be limited to 0.6 μ g/kg.
Description of drawings
Fig. 1 is an immunogene synthetic route of the present invention.
Fig. 2 is an immunogene uv-spectrogram of the present invention.
Fig. 3 is an Oxoquinoxaline-2-carboxylic acid ELISA kit typical curve of the present invention.
Embodiment
The invention will be further described below in conjunction with embodiment, but do not limit the present invention in any form.
1.1 it is immunogenic synthetic
Medicine transformation of the present invention and immunogenic preparation are according to the technology path shown in the accompanying drawing 1.Concrete steps are: get chloride QCA0.0348g and add 1.0mL N, in the dinethylformamide (DMF), dropwise add γ-An Jidingsuan solution, stirring reaction 3h under stirring.The centrifugal sediment of removing, this is an A liquid.(BSA) is dissolved in 5mL physiological saline with the 340mg bovine serum albumin(BSA), and this is a B liquid.The A drop is added B liquid, add N-hydroxy-succinamide (NHS) 0.023g again, N, N-dicyclohexylcarbodiimide (DCC) 0.0434g, 4 ℃ of reactions are spent the night, the centrifugal precipitation of removing, get supernatant and use normal saline dialysis 6 days, every 12h changes dislysate, with the products therefrom low-temperature freeze-dry, packing is stored in-20 ℃ of refrigerators, uses for immunity.Its uv-spectrogram as shown in Figure 2.
1.2 coating antigen is synthetic
Get chloride QCA0.0348g and add 1.0mLN, in the dinethylformamide (DMF), dropwise add γ-An Jidingsuan solution, stirring reaction 3h under stirring.The centrifugal sediment of removing, this is an A liquid.(OVA) is dissolved in 5mL physiological saline with the 180mg oralbumin, and this is a B liquid.The A drop is added B liquid, add N-hydroxy-succinamide (NHS) 0.023g again, N, N-dicyclohexylcarbodiimide (DCC) 0.0434g, 4 ℃ of reactions are spent the night, the centrifugal precipitation of removing, get supernatant and use normal saline dialysis 6 days, every 12h changes dislysate, with the products therefrom low-temperature freeze-dry, packing is stored in-20 ℃ of refrigerators, for coating antigen.
The preparation of embodiment 2, antibody
2.1 immune animal
Adopt new zealand white rabbit as immune animal, with QCA and γ-An Jidingsuan reaction, reaction product and ovalbumin coupling are immunogene, immunizing dose is 0.5～1mg/, Freund's complete adjuvant with immunogene and equivalent when head exempts from is mixed and made into emulsifying agent, the subcutaneous multi-point injection of nape portion, getting the same dose immunogene 2～4 weeks adds equivalent incomplete Freund mixing and emulsifying at interval, booster immunization once, immunity is 6 times altogether, to monitor serum antibody titer and specificity between duration of immunity, not add adjuvant for the last time.Take a blood sample behind last immunity 7～10d, the arteria carotis bloodletting obtains the QCA polyclonal antibody of purifying through ammonium sulfate precipitation.
2.2 antibody purification
Adopt sad-ammonium sulfate salting-out process antibody purification.Get rabbit anteserum 5mL, add the acetate buffer of 0.06mol/L pH5.0, transfer to pH4.5 with the HCl of 0.1mol/L; Under room temperature, add sad 165 μ L, stir 30min; With 44 ℃ of centrifugal 30min of serum 10000r/min, abandon precipitation, with the NaOH solution of 0.1mol/L supernatant pH is transferred to 7.4; Slowly drip equivalent saturated ammonium sulfate solution to supernatant, the final concentration that makes ammonium sulfate is 50%, stirs 20min, and 4 ℃ of centrifugal 30min of 10000r/min abandon supernatant, and precipitation is dissolved among a small amount of 0.01mol/LpH7.2PBS; To precipitate suspending liquid and move into bag filter, and,, put-20 ℃ of refrigerators and preserve the serum packing bottle of purifying with 0.01mol/L pH7.2PBS dialysed overnight.
2.3 antibody titer
Adopting the square formation titrimetry to record antibody titer is 1: 32000.
2.4 antibody sensitivity
The employing indirect competitive ELISA is measured, with antibody I C
50Value (inhibiting rate is 50% pairing drug concentration) is judged antibody sensitivity for index.QCA-ABA is diluted to 0 μ g/L, 0.2 μ g/L, 0.8 μ g/L, 3.2 μ g/L, 12.8 μ g/L, 51.2 μ g/L series concentration, measure light absorption value, the mean value of the standard items light absorption value that is obtained multiply by 100 again divided by the light absorption value of 0 μ g/L standard, and plotting one is the curve map of semilog coordinate system with QCA concentration (μ g/L).The result shows, antibody I C
50Value is 1～10 μ g/L.
2.5 antibody specificity
Adopt the indirect competitive ELISA program determination, the cross reacting rate of calculating antibody according to a conventional method is the specificity that index is judged antibody with the cross reacting rate of antibody.The result is as shown in table 1, and QCA-ABA-BSA antibody removes has cross reaction with MQCA-ABA-BSA (12%), and is not obvious with the cross reaction more than carbadox, the quinoline match, with analogue, catalyzer, derivative reagent no cross reaction.
Table 1 antibody specificity of the present invention
The foundation of embodiment 3, sample-pretreating method
(1) get the equal pledge 2g of pig muscle or liver organization, add 5% metaphosphoric acid, 10% methanol solution 5mL, vibration 2min, the centrifugal 10min of 4800r/min gets supernatant; To precipitate again and repeat to extract once, merge extracted twice liquid by above-mentioned steps;
(2) in the extract of step (1), add ethyl acetate 6mL, vibration 2min, the centrifugal 10min of 4800r/min gets the upper strata ethyl acetate layer; To lower floor's liquid more once, combined ethyl acetate layer by the above-mentioned steps re-extract;
(3) add 0.5mol/L phosphate buffer (pH7.0) 5mL in the ethyl acetate layer of step (2) and strip, vibration 1min leaves standstill 10min, takes off a layer water; Supernatant liquid is repeated to extract once by above-mentioned steps again, merge water;
(4) respectively with methyl alcohol 3mL and distilled water 3mL activation MAX post, flow speed control adds the sample of above-mentioned steps (3) preparation at 1 droplet/second;
(5) treat that sample extracting solution all flows out after, use 0.05mol/L NaOH solution 3mL, methyl alcohol 3mL drip washing MAX post respectively, positive pressure blowing is removed impurity and is disturbed;
(6) with 2% formic acid methanol solution 3mL wash-out and collect eluent, positive pressure blowing is so that collect fully;
(7) 40 ℃ of nitrogen dry up, and add methylene chloride 2mL, whirling motion 30s, dissolved residue in the test tube after drying up;
(8) derivative reagent and each 10 μ L of catalyzer after adding is diluted in test tube respectively put 37 ℃ of water-bath lucifuges reaction 5h;
(10) react the back 47 ℃ of nitrogen that finish and dry up, in test tube, add sample diluting liquid 2mL of the present invention, vibration 30s, dissolved residue as sample solution, is measured for the ELISA method.
The foundation of embodiment 4, detection method
4.1 sensitivity
QCA is diluted to 0ng/mL, 0.2ng/mL, 0.8ng/mL, 3.2ng/mL, 12.8ng/mL, 51.2ng/mL series concentration with methylene chloride, adds 37 ℃ of reactions of 10 μ L butylamine and 20 μ L itrile group diethyl phosphates (DEPC) 5h, 47 ℃ of N
2Dry up, (PBS pH7.4) is settled to original concentration, measures with indirect competitive ELISA with phosphate buffer.The mean value of the standard items light absorption value that is obtained is divided by the light absorption value (inhibiting rate) of 0ng/mL standard, is horizontal ordinate with the logarithm of QCA solution concentration, is ordinate with the inhibiting rate, the drawing standard curve.
Adopt IC
50Estimate sensitivity of the present invention.Measure the IC of 20 typical curves respectively
50(seeing Table 2), the result shows IC
50Mobility scale between 1～10 μ g/L, average is 4.44 μ g/L.Measure 20 parts of blank tissue (pig muscle, liver) samples, its mean value is added 3 times of standard deviations, be and organize lowest detectable limit.Result's (seeing Table 3) shows that pig liver and musculature lowest detectable limit are respectively 0.68 μ g/kg and 0.60 μ g/kg.
Table 2 50% inhibition concentration
Table 3 organize lowest detectable limit (n=20, LOD=X+3SD)
4.2 accuracy
1mg/mLQCA liquid is added in pig muscle or the pig liver tissue, making its final concentration is 0 μ g/kg, 1 μ g/kg, 5 μ g/kg, 20 μ g/kg, 5 repetitions of each concentration, repeat 7 days, extract, purify, derive according to embodiment 3 described methods, adopt QCA concentration in the indirect competitive ELISA working sample, and the calculate recovery rate and the coefficient of variation (the results are shown in Table 4).Add the QCA of 1 μ g/kg, 5 μ g/kg, 20 μ g/kg in pork, the pig liver, all between 60%～115%, interassay coefficient of variation all＜20% for the recovery.
The accuracy of table 4 the inventive method
4.3 precision
The coefficient of variation with the standard items inhibiting rate is an index, estimates precision of the present invention (making a variation between variation and plate in the plate).Result's (table 5) shows, the ELISA method good reproducibility of being set up, Variation Lines number average＜20% in the plate and between plate.
The precision of table 5 the inventive method
The preparation of embodiment 5, enzyme-linked immunologic detecting kit
5.1 the assembling of enzyme-linked immunologic detecting kit
Kit of the present invention mainly is made up of goat anti-rabbit antibody, QCA antibody liquid, substrate colour developing A liquid, substrate colour developing B liquid, stop buffer, concentrated cleaning solution, sample diluting liquid, derivative reagent butylamine, catalyzer itrile group diethyl phosphate (DEPC) and the foam carriage of box body, ELISA Plate, QCA standard items mother liquor (1mg/mL), horseradish peroxidase (HRP) mark
5.2 the preparation of agents useful for same
(1) wraps diluted liquid: get Na
2CO
31.5g, NaHCO
32.9g, Na
2N
30.2g, add distilled water to 1000mL, transfer to pH9.6; (2) confining liquid: get ovalbumin 0.1g and be dissolved in 100mLpH7.4PBS; (3) concentrated cleaning solution: get NaCl80.0g, KH
2PO42g, Na
2HPO
412H2O29g, KCl2g adds tween (Tween)-20 5mL, and thimerosal 0.1g adds distilled water to 1000mL, transfers to pH7.4; (4) concentrating sample dilution: get NaCl80.0g, KH
2PO
42g, Na
2HPO
412H
2O29g, KCl2g, thimerosal 0.1g adds distilled water to 1000mL, transfers to pH7.4; (5) substrate colour developing A liquid: again in the present embodiment, what adopt is the tetramethyl biphenyl amine aqueous solution, promptly get tetramethyl benzidine 200mg, absolute ethyl alcohol (in a further embodiment, can change described tetramethyl benzidine into o-phenylenediamine, absolute ethyl alcohol can change dimethyl sulfoxide into) 100mL, add distilled water to 1000mL; (6) substrate colour developing B liquid: get Na
2HPO
414.60g, citric acid 9.33g, 0.75% hydrogen peroxide urea 6.4mL, add distilled water to 1000mL, transfer to the substrate of pH5.0～5.4:(7) colour developing mixed liquor (being tetramethyl benzidine-hydrogen peroxide urea solution in the present embodiment): develop the color A liquid and substrate colour developing liquid B of substrate mixed and be substrate colour developing liquid mixed liquor in 1: 1 by volume; (8) stop buffer: what adopt in the present embodiment is the sulfuric acid solution of 2mol/L, promptly get 18mol/L concentrated sulphuric acid 100mL, slowly be added drop-wise to distilled water to 900mL, be stop buffer (sulfuric acid solution of 2mol/L can be changed into the hydrochloric acid solution of 4mol/L in a further embodiment).
5.3 the preparation of ELISA Plate
Get 48 or 96 hole ELISA Plate, be cushioned liquid with bag QCA and γ-An Jidingsuan reaction back are diluted to 4 μ g/mL with the ovalbumin conjugate, every hole adds 100 μ L, 4 ℃ are spent the night, the coating buffer that inclines with cleansing solution washing 3 times, pats dry, every then hole adds 200 μ L confining liquids, hatch 1h for 37 ℃, liquid in the hole of inclining, cleansing solution washing 3 times, pat dry, with aluminium film vacuum seal preservation, standby.
5.4 the stability of kit of the present invention
2～8 ℃ of stability tests: place 4 ℃ to preserve down kit, in 0,1,2,3,4,5,6, get kit July respectively, measures IC
50Recovery when addition is 4 μ g/kg in the light absorption value of value, 0 standard solution and the pig liver.With IC
50Value less than 0.6 and the recovery standard for judging that kit lost efficacy in 50%～120% scope not, the results are shown in Table 6 greater than the light absorption value of 10 μ g/L, 0 standard solution.
4 ℃ of stability tests of table 6 kit of the present invention
37 ℃ of accelerated stability tests: place 37 ℃ to preserve down kit, in 0,1,2,3,4,5d gets kit respectively, measures IC
50Recovery when addition is 4 μ g/kg in the light absorption value of value, 0 standard solution and the pig liver.With IC
50Value less than 0.6 and the recovery standard for judging that kit lost efficacy in 50%～120% scope not, the results are shown in Table 7 greater than the light absorption value of 10 μ g/L, 0 standard solution.
37 ℃ of accelerated stability tests of table 7 kit of the present invention
Table 6 and table 7 result show that kit is when being saved in 3rd month for 4 ℃, and all indexs are all in normal fluctuation range.37 ℃ are saved in 5d, and the light absorption value of 0 standard solution drops to 0.66, IC
50Value is 12.38 μ g/L, and the recovery is 108%, illustrates that the part reagent in the kit has begun to lose efficacy.Test method is edited with reference to Tang Weiguo, " preparation of medical test diagnostic reagent and application ", Shanghai scientific and technical literature publishing house, version in 1996.Kit was placed one day at 37 ℃, was equivalent to 2～8 ℃ and preserved one and a half months.The stability test result shows that the storage life of this kit under 4 ℃ of conditions was at least 6 months.
The mensuration program of embodiment 6, enzyme-linked immunologic detecting kit
6.1 working fluid preparation
QCA standard solution working fluid preparation: the QCA mother liquor is diluted to each concentration QCA standard solution (0ng/mL, 0.2ng/mL, 0.8ng/mL, 3.2ng/mL, 12.8ng/mL, 51.2ng/mL) with methylene chloride, mixing, in test tube, add derivative reagent butylamine and each 10 μ L of catalyzer itrile group diethyl phosphate after diluting respectively, put 37 ℃ of water-baths reaction 5h; React the back 47 ℃ of nitrogen that finish and dry up, in test tube, add sample dilution 2mL, vibration 30s, standby as QCA standard sample solution, measure for the ELISA method;
The preparation of 5% metaphosphoric acid, 10% methanol solution: metaphosphoric acid 50g, methyl alcohol 100mL adds distilled water and is settled to 1000mL;
The preparation of 2% formic acid methanol solution: 2mL formic acid adds 98mL methyl alcohol mixing;
0.5mol/LK
2HPO
4Solution preparation: K
2HPO
43H
2O114.11g adds distilled water to 1000mL, uses H
3PO
4Transfer pH to 7.0;
0.05mol/L the preparation of NaOH solution: NaOH2g adds distilled water to 1000mL;
Sample diluting liquid preparation: the concentrating sample dilution that provides in the kit is used after with 10 times of dilutions of distilled water, put 4 ℃ of refrigerators preservations;
Cleansing solution preparation: the concentrated cleaning solution that provides in the kit is used after with 10 times of dilutions of distilled water, put 4 ℃ of refrigerators preservations;
The preparation of the goat anti-rabbit antibody working fluid of horseradish peroxidase (HRP) mark: determine the consumption of required reagent according to each test sample quantity, with the goat anti-rabbit antibody and 1: 10 by volume the dilution proportion of diluted sample solution of HRP mark, mixing, standby;
The preparation of QCA antibody working fluid: according to each institute expense, with QCA antibody liquid and 1: 10 by volume dilution proportion of sample diluting liquid, mixing, standby;
The derivative reagent working fluid: get derivative reagent butylamine 30 μ L that kit provides in the 1mL acetonitrile, making its final concentration is 300mmol/L, and mixing is now with the current;
The catalyzer working fluid: get catalyzer itrile group diethyl phosphate (DEPC) 50 μ L that kit provides in 500 μ L acetonitriles, making its final concentration is 600mmol/L, and mixing is now with the current;
Substrate colour developing A liquid preparation: in substrate colour developing A liquid bottle, add 10mL ethanol, put 4 ℃ of refrigerators and preserve January.Before facing usefulness,, get the 10 times of dilutions of distilled water of appropriate amount of substrate A stock solution according to each institute expense;
Substrate colour developing B liquid stock solution preparation: in substrate colour developing B liquid bottle, add 2mL distilled water, put 4 ℃ of refrigerators and can preserve January.Before facing usefulness, according to each institute expense, substrate colour developing B liquid adds substrate colour developing B liquid stock solution 6.4 μ L by every milliliter of citrate buffer solution;
Substrate mixed liquor preparation:,, now with the current with substrate develop the color A liquid and by volume 1: 1 mixing of substrate colour developing B liquid according to the consumption that how much determines required reagent of each test set tissue samples.
6.2 determination step
Get capillary strip: the hole bar of enough standard items and the used quantity of sample is inserted the micropore frame, and standard items and sample are done two parallel experiments, write down the position of standard items and sample;
With titer or testing sample: add standard items or the sample liquid 30 μ L that handle well in micropore, note changing the suction nozzle of pipettor when every hole adds sample or standard items;
Add antibody: add QCA antibody working fluid 70 μ L and in each micropore, fully mix, hatch 1h (37 ℃), cover upper film (avoiding evaporating) at incubator;
Washing: pour out the liquid in the hole, wash 3 times and pat dry;
The goat anti-rabbit antibody that adds the HRP mark: the goat anti-rabbit antibody working fluid 100 μ L that add the HRP mark fully mix in each micropore, hatch 1h (37 ℃) at incubator, cover upper film (avoiding evaporating);
Washing: pour out the liquid in the hole, wash 5 times and pat dry;
Add substrate: add substrate mixed liquor 100 μ L in each micropore, fully mix being incorporated in 37 ℃ of dark places and hatching 15min;
Stop: add reaction terminating liquid 50 μ L in each micropore;
Measure: measuring absorbance at the 450nm place, is blank with the air, must read light absorption value in the 60min after adding stop buffer.
6.3 the result judges
The mean value of standard items that obtained and sample light absorption value multiply by 100 again divided by the light absorption value of first standard (0 standard), with the inhibiting rate is ordinate, logarithm with QCA concentration is that horizontal ordinate is made typical curve, curve is the convergence straight line in 0.1～51.2ng/mL scope, and the concentration of corresponding each sample (ng/g) can be read from typical curve.
The examination of embodiment 7, kit of the present invention and application
7.1 the advance of kit of the present invention
Kit of the present invention and HPLC method are compared, and result's (seeing Table 8) shows that ELISA has good sensitivity and specificity, and is good with HPLC method correlativity.
Table 8HPLC method and comparison of the present invention
7.2 the application of kit of the present invention
With 21 45 ages in days, body weight 15.0 ± 2.0kg kind is for " growing up, " the healthy piglet of two-way cross castration is divided into 2 groups at random, and one group is blank group (3), and another group is test group (12).The carbadox 7d that test group is fed and contained 50mg/kg, butcher 3 respectively in drug withdrawal 0d, 4d, 10d, 14d, the blank group is respectively butchered 1 at drug withdrawal 0d, 4d, 10d respectively, adopts the content of QCA in ELISA method and hplc simultaneous determination pig liver, the muscle.Measurement result (table 9) shows: drug withdrawal 0 day, QCA content is respectively 82ng/g and 7.6ng/g in ELISA method mensuration liver, the muscle, and HPLC method testing result is respectively 88ng/g and 6.9ng/g, drug withdrawal 14 days, be respectively 4.8ng/g and 4.6ng/g with QCA content in ELISA method and the HPLC method detection liver, all do not detect in the muscle.Two kinds of method measurement results can both reflect that the prolongation in time of the residual quantity of QCA in liver and muscle reduces gradually, negative control group measurement result all negative (＜1 μ g/kg), dosing group all positive (＞1 μ g/kg), illustrate that kit of the present invention has the good actual ability of detecting, can filter out positive (＞1 μ g/kg).
QCA Determination on content in table 9 pig liver and the musculature
Annotate: "-" expression will not be calculated
Claims (3)
1. an Oxoquinoxaline-2-carboxylic acid remained detection enzyme-linked immunoassay method comprises the preparation of medicine transformation, immunogene and coating antigen, the preparation and the sample pre-treatments of antibody, it is characterized in that according to following steps:
(1) obtains product QCA-ABA Jiang Oxoquinoxaline-2-carboxylic acid (QCA) and γ-An Jidingsuan (ABA) reaction;
(2) with the described product QCA-ABA of step (1) and ovalbumin mutually coupling obtain coating antigen;
(3) just the described product of step (1) and bovine serum albumin(BSA) mutually coupling obtain immunogene;
(4) the immunogen immune rabbit with step (3) obtains the specificity rabbit polyclonal antibody;
(5) use the coating antigen bag of step (2) by solid phase carrier;
(6) with pork or pig liver earlier behind 5% metaphosphoric acid, 10% methanol extraction after the MAX column purification, add the derivative reagent butylamine at last and catalyzer itrile group diethyl phosphate is handled, obtain product to be measured;
(7) product to be measured with step (6) carries out enzyme linked immunosorbent detection.
2. method according to claim 1 is characterized in that described solid phase carrier is an ELISA Plate.
3. be applicable to the enzyme-linked immunologic detecting kit of claim 1 or 2 described methods, comprise box body, be located at the ELISA Plate in the box body and be located at the interior reagent of box body, it is characterized in that, in every hole of ELISA Plate, be coated with described coating antigen; Described reagent comprises anti-Oxoquinoxaline-2-carboxylic acid specificity rabbit polyclonal antibody, horseradish peroxidase mark goat anti-rabbit antibody, concentrated cleaning solution, concentrating sample dilution ， Oxoquinoxaline-2-carboxylic acid standard solution, substrate colour developing A liquid, substrate colour developing B liquid, stop buffer, derivative reagent and catalyzer;
Wherein
Described substrate colour developing A liquid is tetramethyl benzidine or o-phenylenediamine;
Described substrate colour developing B liquid is hydrogen peroxide or urea peroxide;
Described stop buffer is sulfuric acid solution or hydrochloric acid solution;
Described concentrated cleaning solution is the phosphate buffer that contains 0.05～0.1% Tween-20;
Described concentrating sample dilute solution is the phosphate buffer of pH7.4.
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| CN101144816B (en) * | 2007-10-31 | 2011-05-11 | 江南大学 | 3-methyl-quinoline-2-carboxylic acid immunomagnetic bead detection method |
| CN102590498A (en) * | 2012-01-13 | 2012-07-18 | 重庆市科学技术研究院 | Immune colloidal gold test paper for detecting quinoxaline-2-carboxylic acid residue and preparation method of immune colloidal gold test paper |
| CN102621322B (en) * | 2012-03-29 | 2014-04-23 | 北京维德维康生物技术有限公司 | Kit for detecting 3-methyl-quinoxaline-2-carboxylic acid |
| CN102654500A (en) * | 2012-05-17 | 2012-09-05 | 重庆市科学技术研究院 | Detecting reagent kit used for detecting quinoxalinone-2-carboxylic acid and method |
| CN102854309B (en) * | 2012-08-24 | 2014-09-10 | 镇江出入境检验检疫局检验检疫综合技术中心 | ELISA kit for praziquantel content of animal-derived food and detection method thereof |
| CN104597178B (en) * | 2015-01-14 | 2016-05-11 | 华中农业大学 | A kind of 3-Jia based quinoxaline-2 carboxylic acid immune affinity column and preparation method thereof |
| CN108226499A (en) * | 2016-12-13 | 2018-06-29 | 丹阳亿太生物科技发展有限公司 | Detect the time-resolved fluoroimmunoassay kit of Madumycin |
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| US6689886B2 (en) * | 2000-02-04 | 2004-02-10 | Pfizer Inc. | Quinoxalinyl carboxylic acid derivatives |
| CN1811437A (en) * | 2006-02-16 | 2006-08-02 | 中国农业大学 | Method for assaying sulfaquinoxaline and special enzyme-linked immune reagent kit |
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Patent Citations (2)
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| US6689886B2 (en) * | 2000-02-04 | 2004-02-10 | Pfizer Inc. | Quinoxalinyl carboxylic acid derivatives |
| CN1811437A (en) * | 2006-02-16 | 2006-08-02 | 中国农业大学 | Method for assaying sulfaquinoxaline and special enzyme-linked immune reagent kit |
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| Hutchinson MJ,et al.Development and validation of an improved method for confirmation of the carbadox metabolite,quinoxaline-2-carboxylic acid, in porcine liver using LC-electrospray MS-MS according to revised EU criteria for veterinary drug residue analysis.《ANALYST》.2002,第127卷(第3期),342-346. * |
| 邱银生,等.猪食用组织中喹喔啉-2-羧酸的高效液相色谱检测方法.《中国兽医学报》.2003,第23卷(第3期),286-288. * |
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| CN1971279A (en) | 2007-05-30 |
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