CN1971279B - Oxoquinoxaline-2-carboxylic acid remained enzyme linked immunity detecting method and reagent kits - Google Patents

Abstract

The invention belongs to the technical field of immunochemical analysis. Disclosed is an enzyme-linked immunosorbent method and kit for the detection of quinoxaline-2-carboxylic acid residues. The method of the invention mainly includes drug transformation, preparation of immunogen, coating agent, antibody, sample pretreatment and ELISA Establishment of detection methods. The kit of the present invention is mainly composed of quinoxaline-2-carboxylic acid (QCA) specific antibody, QCA standard product, and an enzyme label plate coated with ovalbumin conjugate after QCA reacts with γ-aminobutyric acid . During detection, the sample was hydrolyzed with metaphosphoric acid to release QCA, purified by MAX column, derivatized with butylamine, and determined by indirect competitive ELISA method. The method and the kit of the invention have the advantages of simplicity, speed, sensitivity, accuracy, etc., and can quickly detect a large number of samples at the same time, and the lowest detection limit of tissue is 0.6 μg/kg.

Description

Enzyme-linked immunoassay method and kit for quinoxaline-2-carboxylic acid residues

technical field

The invention belongs to the technical field of food immunochemical analysis. It specifically relates to an enzyme-linked immunosorbent method and kit for the detection of quinoxaline-2-carboxylic acid (QCA) residues, which is suitable for the determination of quinoxaline, a marker for carbadoxy residues, in edible tissues of food animals such as muscle and liver - Residual amount of 2-carboxylic acid (QCA).

Background technique

Carbadox is a quinoxaline drug, which is a synthetic broad-spectrum antibacterial drug, and has been widely used as a growth-promoting agent in livestock and poultry breeding. Toxicology studies have found that carbadox is carcinogenic and mutagenic. In order to protect the health of consumers and expand the trade of animal foods, it is imperative to strengthen the detection of carbadox residues in animal foods.

Carbadox is metabolized into quinoxaline-2-carboxylic acid (QCA) in animals. QCA combined with tissue protein has a long residence time in the body and is regarded as a residual marker of carbadox. Therefore, analysis of carbadox residues usually analyzes its metabolism. Product QCA. The current methods for detecting QCA residues include high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), and gas chromatography-mass spectrometry (GC-MS).

Commonly used instrumental methods (HPLC, LC-MS, GC-MS) require expensive instruments, require high technical requirements for operators, take a long time to analyze, cannot perform high-throughput sample screening, and are not suitable for the majority of grassroots testing units application, difficult to promote. Immunochemical analysis, especially enzyme-linked immunosorbent assay (ELISA), has the advantages of rapidity, high sensitivity, simple operation, and good specificity, and is suitable for high-throughput sample screening. At present, there are no reports on the ELISA detection method and kit of carbadox residue marker quinoxaline-2-carboxylic acid (QCA) at home and abroad.

The enzyme-linked immunosorbent method and kit provided by the present invention adopt the inter-press competition ELISA method, which has a wide detection range, low false positive rate, reliable and accurate detection results, and is suitable for QCA detection in edible tissues of food animals such as muscle and liver , has very important economic and social significance.

Contents of the invention

The first object of the present invention is to provide an enzyme-linked immunosorbent detection method for quinoxaline-2-carboxylic acid (QCA) residues.

The second object of the present invention is to provide an enzyme-linked immunoassay kit for quinoxaline-2-carboxylic acid (QCA) residues.

The method and kit of the present invention are specially used for enzyme-linked immunoassay detection of quinoxaline-2-carboxylic acid (QCA) residues in animal edible tissues. Compared with existing methods, the method and kit of the present invention have high sensitivity, It has the outstanding advantages of fast detection, convenient use and low detection cost.

Technical scheme of the present invention is:

An enzyme-linked immunosorbent method for detecting quinoxaline-2-carboxylic acid residues, the steps of which include drug modification, preparation of immunogen and coating source, preparation of antibody and sample pretreatment, characterized in that the following steps are followed:

(1) reacting quinoxaline-2-carboxylic acid (QCA) with gamma-aminobutyric acid (ABA) to obtain product QCA-ABA;

(2) Coupling the product QCA-ABA described in step (1) with ovalbumin to obtain the coating former;

(3) coupling the product described in step (1) with bovine serum albumin to obtain an immunogen;

(4) immunizing rabbits with the immunogen of step (2) to obtain specific rabbit polyclonal antibodies;

(5) Coating the solid phase carrier with the coating original of step (1);

(6) Purify the sample through MAX column after acid hydrolysis and extraction, and finally add derivatization reagent and catalyst for treatment to obtain the product to be tested;

(7) The product to be tested in step (6) is subjected to enzyme-linked immunoassay.

in:

The solid phase carrier is a microtiter plate. For example, a 48- or 96-well ELISA plate can be used as the solid phase carrier.

The derivatizing reagent is butylamine.

The catalyst is diethyl cyanophosphate.

An enzyme-linked immunoassay kit based on the above method, comprising a box body, a hole enzyme label pole arranged in the box body, and reagents arranged in the box body, and each hole of the enzyme-linked plate is coated with the described coating The original; the reagents include anti-quinoxaline-2-carboxylic acid-specific rabbit polyclonal antibody, horseradish peroxidase-labeled goat anti-rabbit antibody, concentrated washing solution, concentrated sample diluent, quinoxaline-2- Carboxylic acid standard solution, substrate chromogenic solution A, substrate chromogenic solution B, stop solution, derivatization reagent and catalyst.

in:

The substrate color developing liquid A is tetramethylbenzidine or o-phenylenediamine.

The substrate color B solution is hydrogen peroxide or carbamide peroxide.

The stop solution is sulfuric acid solution or hydrochloric acid solution.

The concentrated washing liquid is a phosphate buffer containing 0.05-0.1% Tween-20.

The concentrated sample dilution solution is a phosphate buffer solution with a pH of 7.4.

The ELISA method and kit of the present invention mainly adopt indirect competitive ELISA method to qualitatively or quantitatively detect QCA residues in animal edible tissues such as liver and muscle. It uses highly specific QCA polyclonal antibody, which has the characteristics of high specificity, high sensitivity, high precision, high accuracy, etc., and the minimum detection limit of tissue is 0.6 μg/kg.

Description of drawings

Fig. 1 is the immunogen synthesis route of the present invention.

Fig. 2 is the ultraviolet spectrum of the immunogen of the present invention.

Fig. 3 is the standard curve of the quinoxaline-2-carboxylic acid ELISA kit of the present invention.

Detailed ways

The present invention will be further described below in conjunction with embodiment, but does not limit the present invention in any form.

Embodiment 1, antigen preparation

1.1 Synthesis of immunogen

The pharmaceutical transformation and immunogen preparation of the present invention follow the technical route shown in Figure 1. The specific steps are: add 0.0348g of acid chloride QCA to 1.0mL N,N-dimethylformamide (DMF), add γ-aminobutyric acid solution dropwise under stirring, and stir for 3h. Centrifuge to remove the precipitate, this is liquid A. Dissolve 340mg of bovine serum albumin (BSA) in 5mL of physiological saline, which is liquid B. Add liquid A dropwise to liquid B, then add 0.023g of N-hydroxysuccinimide (NHS), 0.0434g of N,N-dicyclohexylcarbodiimide (DCC), react overnight at 4°C, centrifuge to remove the precipitate, and take The supernatant was then dialyzed with normal saline for 6 days, and the dialysate was changed every 12 hours. The resulting product was freeze-dried and stored in a -20°C refrigerator for immunization. Its UV spectrum is shown in Figure 2.

1.2 Synthesis of Coating Progen

Add 0.0348g of acid chloride QCA to 1.0mL N,N-dimethylformamide (DMF), add γ-aminobutyric acid solution dropwise under stirring, and stir for 3h. Centrifuge to remove the precipitate, this is liquid A. Dissolve 180mg of ovalbumin (OVA) in 5mL of normal saline, which is liquid B. Add liquid A dropwise to liquid B, then add 0.023g of N-hydroxysuccinimide (NHS), 0.0434g of N,N-dicyclohexylcarbodiimide (DCC), react overnight at 4°C, centrifuge to remove the precipitate, and take The supernatant was dialyzed with normal saline for 6 days, and the dialysate was replaced every 12 hours. The resulting product was lyophilized at low temperature, and stored in a -20°C refrigerator for packaging.

Embodiment 2, the preparation of antibody

2.1 Immunization of animals

New Zealand white rabbits were used as immunized animals, and QCA was reacted with γ-aminobutyric acid, and the reaction product was coupled with ovalbumin to form an immunogen. Freund's complete adjuvant was mixed to make an emulsifier, and it was injected subcutaneously at multiple points on the back of the neck. The same dose of immunogen plus an equal amount of Freund's incomplete adjuvant was mixed and emulsified at an interval of 2 to 4 weeks, and the booster immunization was performed once, and a total of 6 immunizations were performed. To monitor serum antibody titer and specificity, no adjuvant was added for the last time. Blood was collected 7-10 days after the last immunization, blood was exsanguinated from the carotid artery, and the purified QCA polyclonal antibody was obtained by fractional precipitation with ammonium sulfate.

2.2 Antibody purification

Antibodies were purified by octanoic acid-ammonium sulfate salting-out method. Take 5 mL of rabbit serum, add 0.06 mol/L acetic acid buffer solution with pH 5.0, adjust the pH to 4.5 with 0.1 mol/L HCl; add 165 μL of octanoic acid at room temperature, stir for 30 min; centrifuge the serum at 10,000 r/min at 44°C for 30 min , discard the precipitate, and adjust the pH of the supernatant to 7.4 with 0.1mol/L NaOH solution; slowly add an equivalent amount of saturated ammonium sulfate solution to the supernatant to make the final concentration of ammonium sulfate 50%, stir for 20min, 4°C 10000r Centrifuge for 30 min at 1/min, discard the supernatant, dissolve the precipitate in a small amount of 0.01mol/L pH7.2PBS; transfer the precipitate suspension into a dialysis bag, dialyze overnight with 0.01mol/L pH7.2PBS, and divide the purified serum into vials. Store in -20°C refrigerator.

2.3 Antibody titer

The antibody titer measured by the square array titration method was 1:32000.

2.4 Antibody sensitivity

The indirect competitive ELISA was used to determine the sensitivity of the antibody by using the antibody IC ₅₀ value (drug concentration corresponding to an inhibition rate of 50%) as an index. Dilute QCA-ABA to 0μg/L, 0.2μg/L, 0.8μg/L, 3.2μg/L, 12.8μg/L, 51.2μg/L serial concentrations, measure the absorbance value, and obtain the average absorbance value of the standard The value was divided by the absorbance value of the 0 μg/L standard and multiplied by 100 to draw a graph with QCA concentration (μg/L) as the semi-logarithmic coordinate system. The results showed that the IC ₅₀ value of the antibody was 1-10 μg/L.

2.5 Antibody specificity

The indirect competitive ELISA program was used to determine the cross-reactivity rate of the antibody according to the conventional method, and the specificity of the antibody was determined by the cross-reaction rate of the antibody as an index. The results are shown in Table 1. Except that the QCA-ABA-BSA antibody has a cross-reaction with MQCA-ABA-BSA (12%), the cross-reaction with carbadoxy and cydox is not obvious. No cross-reactivity.

Table 1 Antibody specificity of the present invention

Embodiment 3, establishment of sample pretreatment method

(1) Take 2 g of porcine muscle or liver tissue homogenate, add 5 mL of 5% metaphosphoric acid and 10% methanol solution, shake for 2 min, centrifuge at 4800 r/min for 10 min, and take the supernatant; repeat the extraction once again according to the above steps, and combine two extracts;

(2) Add 6 mL of ethyl acetate to the extract of step (1), shake for 2 minutes, centrifuge at 4800 r/min for 10 minutes, take the upper layer of ethyl acetate layer; repeat the extraction once for the lower layer of liquid according to the above steps, and combine the ethyl acetate layer ;

(3) Add 5mL of 0.5mol/L phosphate buffer (pH7.0) to the ethyl acetate layer of step (2) for back extraction, shake for 1min, let stand for 10min, take the lower aqueous phase; The above steps were repeated for extraction once, and the aqueous phase was combined;

(4) Activate the MAX column with 3mL of methanol and 3mL of distilled water respectively, the flow rate is controlled at 1 drop/s, and the sample prepared in the above step (3) is added;

(5) After all the sample extracts flow out, wash the MAX column with 3mL of 0.05mol/L NaOH solution and 3mL of methanol respectively, and blow dry under positive pressure to remove the interference of impurities;

(6) Elute with 3mL of 2% methanol solution of formic acid and collect the eluent, blow dry under positive pressure to collect completely;

(7) Blow dry with nitrogen at 40°C, add 2 mL of dichloromethane into the dried test tube, vortex for 30 seconds, and dissolve the residue;

(8) Add 10 μL each of the diluted derivatization reagent and catalyst to the test tube, and place in a water bath at 37°C in the dark for 5 hours;

(10) After the reaction is completed, blow dry with nitrogen at 47°C, add 2 mL of the sample diluent of the present invention to the test tube, shake for 30 s, dissolve the residue, and use it as a sample solution for ELISA determination.

Embodiment 4, establishment of detection method

4.1 Sensitivity

Dilute QCA with dichloromethane to a series of concentrations of 0 ng/mL, 0.2 ng/mL, 0.8 ng/mL, 3.2 ng/mL, 12.8 ng/mL, 51.2 ng/mL, add 10 μL butylamine and 20 μL diethyl cyanophosphate The ester (DEPC) was reacted at 37°C for 5h, dried under _N2 at 47°C, and then adjusted to the original concentration with phosphate buffer (PBS, pH7.4), and determined by indirect competitive ELISA. The average absorbance value of the standard obtained was divided by the absorbance value (inhibition rate) of the 0ng/mL standard, and the logarithm of the QCA solution concentration was taken as the abscissa, and the inhibition rate was used as the ordinate to draw a standard curve.

The sensitivity of the present invention was evaluated using _IC50 . The IC ₅₀ of the standard curve was measured 20 times (see Table 2), and the results showed that the IC ₅₀ ranged from 1 to 10 μg/L, with an average value of 4.44 μg/L. Determination of 20 blank tissue (porcine muscle, liver) samples, the average value plus 3 times the standard deviation is the lowest detection limit of the tissue. The results (see Table 3) showed that the lowest detection limits of pig liver and muscle tissue were 0.68 μg/kg and 0.60 μg/kg, respectively.

Table 2 50% inhibitory concentration

Table 3 The lowest detection limit of tissue (n=20, LOD=X+3SD)

4.2 Accuracy

Add 1 mg/mL QCA solution to porcine muscle or porcine liver tissue, so that the final concentration is 0 μg/kg, 1 μg/kg, 5 μg/kg, 20 μg/kg, and each concentration is repeated 5 times for 7 days, according to the example The method described in 3 was extracted, purified, and derived, and the concentration of QCA in the sample was determined by indirect competitive ELISA, and the recovery rate and coefficient of variation were calculated (results are shown in Table 4). When 1μg/kg, 5μg/kg, and 20μg/kg of QCA were added to pork and pig liver tissues, the recoveries were all between 60% and 115%, and the coefficients of variation between batches were all <20%.

The accuracy of table 4 the inventive method

4.3 Precision

Take the coefficient of variation of the inhibition rate of the standard substance as an index to evaluate the precision of the present invention (intra-plate variation and inter-plate variation). The results (Table 5) showed that the established ELISA method had good repeatability, and the coefficients of variation both within and between plates were less than 20%.

The precision of table 5 the inventive method

Embodiment 5, the preparation of enzyme-linked immunoassay kit

5.1 Assembly of enzyme-linked immunoassay kit

The kit of the present invention mainly consists of a box body, an ELISA plate, QCA standard product mother solution (1mg/mL), horseradish peroxidase (HRP)-labeled goat anti-rabbit antibody, QCA antibody solution, substrate chromogenic A solution, Substrate Chromogenic Solution B, Stop Solution, Concentrated Wash Solution, Sample Diluent, Derivatization Reagent Butylamine, Catalyst Diethyl Nitrile Phosphate (DEPC) and Foam Holder

5.2 Preparation of reagents used

(1) Coating diluent: Take 1.5g of Na ₂ CO ₃ , 2.9g of NaHCO ₃ , 0.2g of Na ₂ N ₃ , add double distilled water to 1000mL, and adjust to pH 9.6; (2) Blocking solution: Take egg white Protein _0.1g was dissolved in 100mLpH7.4PBS; (3) Concentrated washing solution: take NaCl80.0g, _KH2PO42g , _Na2HPO4 12H2O29g, KCl2g, add Tween (Tween)-20 5mL, thimerosal 0.1g, add bis Distilled water to 1000mL, _adjusted to pH7.4; (4) concentrated sample diluent: take _NaCl80.0g , _KH2PO42g , _Na2HPO4 · _12H2O29g , KCl2g, thimerosal 0.1g, add double distilled water to 1000mL, adjusted to pH 7.4; (5) Substrate chromogenic solution A: in this embodiment, tetramethylbenzidine solution was used, that is, 200 mg of tetramethylbenzidine, absolute ethanol (in another In the embodiment, the tetramethylbenzidine can be changed to o-phenylenediamine, and the absolute ethanol can be changed to dimethyl sulfoxide) 100mL, and double distilled water is added to 1000mL; (6) Substrate chromogenic liquid B : Take Na ₂ HPO ₄ 14.60g, citric acid 9.33g, 0.75% hydrogen peroxide urea 6.4mL, add double distilled water to 1000mL, adjust to pH5.0～5.4: (7) Substrate chromogenic mixture (in this In the embodiment, it is tetramethylbenzidine-hydrogen peroxide urea solution): the substrate chromogenic solution A and the substrate chromogenic solution B are mixed in a volume ratio of 1:1 to obtain the substrate chromogenic solution mixed solution; (8 ) Termination solution: What adopted in the present embodiment is the sulfuric acid solution of 2mol/L, namely take 100mL of 18mol/L concentrated sulfuric acid, slowly add dropwise to double distilled water to 900mL, be the termination solution (in another embodiment can be Change the sulfuric acid solution of 2mol/L to the hydrochloric acid solution of 4mol/L).

5.3 Preparation of ELISA plate

Take a 48- or 96-well ELISA plate, react QCA with γ-aminobutyric acid with coating buffer, dilute it with ovalbumin conjugate to 4 μg/mL, add 100 μL to each well, overnight at 4 °C, pour off the coating solution, washed 3 times with washing solution, patted dry, then added 200 μL of blocking solution to each well, incubated at 37°C for 1 h, poured off the liquid in the well, washed 3 times with washing solution, patted dry, and sealed with aluminum film for storage until use.

5.4 Stability of the kit of the present invention

Stability test at 2-8°C: store the kit at 4°C, take out the kit at 0, 1, 2, 3, 4, 5, 6, and July, and measure the IC ₅₀ value and the absorbance of the 0 standard solution Value and the recovery rate when the addition amount in pig liver was 4μg/kg. The criteria for judging the failure of the kit were the _IC50 value greater than 10 μg/L, the absorbance value of the 0 standard solution less than 0.6, and the recovery rate not within the range of 50% to 120%. The results are shown in Table 6.

Table 6 4 ℃ stability test of kit of the present invention

Accelerated stability test at 37°C: store the kit at 37°C, take out the kit at 0, 1, 2, 3, 4, and 5 days, measure the IC ₅₀ value, the absorbance value of the 0 standard solution, and the pig liver added The recovery rate when the amount is 4 μg/kg. The criteria for judging the failure of the kit were the _IC50 value greater than 10 μg/L, the absorbance value of the 0 standard solution less than 0.6, and the recovery rate not within the range of 50% to 120%. The results are shown in Table 7.

Table 7 37 ℃ accelerated stability test of kit of the present invention

The results in Table 6 and Table 7 show that when the kit is stored at 4°C until the third month, all indicators are within the normal fluctuation range. After being stored at 37°C until the 5th day, the absorbance value of the 0 standard solution dropped to 0.66, the IC ₅₀ value was 12.38 μg/L, and the recovery rate was 108%, indicating that some reagents in the kit had begun to fail. The test method refers to "Preparation and Application of Medical Laboratory Diagnostic Reagents" edited by Tang Weiguo, Shanghai Science and Technology Literature Publishing House, 1996 edition. The kit is stored at 37°C for one day, which is equivalent to one and a half months at 2-8°C. The results of the stability test show that the shelf life of the kit at 4°C is at least 6 months.

Embodiment 6, the assay procedure of enzyme-linked immunoassay kit

6.1 Preparation of working solution

Preparation of QCA standard solution working solution: Dilute the QCA mother solution with dichloromethane to each concentration of QCA standard solution (0ng/mL, 0.2ng/mL, 0.8ng/mL, 3.2ng/mL, 12.8ng/mL, 51.2ng/mL mL), mix well, add 10 μL each of the diluted derivative reagent butylamine and the catalyst diethyl cyanophosphate to the test tube, and put it in a 37°C water bath for 5 hours; after the reaction is completed, dry it with nitrogen at 47°C, and add Sample diluent 2mL, shake for 30s, as QCA standard sample solution, spare, for ELISA method determination;

Preparation of 5% metaphosphoric acid and 10% methanol solution: 50g metaphosphoric acid, 100mL methanol, add double distilled water to make up to 1000mL;

2% formic acid methanol solution preparation: add 2mL formic acid to 98mL methanol and mix well;

0.5mol/L K ₂ HPO ₄ solution preparation: K ₂ HPO ₄ 3H ₂ O114.11g, add double distilled water to 1000mL, adjust pH to 7.0 with H ₃ PO ₄ ;

0.05mol/L NaOH solution preparation: NaOH 2g, add double distilled water to 1000mL;

Preparation of sample diluent: Dilute the concentrated sample diluent provided in the kit 10 times with distilled water before use, and store in a refrigerator at 4°C;

Washing solution preparation: Dilute the concentrated washing solution provided in the kit 10 times with distilled water before use, and store in a refrigerator at 4°C;

Preparation of horseradish peroxidase (HRP)-labeled goat anti-rabbit antibody working solution: determine the amount of reagents required according to the number of samples tested each time, and mix the HRP-labeled goat anti-rabbit antibody and sample dilution solution at a volume ratio of 1:10 Dilute in proportion, mix well, and set aside;

Preparation of QCA antibody working solution: according to the required amount each time, dilute the QCA antibody solution and sample diluent in a volume ratio of 1:10, mix well, and set aside;

Derivatization reagent working solution: Take 30 μL of the derivatization reagent butylamine provided in the kit in 1 mL of acetonitrile to make the final concentration 300 mmol/L, mix well, and prepare immediately for use;

Catalyst working solution: Take 50 μL of the catalyst diethyl cyanophosphate (DEPC) provided in the kit and add it to 500 μL of acetonitrile to make the final concentration 600 mmol/L, mix well, and prepare and use immediately;

Preparation of Substrate Chromogenic Solution A: Add 10mL of ethanol to the bottle of Substrate Chromogenic Solution A, store in a refrigerator at 4°C for one month. Before use, according to the required amount each time, take an appropriate amount of substrate A stock solution and dilute it 10 times with distilled water;

Preparation of substrate chromogenic solution B stock solution: Add 2 mL of distilled water to the bottle of substrate chromogenic B solution, and store in a refrigerator at 4°C for one month. Before use, according to the required amount each time, add 6.4 μL of substrate chromogenic solution B stock solution per milliliter of citric acid buffer solution;

Substrate mixed solution preparation: Determine the amount of this reagent required according to the number of tissue samples to be tested each time. Mix the substrate chromogenic solution A and the substrate chromogenic solution B in a volume ratio of 1:1, and prepare and use immediately.

6.2 Determination steps

Take microwell strips: insert enough well strips for the standard and sample into the microwell rack, do two parallel experiments for the standard and sample, and record the positions of the standard and sample;

Add standard solution or sample to be tested: Add 30 μL of standard product or processed sample solution into the microwell, pay attention to replace the tip of the pipette when adding sample or standard product to each well;

Add antibody: Add 70 μL of QCA antibody working solution to each microwell, mix well, incubate in the incubator for 1 hour (37°C), and cover with a film (to prevent evaporation);

Washing: pour out the liquid in the well, wash 3 times and pat dry;

Add HRP-labeled goat anti-rabbit antibody: Add 100 μL of HRP-labeled goat anti-rabbit antibody working solution to each microwell, mix well, incubate in an incubator for 1 hour (37°C), and cover with a film (to prevent evaporation);

Washing: pour out the liquid in the well, wash 5 times and pat dry;

Add substrate: Add 100 μL of substrate mixture to each microwell, mix well and incubate at 37°C in the dark for 15 minutes;

Termination: Add 50 μL of reaction termination solution to each microwell;

Determination: Measure the absorbance value at 450nm, use air as a blank, and read the absorbance value within 60 minutes after adding the stop solution.

6.3 Result Judgment

The average value of the obtained standard substance and sample absorbance value is divided by the absorbance value of the first standard (0 standard) and multiplied by 100, and the inhibition rate is used as the vertical axis, and the logarithm of the QCA concentration is used as the horizontal axis to make the standard curve. The curve approaches a straight line in the range of 0.1-51.2ng/mL, and the concentration (ng/g) corresponding to each sample can be read from the standard curve.

Embodiment 7, assessment and application of the kit of the present invention

7.1 Advancement of the kit of the present invention

Comparing the kit of the present invention with the HPLC method, the results (see Table 8) show that the ELISA has good sensitivity and specificity, and is well correlated with the HPLC method.

Table 8HPLC method and the comparison of the present invention

7.2 Application of the kit of the present invention

21 45-day-old piglets with a body weight of 15.0 ± 2.0 kg were "growth" binary hybrid castrated healthy piglets, and were randomly divided into two groups, one group was the blank control group (3 pigs), and the other group was the test group ( 12 heads). The test group was fed with 50 mg/kg of carbadox for 7 days, and 3 heads were slaughtered on the 0d, 4d, 10d, and 14d of the withdrawal of the drug. The blank control group was slaughtered on the 0d, 4d, and 10d of the drug withdrawal. Simultaneous determination of QCA content in pig liver and muscle by liquid chromatography. Determination result (table 9) shows: drug withdrawal 0 days, ELISA method measures the QCA content in liver, muscle and is respectively 82ng/g and 7.6ng/g, and HPLC method detection result is respectively 88ng/g and 6.9ng/g, stops. After 14 days of taking the drug, the QCA content in the liver was detected by ELISA method and HPLC method to be 4.8ng/g and 4.6ng/g respectively, and it was not detected in the muscle. The results of the two methods can reflect that the residual amount of QCA in the liver and muscle gradually decreases with the prolongation of time. ), indicating that the kit of the present invention has good actual detection ability and can screen out positive samples (>1 μg/kg).

Determination of QCA content in table 9 pig liver and muscle tissue

Note: "-" means not counted

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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