CN109734675B - Method and product suitable for detecting olaquindox content in veterinary drug preparation - Google Patents

Abstract

The invention discloses a method for detecting olaquindox content in a veterinary drug preparation and a product. The product used for detection is a olaquindox hapten compound shown in formula I. The invention designs and synthesizes small molecule target analyte hapten by depending on immunology, immunochemistry basic principle and residue analysis technical means, and couples with carrier protein to prepare effective artificial antigen, and immunizes animals to prepare specific antibody aiming at small molecule analyte. The method utilizes the specific immunological reaction of the antigen and the antibody to quantitatively detect trace micromolecule target analytes in a sample, and has the characteristics of specificity, sensitivity, accuracy, rapidness, convenience, low price and the like.

Description

Method and product suitable for detecting olaquindox content in veterinary drug preparation

Technical Field

The invention belongs to the field of detection, and relates to a method and a product for detecting the olaquindox content in a veterinary drug preparation.

Background

Olaquindox (OLA quindox, OLA) is a quinoxaline broad-spectrum antibacterial drug, has the function of promoting protein assimilation, can obviously improve feed reward, and is widely applied to the breeding industry as an early livestock and poultry feed additive. However, olaquindox has obvious accumulative toxicity and certain genetic toxicity in vivo, has obvious teratogenic effect on most animals, and has potential tripathogenicity on human. The pharmacopoeia of the people's republic of China (2005 edition) states that olaquindox is prohibited for use in poultry, aquatic animals, and pigs of 35kg or more. The pharmacopoeia office of the domestic animal in 2017 bans olaquindox as a pharmaceutical feed additive for food animals. However, due to their good antibacterial and growth-promoting effects, some lawless persons are often given the benefit of illegally adding olaquindox to some veterinary formulations to enhance the efficacy. Illegal addition can cause farmers to unknowingly use antibacterial drugs, not only induce drug resistance, but also cause drug residues, the residual drugs enter human bodies through animal foods and cause poisoning after long-term accumulation, and large doses can cause pathological changes of human livers and kidneys and even harm life and health.

At present, home and abroad related olaquindox detection methods mainly comprise high performance liquid chromatography, a gas-mass combination method, a liquid-mass combination method and the like. Although these methods have strong specificity and high sensitivity, they have the disadvantages of complicated treatment process, poor purification effect, much waste of organic solvent, long time required, and the like. At present, no related method report aiming at illegally adding olaquindox in veterinary drug preparations exists, the detection method is researched, an immunochemical analysis method has unique advantages in qualitative and quantitative aspects of antigen and antibody, the operation is simple, convenient and rapid, the cost is low, the sensitivity is higher, the analysis sample amount is large, the defects of physicochemical analysis are overcome, and the method has great significance for scientific use of antibacterial drugs, inhibition of bacterial drug resistance, public health safety and food safety.

Disclosure of Invention

The invention aims to provide a method and a product for detecting the olaquindox content in a veterinary drug preparation, and the method and the product have the advantages of high sensitivity, strong accuracy, high sensitivity and simple and convenient operation.

The invention aims to provide a olaquindox hapten compound, which has a structural formula shown as a formula I:

it is another object of the present invention to provide a method for preparing said olaquindox hapten compounds of formula I, comprising:

uniformly mixing the compound shown in the formula II with succinic anhydride to perform nucleophilic substitution reaction to obtain the compound;

in the nucleophilic substitution reaction step of the method, the temperature is 70-90 ℃; in particular 80 ℃;

the feeding molar ratio of the compound shown in the formula II to the succinic anhydride is 1: 0.5 to 1; specifically, 1: 0.8;

the nucleophilic substitution reaction is carried out in an organic solvent; the organic solvent is specifically selected from at least one of pyridine, triethylamine and acetone.

In addition, the application of the olaquindox hapten compound shown in the formula I in preparing olaquindox antigen, the olaquindox antigen containing the olaquindox hapten compound shown in the formula I, the application of the olaquindox hapten compound shown in the formula I or the olaquindox antigen in preparing antibody, and the antibody prepared from the olaquindox hapten compound shown in the formula I or the olaquindox antigen serving as immunogen also belong to the protection scope of the invention.

Wherein the olaquindox hapten compound shown in the formula I is coupled with carrier protein; the coupling molar ratio of the olaquindox hapten compound shown in the formula I to the carrier protein is 1-1.5: 1; specifically 1.09: 1; the carrier protein is specifically BSA or OVA.

The antibody may specifically be a monoclonal antibody.

In addition, the application of the olaquindox hapten compound shown in the formula I or the olaquindox antigen or the antibody in detection of olaquindox also belongs to the protection scope of the invention.

The invention also claims a method for detecting olaquindox in a veterinary agent, comprising: detecting a veterinary drug reagent by using the olaquindox antigen or the antibody;

or, the kit for detecting olaquindox is used for detecting veterinary drug reagents.

The kit also comprises a substrate color development solution, a stop solution, a sample diluent and a concentrated washing solution, and also can comprise an enzyme-labeled antibody working solution;

specifically, the enzyme-labeled antibody working solution is a goat anti-mouse antibody labeled by horseradish peroxidase; the specification is 1 bottle (12 mL);

the sample diluent was 0.01M PBS, pH7.4, 1 vial (10X, 15 mL);

the concentrated wash was 0.01M PBST solution pH7.4 in 1 vial (20X, 25 mL);

the substrate color developing solution is 1 bottle (7mL) of each of a substrate A solution and a substrate B solution; specifically, the substrate A is 2% carbamide peroxide aqueous solution; the substrate B is 1% tetramethyl benzidine aqueous solution; the stop solution is 2M H₂SO₄The specification of the solution was 1 bottle (7 mL).

The invention designs and synthesizes small molecule target analyte hapten by depending on immunology, immunochemistry basic principle and residue analysis technical means, and couples with carrier protein to prepare effective artificial antigen, and immunizes animals to prepare specific antibody aiming at small molecule analyte. The method utilizes the specific immunological reaction of the antigen and the antibody to quantitatively detect trace micromolecule target analytes in a sample, and has the characteristics of specificity, sensitivity, accuracy, rapidness, convenience, low price and the like.

Drawings

FIG. 1 is a mass spectrum of olaquindox hapten.

FIG. 2 is a MALDI-TOF-MAS diagram of BSA.

FIG. 3 is a MALDI-TOF-MAS diagram of olaquindox BSA complex.

FIG. 4 is a standard curve diagram of olaquindox kit.

Detailed Description

The present invention will be further illustrated with reference to the following specific examples, but the present invention is not limited to the following examples. The method is a conventional method unless otherwise specified. The starting materials are commercially available from the open literature unless otherwise specified.

Example 1 preparation of immunogen and coatingen

Preparation of quindox hapten

A50 ml round bottom flask was rinsed clean, blown dry with ethanol, mounted on a stirrer and a stir bar was added. 500mg (1.9mmol) of the compound of formula II is weighed out, dissolved in 5ml of pyridine, stirred at RT for 5min, 155mg (1.55mmol) of succinic anhydride are added, heated to 80 ℃ and reacted, the reaction is monitored on a dot-and-dot plate for 1 h/time.

Completion of the reaction was detected by TLC, treated, adjusted to neutral PH with 2M HCl, extracted with 10ml ethyl acetate water 3, dried over anhydrous sodium sulfate and concentrated, added 1.5g100 mesh silica gel, mixed and stirred, packed with 10g200 mesh silica gel, subjected to column chromatography, purified with ethyl acetate: and (3) carrying out gradient elution by using petroleum ether 10:1, collecting the required solution, spin-drying and collecting to obtain the compound shown in the formula I.

The reaction equation is as follows:

structural identification of di-and quindox hapten

Mass spectrum detection is carried out on the obtained product (figure 1), and the result shows that the chemical structural formula of the product is shown as a formula I, namely the olaquindox hapten.

Example 2 preparation and Structure identification of olaquindox Artificial antigen

Preparation of quindox artificial antigen

1. Synthesis of immunogens

(1) 18.4mg of hapten was dissolved in 1.5ml of DMF, stirred at 200rpm for 10min, added with 7.25mg of CDI to dissolve, and stirred at room temperature (500rpm) for activation for 2-3 h.

(2) Weighing 50mg of BSA, dissolving in 3.5ml of CB solution, stirring at 200rpm for 10min to fully dissolve the BSA, cooling in an ice bath at 0-4 ℃, dropwise adding the reaction solution in the step 1 (1ml/min) under stirring at 1000rpm, and stirring at 500rpm for reaction for 24h.

(3) The reaction product was put into a dialysis bag (10cm) washed clean with distilled water, dialyzed at 1L0.01M PBS (1X, pH7.2) with stirring (100rpm) at 4 ℃ for 3d, the solution was changed 3 times a day (once in the morning, at night), the total of the solutions was changed 9 times, the dialyzed product was centrifuged at 5000rpm for 6min, and 1.5 ml/tube was filled, and the antigen was numbered and stored at-20 ℃ for further use.

2. Synthesis of coatingen

(1) 27.4mg of hapten was dissolved in 1.5ml of DMF, stirred at 200rpm for 10min, added with 10.8mg of CDI to dissolve, and stirred at room temperature (500rpm) for activation for 2-3 h.

(2) Weighing OVA 50mg, dissolving in CB solution 3.5ml, stirring at 200rpm for 10min to fully dissolve, cooling at 0-4 ℃ in an ice bath, dropwise adding the reaction solution in the step 1 (1ml/min) under stirring at 1000rpm, and stirring at 500rpm for reaction for 24h.

(3) The reaction product was put into a dialysis bag (10cm) washed clean with distilled water, dialyzed at 1L0.01M PBS (1X, pH7.2) with stirring (100rpm) at 4 ℃ for 3d, the solution was changed 3 times a day (once in the morning, at night), the total of the solutions was changed 9 times, the dialyzed product was centrifuged at 5000rpm for 6min, and 1.5 ml/tube was filled, and the antigen was numbered and stored at-20 ℃ for further use.

Identification of di-and quindox artificial antigens

The identification result of the immunogen MALDI-TOF-MS shows that the coupling ratio is as follows: r ═ (66740.772-66294.114)/411.37 ═ 1.09 (fig. 2 and 3). Namely, in the immunogen, the molar ratio of coupling of the olaquindox hapten (shown in a formula I) and Bovine Serum Albumin (BSA) is 1.09: 1.

EXAMPLE 3 preparation of monoclonal antibody by Immunity of olaquindox Artificial antigen to animal

First, animal immunization

Dissolving 100 mu g of immunogen (olaquindox-BSA) prepared in example 2 by using physiological saline, uniformly mixing the immunogen and Freund's complete adjuvant in equal volume, injecting and immunizing Balb/c female mice with 6-8 weeks old by subcutaneous injection at the neck and back, uniformly mixing the immunogen and Freund's incomplete adjuvant in equal volume at 7, 14 and 28 days after primary immunization, performing additional immunization once respectively, and performing additional immunization once by using 100 mu g of immune complex 3 days before fusion without adding Freund's adjuvant.

Second, cell fusion and cloning

Mixing splenocytes of immunized mice with myeloma cells of mice (SP2/0) in logarithmic growth phase, slowly adding preheated fusion agent (PEG4000) within 45s for fusion, suspending with HAT medium, adding appropriate amount of feeder cells, culturing in 96-well culture plate at 37 deg.C and 5% CO₂Culturing in an incubator, half-changing the culture medium with HT after 5 days, and completely changing the culture medium after 9 days.

3. After cell fusion, when the cells grow to 1/4 of the culture hole area, hybridoma cells are screened by a step screening method. The primary selection adopts an indirect ELISA method, an enzyme label plate is coated with coating antigen (the optimal coating concentration and the positive serum dilution are titrated conventionally by a square matrix method in advance), culture supernatant of a detected hole is added, incubation and washing are carried out, and then goat anti-mouse IgG-HRP, IgM-HRP and OPD are added for color reaction. The screened positive hole is screened by an indirect competitive ELISA method, the cell supernatant is mixed with 100 mu g/mL olaquindox with equal volume, the mixture is taken in water bath at 37 ℃ for 30min, and then the mixture is added into a coated enzyme label plate. Meanwhile, PBS was used instead of olaquindox as a control, and the rest steps were as above. OD if after olaquindox blocking₄₅₀And (3) judging the wells to be positive when the nm value is reduced to below 50% of the control wells, and subcloning the wells which are positive after 2-3 detections by using a limiting dilution method immediately.

Preparation and purification of monoclonal antibody

Carrying out expanded culture on the hybridoma after 2-3 times of subcloning and strain establishment, collecting supernatant, measuring titer by using indirect ELISA, and freezing and storing; and injecting 0.5 mL/mouse of liquid paraffin into the abdominal cavity of a Balb/c mouse aged 8-10 weeks, injecting 1-2 multiplied by 105/mouse of hybridoma into the abdominal cavity after 7-10 days, and extracting ascites of the mouse after 7-10 days. Collecting cell supernatant or ascites, measuring the titer by an indirect ELISA method (the titer is measured by the maximum dilution multiple of the cell supernatant or the ascites with P/N > 2.1), and the result shows that the titer of the cell supernatant is 1: 10000, ascites titer 1: 50000. then, the mixture is purified by an octanoic acid-saturated ammonium sulfate method and is placed into a-20 ℃ environment for storage after purification.

Example 4 preparation of antiserum by immunizing animal with olaquindox Artificial antigen

First, animal immunization

The olaquindox artificial antigen "olaquindox-BSA" obtained in step example 2 was used as an immunogen to immunize New Zealand white rabbits. The immunization dose is 100-200 mug each time, the immunization mode is double shoulders and back thighs are injected in multiple points subcutaneously, and each area is approximately used with 1/4 immunogen. Diluting the immunogen with normal saline during first immunization, mixing the diluted immunogen with Freund incomplete adjuvant in a volume ratio of 1:1 to prepare an emulsifier, taking the same dose of immunogen and the same volume of Freund incomplete adjuvant every 2 weeks, mixing and emulsifying the mixture, enhancing immunity once, adding the immunization once in the way, taking the same dose of immunogen and the same volume of Freund incomplete adjuvant every 3 weeks for last immunization, taking blood from an ear artery, and detecting the titer of the antibody. And 7-10 days after the last immunization, bleeding by using carotid artery, wherein the blood obtained from each rabbit is about 100-120ml, placing the obtained blood in a refrigerator at 4 ℃ for 3-4 hours, centrifuging, and separating out serum.

Second, measurement of antiserum titer

And (3) measuring the antibody titer of the serum obtained in the step one by adopting an indirect ELISA method, which comprises the following specific steps:

1) coating: to a 96-well plate, 100. mu.L of a 2. mu.g/mL "olaquindox-OVA" solution (diluted with coating buffer) was added, while a control not coated with the antigen was set, coated overnight at 4 ℃ and washed 3 times with PBS buffer.

Coating buffer solution: 0.05M sodium carbonate-sodium bicarbonate buffer (water as solvent, with Na as solute and concentration)₂CO₃1.59g/L and NaHCO₃2.93g/L)。

2) And (3) sealing: add 150. mu.L/well blocking solution, incubate at 37 ℃ for 2h, discard blocking solution, wash 3 times, pat dry. Storing in a refrigerator at 4 deg.C.

Sealing liquid: phosphate buffer containing 0.5% (volume percent) calf serum, 3% (3g/100ml) casein, pH 7.4.

3) Adding a sample to be tested: sucking 100 mul of serum to be detected with different dilutions, adding the serum to the corresponding enzyme label plate, incubating for 30min at 37 ℃, washing the plate for 4 times, and patting to dry.

Simultaneously setting a control of the rabbit serum without immunization; the control of the sample to be tested (negative control well) was replaced with PBS.

4) Adding an enzyme-labeled secondary antibody: taking a goat anti-rabbit IgG antibody marked by horseradish peroxidase, and mixing the raw materials in a volume ratio of 1: after 5000-fold dilution, 100. mu.l/well, incubation at 37 ℃ for 20 to 30min, washing 4 times, patting dry.

5) Color development: 20 XTMB was diluted to 1 XTMB, added at 100. mu.l/well, and developed at 37 ℃ for 15-30 min.

6) And (4) terminating: adding stop solution (2 MH)₂SO₄)50 μ l/well.

7) Reading: the OD value of each well is measured by a single wavelength of 450nm, and the ratio (P/N) of the OD value of each well to the OD value of a negative control well (a control of a sample to be measured is replaced by PBS) is more than 2.1 and is used as a critical point for judging the serum titer.

The ELISA result determination method comprises the following steps: expressed as the maximum dilution of serum with P/N > 2.1.

The results show that the antibody titer in serum is 1: 16000.

example 5 detection of olaquindox with olaquindox ELISA kit

Assembly of olaquindox enzyme-linked immunosorbent assay kit

1. The olaquindox enzyme-linked immunoassay kit comprises the following components:

(1) olaquindox standard working solution: 6 bottles, 1.5 mL/bottle, the concentration is 0 mug/kg, 0.1 mug/kg, 0.3 mug/kg, 0.9 mug/kg, 2.7 mug/kg, 8.1 mug/kg;

(2) olaquindox enzyme label plate: 1 piece (8 holes × 12 strips) of ELISA plate coated with "olaquindox-OVA" prepared in example 2;

(3) olaquindox antibody working solution: 1 bottle (7mL), diluting the antibody with an antibody diluent at a ratio of 1:8000, wherein the antibody diluent is 0.2MPBS containing 6% (volume fraction) of goat serum, and the olaquindox antibody is an antibody purified from the antiserum prepared in example 3; (4) enzyme marker working solution: 1 bottle (12mL), wherein the enzyme marker is a goat anti-mouse antibody marked by horseradish peroxidase;

(5) sample diluent: 1 vial (10X, 15mL) of 0.01M PBS, pH 7.4;

(6) washing liquid: 1 vial (20X, 25mL) of 0.01M PBST solution, pH 7.4;

(7) each of the substrate A solution and the substrate B solution was 1 bottle (7 mL). Wherein the substrate A is 2% carbamide peroxide aqueous solution. The substrate B is 1% tetramethyl benzidine aqueous solution;

(8) stopping liquid: 1 bottle (7mL) of 2M H₂SO₄A solution;

(9) a cover plate film;

(10) a valve bag.

2. Equipment and materials not provided for

(1) Device

A microplate reader (detection wavelength 450nm, reference wavelength 630nm), a balance (precision: 0.01g), a vortex oscillator, a centrifuge (4000g), a micropipette and a timer.

(2) Reagent

Deionized water.

3. Principle of detection of kit

And (3) the olaquindox in the sample competes with the antigen specificity competitive antibody fixed on the enzyme label plate, an enzyme label is added, the substrate is catalyzed to develop color, and the content of the olaquindox in the sample is judged according to the color depth of the color development. Dark color, low content, light color and high content.

Use method of di-olaquindox enzyme-linked immunosorbent assay kit

1. Pretreatment of preparation, traditional Chinese medicine and chemical medicine samples (dilution factor: 1000)

a) Weighing 0.5 +/-0.05 g/mL of sample in a 50mL centrifuge tube;

b) adding 5mL of sample diluent (see 7.1), and fully whirling for 3 min;

c) centrifuging the solid sample for 5min or standing for 10min, wherein the solid sample needs more than 4000 g; the liquid sample does not need to be centrifuged or kept still;

d) adding 10 mu L of sample liquid into 990 mu L of sample diluent, and whirling for 30 s;

e) 50. mu.L of the suspension was taken for detection.

2. Detection step

(1) Inserting the lath into the ELISA plate frame, recording the positions of each standard product and each sample, suggesting that the two holes are parallel, sealing the unused lath by a self-sealing bag, and immediately storing in an environment of 2-8 ℃;

(2) respectively adding 50 mu L of olaquindox standard substance working solution (or sample solution to be detected) with each concentration into the corresponding standard substance (or sample hole to be detected);

(3) adding 50 mu L of olaquindox antibody working solution into each hole;

(4) covering a cover plate film, slightly oscillating the ELISA plate for 10s, fully and uniformly mixing, and reacting for 20min at room temperature (25 +/-2 ℃) in a dark place;

(5) uncovering the cover plate film;

(6) pouring out liquid in the plate holes, adding 260 mu L of washing working solution into each hole, and fully washing for 4 times, wherein each time of soaking is 15-30 s;

(7) pouring out liquid in the plate hole, pouring the enzyme label plate on absorbent paper, and patting dry;

(8) immediately adding 100 mu L of enzyme marker working solution into each hole;

(9) covering a cover plate film, slightly oscillating the ELISA plate for 10s, fully and uniformly mixing, and reacting for 20min at room temperature (25 +/-2 ℃) in a dark place;

(10) repeating steps (5) - (7);

(11) immediately adding 100 μ L of a mixture of the substrate A solution and the substrate B solution to each well (the substrate A solution and the substrate B solution were mixed at a volume of 1: 1);

(12) covering a cover plate film, slightly oscillating the ELISA plate for 10s, fully and uniformly mixing, and reacting for 10-15min at room temperature (25 +/-2 ℃) in a dark place;

(13) uncovering the cover plate membrane, adding 50 mu L of stop solution into each hole, slightly oscillating the ELISA plate for 10s, and fully and uniformly mixing;

(14) and reading the absorbance value of the ELISA plate by using an ELISA reader at the dual wavelength of 450nm and 630nm within 5min after termination.

3. Result calculation or determination

(1) The average absorbance value of each standard (or sample to be measured) is divided by the absorbance value of the zero standard (standard with the concentration of 0 mug/kg), and multiplied by 100 to obtain the percentage of absorbance corresponding to each standard, namely the percent absorbance value.

(2) And (3) drawing a standard curve by taking the percent absorbance value of each standard as a vertical coordinate and the corresponding olaquindox concentration as a horizontal coordinate.

(3) Substituting the percent absorbance value of the sample to be detected into a standard curve equation to obtain the corresponding concentration of the sample to be detected, and multiplying the dilution multiple of the corresponding sample to obtain the actual content of the olaquindox in the original sample to be detected.

Enzyme linked immunosorbent assay kit for detecting olaquindox

1. Specificity detection

The specificity of the olaquindox enzyme linked immunosorbent assay kit is determined by carrying out a cross reaction test with a corresponding substance. The smaller the cross-reaction, the better the specificity.

Respectively carrying out serial dilution on the olaquindox and other analogues (3-methyl quinoxaline-2-carboxylic acid, quinoxaline-2-carboxylic acid and carbalox), respectively operating according to the step two 2, replacing 'olaquindox standard work solution' with serial dilution of the olaquindox and other analogues, making a standard curve, and finding out respective 50% Inhibition Concentrations (IC) on the curve₅₀) The specific method comprises the following steps: the corresponding olaquindox concentration (mug/kg) with a value on the ordinate equal to 50%, i.e. IC, is obtained₅₀The value is obtained. The cross-reactivity of the kit to olaquindox and each analogue was calculated using the following formula.

Cross-reactivity (%). ratio (concentration of olaquindox causing 50% inhibition/concentration of olaquindox analogue causing 50% inhibition) × 100%

The results are shown in table 1, and it can be seen from table 1 that the cross-reactivity of the olaquindox elisa kit to various analogues is less than 0.1%. The result shows that the olaquindox enzyme linked immunosorbent assay kit has extremely high specificity to olaquindox, can effectively eliminate the interference of other analogues, and can be specially used for the detection of olaquindox.

TABLE 1 specificity of olaquindox enzyme-linked immunosorbent assay kit

2. Determination of minimum detection limit

And (3) taking 20 blank samples for detection, calculating a measured value according to a standard curve, calculating an average value of the measured value, and adding 3 times of standard deviation to obtain the lowest detection limit.

TABLE 2 statistical table of measurement results of blank samples (. mu.g/kg)

The result shows that in order to prevent the occurrence of false positive, the detection limit of olaquindox in the kit corresponding to injection, powder, pill and paste medicament selling preparations can be defined as 1 mug/kg.

3. Determination of accuracy and precision of olaquindox enzyme-linked immunosorbent assay kit

The accuracy and precision of the olaquindox enzyme-linked immunoassay kit are respectively determined. The adding concentrations of two olaquindox standard substances are respectively 1 mug/kg and 2 mug/kg, and each adding amount is 5 in parallel. The results of the measurements with the 3 batches of kits were shown in Table 3, where the addition recovery and the intra-batch inter-batch coefficient of variation were calculated.

TABLE 3 kit accuracy and precision (μ g/kg)

As can be seen from Table 3, the recovery rates of the respective concentrations were 86.00% -115.00%, the intra-batch coefficient of variation was less than 10%, and the inter-batch coefficient of variation was less than 15%.

Claims (13)

1. A olaquindox hapten compound shown in a formula I,

2. the use of a olaquindox hapten compound of formula I as defined in claim 1 for the preparation of olaquindox antigens.

3. A olaquindox antigen comprising a olaquindox hapten compound of formula I as defined in claim 1.

4. The use or a olaquindox antigen as claimed in claim 2 or 3, wherein: the olaquindox hapten compound shown in the formula I is coupled with carrier protein;

the coupling molar ratio of the olaquindox hapten compound shown in the formula I to the carrier protein is 1-1.5: 1.

5. the use or a olaquindox antigen as claimed in claim 4, wherein: the coupling molar ratio of the olaquindox hapten compound shown in the formula I to the carrier protein is 1.09: 1;

the carrier protein is BSA or OVA.

6. Use of a olaquindox hapten compound of formula I according to claim 1 or of an olaquindox antigen according to claim 3 for the preparation of antibodies.

7. An antibody produced from a olaquindox hapten compound of formula I as defined in claim 1 or from an olaquindox antigen as defined in claim 3 as an immunogen.

8. Use of a olaquindox hapten compound of formula I as defined in claim 1 or an olaquindox antigen as defined in claim 3 or an antibody as defined in claim 7 for the detection of olaquindox.

9. A kit for detecting olaquindox comprising an antigen of olaquindox as claimed in claim 3 and an antibody as claimed in claim 7.

10. A method for detecting olaquindox in a veterinary agent comprising: detecting a veterinary agent with the olaquindox antigen of claim 3 or the antibody of claim 7;

alternatively, the kit for detecting olaquindox of claim 9 is used for detecting veterinary agents.

11. The kit of claim 9 or the method of claim 10, characterized in that: the kit also comprises a substrate color development solution, a stop solution, a sample diluent and a concentrated washing solution.

12. The kit or method of claim 11, wherein: the substrate color developing solution is a substrate solution A and a substrate solution B;

the stop solution is 2M H₂SO₄A solution;

the sample diluent is 0.01M PBS with pH7.4;

the concentrated washings were 0.01M PBST solution pH 7.4.

13. The kit or method of claim 12, wherein: the substrate A is 2% carbamide peroxide aqueous solution; the substrate B is 1% tetramethyl benzidine aqueous solution.

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