Antibody-fluorescent nanospheres conjugate for synchronous detection of antibiotics enrofloxacin and ciprofloxacin and preparation method thereof

Abstract

Antibody-fluorescent nanospheres conjugate for synchronous detection of antibiotics enrofloxacin and ciprofloxacin and preparation method thereof, the steps are as follows: carboxylate-modify polystyrene microspheres on the surface by hydrolysis, and then embed rare earth europium elements inside the microspheres by embedding method to form a combination of polystyrene microspheres and europium elements to obtain fluorescent microspheres; construct a pretreatment system for the fluorescent microspheres, and clean and activate the fluorescent microspheres; couple the fluorescent microspheres with monoclonal antibodies against enrofloxacin or ciprofloxacin; the prepared product is stored in the dark at 2-8°C. The conjugate prepared by the method of the present invention can not only greatly improve the detection sensitivity of the target antibiotics, but also completely shield the cross-interference of the two antibiotics during synchronous detection, solving the problem in the prior art that the detection of enrofloxacin and ciprofloxacin requires separate detection of two indicators, which is both laborious and costly.

Description

Technical Field

[0001] The present invention relates to the technical field of rapid detection and analysis in industries such as food, agriculture, and environment, and specifically relates to an antibody-fluorescent nanospheres conjugate for simultaneous detection of antibiotics enrofloxacin and ciprofloxacin and a preparation method thereof. Background Art

[0002] Enrofloxacin is a third-generation fluoroquinolone broad-spectrum antibacterial drug. Due to the gradual change of alkaline molecules, the antibacterial performance of enrofloxacin has been greatly improved, and it is widely used in the prevention and treatment of bacterial and mycoplasma infections in animals such as livestock, poultry, and aquatic products. After being used, enrofloxacin can be metabolized into ciprofloxacin in the animal body. According to the national veterinary drug residue limit standard in food, the residue limit of enrofloxacin in the animal body refers to the sum of enrofloxacin itself and its metabolite ciprofloxacin, and its maximum residue limit value is 0.1 mg / kg. The unscientific abuse of enrofloxacin will cause drug residues, which will cause great harm to human health through the food chain. Therefore, detecting the residue amount of enrofloxacin in food animals during their market period has always been an essential daily task for the agricultural and rural departments and market supervision departments to ensure food quality and safety.

[0003] Currently, the detection methods for enrofloxacin and ciprofloxacin residues in food animals are based on enzyme-linked immunosorbent assay and colloidal gold methods. The detection steps of these methods are "one index, one extraction, and one reading". The judgment of one residue limit requires the detection of two indicators separately, which is both laborious and costly. Based on the immune reaction principle of specific binding between antigen and antibody, the present invention conjugates fluorescent nanospheres with antibodies of two antibiotics respectively to form conjugates, and simultaneously detects the fluorescence signal intensities of the two conjugates to achieve the simultaneous rapid quantitative detection of two antibiotics, enrofloxacin and ciprofloxacin. Summary of the Invention

[0004] Technical Problem to be Solved: Aiming at the problem that the detection of enrofloxacin and ciprofloxacin in the prior art requires the separate detection of two indicators, which is both laborious and costly, the present invention provides an antibody-fluorescent nanospheres conjugate for simultaneous detection of antibiotics enrofloxacin and ciprofloxacin and a preparation method thereof. The fluorescent nanospheres are conjugated with antibodies of two antibiotics respectively to form conjugates, and the fluorescence signal intensities of the two conjugates are detected simultaneously, so as to achieve the simultaneous rapid quantitative detection of two antibiotics, enrofloxacin and ciprofloxacin.

[0005] Technical Solution: The preparation method of an antibody-fluorescent nanospheres conjugate for simultaneous detection of antibiotics enrofloxacin and ciprofloxacin comprises the following steps:

[0006] Step 1. Prepare fluorescent microspheres. Polystyrene microspheres with a particle size of 100 - 500 nm are carboxylated on the surface by hydrolysis, and then rare earth europium elements are embedded inside the microspheres by an embedding method to form a combination of polystyrene microspheres and europium elements to obtain fluorescent microspheres. Through detection, the properties of the fluorescent microspheres are as follows: excitation wavelength 360 to 365 nm, emission wavelength 610 to 620 nm; good dispersion in the aqueous phase;

[0007] Step 2. Construct a pretreatment system for fluorescent microspheres. Clean and activate the fluorescent microspheres. When cleaning, transfer 100 μL of fluorescent microspheres to a 2 mL centrifuge tube, add 400 - 900 μL of cleaning solution, and ultrasonically mix evenly for 5 - 10 min under the conditions of a power of 300 W and a frequency of 40 KHz. After that, centrifuge at a speed of 5000 - 20000 r / min for 10 - 20 min to remove the supernatant, then add 400 - 900 μL of cleaning solution to the centrifuge tube, and ultrasonically mix evenly for 5 - 8 min under the conditions of a power of 300 W and a frequency of 40 KHz. Subsequently, for activation, add 20 - 100 μL of activation solution to the centrifuge tube obtained in the cleaning step, vortex and mix evenly, and place it on a mixer for rotary incubation at room temperature for 20 - 40 min to obtain a centrifuge tube containing activated fluorescent microspheres;

[0008] Step 3. Couple the fluorescent microspheres with antibodies. Centrifuge the centrifuge tube containing activated fluorescent microspheres at a speed of 5000 - 20000 r / min for 10 - 20 min to remove the supernatant. Repeat the above cleaning steps twice, then add 400 - 900 μL of microsphere coupling buffer, ultrasonically mix evenly for 5 - 10 min, and then add 0.05 - 0.1 mg of anti - enrofloxacin or anti - ciprofloxacin monoclonal antibody, vortex and mix evenly, and place it on a mixer for rotary incubation at room temperature for 1 - 2 h. After that, centrifuge at a speed of 5000 - 20000 r / min for 10 - 20 min to remove the supernatant, then add 1 mL of microsphere blocking solution, and place it on a mixer for rotary incubation at room temperature for 1 - 2 h. After that, centrifuge at a speed of 5000 - 20000 r / min for 10 - 20 min to remove the supernatant. Repeat the above steps to wash the precipitate once more with the microsphere blocking solution. Then, add 1 - 2 mL of microsphere suspension, mix evenly, and disperse ultrasonically for 5 min under the conditions of a power of 300 W and a frequency of 40 KHz. Thus, the coupling of the fluorescent microspheres with the anti - enrofloxacin monoclonal antibody or the anti - ciprofloxacin monoclonal antibody is completed, and the enrofloxacin antibody - fluorescent microsphere conjugate and the ciprofloxacin antibody - fluorescent microsphere conjugate are respectively prepared;

[0009] Step 4. Store the prepared product in the dark at 2 - 8 °C.

[0010] Preferably, the specific processes of hydrolysis and embedding in Step 1 are as follows: Add 25 mg / mL of polystyrene microspheres, 7 mg / mL of sodium dodecyl sulfonate, and 15 mg / mL of potassium persulfate into a 100 mL glass container in sequence, dissolve them with deionized water, mix evenly by ultrasonic treatment, with the total volume being 20 mL, then add 1.5 mL of methanol and 1.0 mL of undecylenic acid, and mix well; Pass nitrogen, seal, and place it in a constant temperature water bath at 70 °C for oscillating reaction for 4 h. After the reaction ends, centrifuge the microspheres, wash them three times with alcohol and three times with water respectively, and preserve them by vacuum drying to obtain carboxylated polystyrene microspheres; Take 0.1 g of the carboxylated polystyrene microspheres prepared in the above process, add 10 mL of a deionized water / acetone mixed solution (v / v, 1:1), oscillate at 28 °C for 10 h, then add 0.5 mL of 0.25 M europium trichloride-ethanol solution, and oscillate at 28 °C for reaction for 20 h. Finally, remove the organic solvent by rotary evaporation, centrifuge the product at 5000 - 10000 r / min for 3 min, wash it three times with deionized water and ethanol, and finally redissolve it with water containing 0.05 wt% sodium azide and preserve it at 4 °C to obtain fluorescent microspheres.

[0011] Preferably, the preparation method of the cleaning solution in Step 2 is as follows: Dissolve 2-(N-morpholino)ethanesulfonic acid in ultrapure water, with the ratio of 2-(N-morpholino)ethanesulfonic acid to ultrapure water being 1.066 g:100 mL, adjust the pH to 6.0 with 5 M NaOH solution to obtain a cleaning solution with a final concentration of 50 mM and a pH of 6.0.

[0012] Preferably, the activation solution in Step 2 includes Solution A and Solution B. Solution A is an ethanol solution of N-hydroxysuccinimide, with the ratio of N-hydroxysuccinimide to ethanol being 10 mg:1 mL, and Solution B is an ethanol solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, with the ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to ethanol being 10 mg:1 mL.

[0013] Preferably, add 10 - 50 μL of activation solution A and 10 - 50 μL of activation solution B into the centrifuge tubes obtained in the cleaning step respectively, and mix well by vortexing.

[0014] Preferably, the preparation method of the microsphere coupling buffer in step three is as follows: Dissolve 2-(N-morpholino)ethanesulfonic acid in ultrapure water, with the ratio of 2-(N-morpholino)ethanesulfonic acid to ultrapure water being 0.213 g:100 mL, adjust the pH to 6.0 with 5 M NaOH solution to obtain a microsphere coupling buffer with a final concentration of 10 mM and a pH of 6.0; the preparation method of the microsphere blocking solution is as follows: Dissolve BSA in the microsphere coupling buffer, with the ratio of BSA to the microsphere coupling buffer being 50 mg:10 mL; the preparation method of the microsphere suspension is as follows: Respectively take trehalose, BSA and polyvinylpyrrolidone PVP-K30 and dissolve them in the microsphere coupling buffer, then add Tween-20 and ProClin300 preservatives and mix evenly. The ratio of trehalose, BSA, polyvinylpyrrolidone PVP-K30, microsphere coupling buffer, Tween-20 and ProClin300 preservatives is 0.5-1 g:0.1-0.5 g:0.1-0.5 g:10 mL:10-50 μL:10 μL.

[0015] An antibody-fluorescent nanomicrosphere conjugate for the simultaneous detection of antibiotics enrofloxacin and ciprofloxacin prepared based on the above method.

[0016] The application of the above antibody-fluorescent nanomicrosphere conjugate in the simultaneous detection of antibiotics enrofloxacin and ciprofloxacin.

[0017] The specific detection method is as follows: (1) Prepare an immunofluorescence chromatography test card for the simultaneous detection of antibiotics enrofloxacin and ciprofloxacin. The test card includes a bottom plate, a sample pad, a nitrocellulose membrane and a blotting paper pad arranged on the bottom plate. On the upper surface of the nitrocellulose membrane, three lines are drawn from bottom to top, namely ciprofloxacin-coated antigen (test line T2), enrofloxacin-coated antigen (test line T1) and rabbit anti-chicken IgY (control line C). The two ends of the nitrocellulose membrane are respectively lapped with a sample pad and a blotting paper pad. Among them, one end of the nitrocellulose membrane is pasted with a blotting paper pad by pressing tightly 2 mm from the edge of the nitrocellulose membrane, and the other end of the nitrocellulose membrane is pasted with a sample pad by pressing tightly 2 mm from the edge of the nitrocellulose membrane, and then cut into a test strip with a width of 4 mm and placed in the test strip slot of the lower cover of the test card, and then cover the upper cover of the test card;

[0018] (2) Prepare a working solution of a certain multiple by mixing three kinds of microspheres, namely ciprofloxacin antibody-fluorescent nanomicrospheres, enrofloxacin antibody-fluorescent nanomicrospheres and rabbit anti-chicken IgY polyclonal antibody-fluorescent nanomicrospheres, with a 1wt% Tween-20-PBS solution.

[0019] (3) Preparation of Detection Standard Curve Preparation of a series of standard products: Take enrofloxacin standard and ciprofloxacin standard, and prepare a series of concentrations of enrofloxacin / ciprofloxacin standard with the sample diluent prepared in (2) above: 0 / 0, 0.05 / 0.25, 0.45 / 2.25, 4.05 / 20.25 ng / mL. Take 100 μL and add it to the test card, let it stand at room temperature (about 25 °C) for 10 min, and then read with a time-resolved fluorescence immunoassay analyzer to obtain the ratio of the test line T to the quality control C line (T / C).

[0020] Beneficial effects: The present invention provides an antibody-fluorescent nanospheres conjugate for the synchronous detection of antibiotics enrofloxacin and ciprofloxacin and a preparation method thereof. The conjugate prepared by this method can not only greatly improve the detection sensitivity of target antibiotics, but also completely shield the cross-interference of the two antibiotics during synchronous detection. Detailed implementation mode

[0021] The present invention will be further described below in conjunction with specific embodiments.

[0022] Example 1

[0023] Select polystyrene microspheres (purchased from Suzhou Zhiyi Microsphere Technology Co., Ltd.), with a particle size requirement of 100 to 500 nm. Through the hydrolysis of PS / MMA, carboxylation modification is carried out on the surface of the microspheres, and then the rare earth europium element is embedded inside the microspheres by an embedding method to form a combination of polystyrene microspheres and europium element, which is called fluorescent microspheres in the present invention.

[0024] The specific steps are as follows: Add 25 mg / mL of polystyrene microspheres, 7 mg / mL of sodium dodecyl sulfate, and 15 mg / mL of potassium persulfate to a 100 mL glass container in sequence, dissolve with deionized water, mix evenly by ultrasonic wave, with a total volume of 20 mL, then add 1.5 mL of methanol and 1.0 mL of undecylenic acid, and mix well; Pass nitrogen, seal, and place it in a 70 °C constant temperature water bath oscillator for reaction for 4 h. After the reaction, centrifuge the microspheres, wash with alcohol and water three times each, and store them in vacuum drying to obtain carboxylated polystyrene microspheres; Take 0.1 g of the carboxylated polystyrene microspheres prepared in the above process, add 10 mL of deionized water / acetone mixture (v / v, 1:1), oscillate at 28 °C for 10 h, then add 0.5 mL of 0.25 M europium trichloride-ethanol solution, oscillate and react at 28 °C for 20 h, finally remove the organic solvent by rotary evaporation, centrifuge the product at 10000 r / min for 3 min, wash with deionized water and ethanol three times, and finally redissolve with water containing 0.05 wt% sodium azide and store at 4 °C to obtain fluorescent microspheres.

[0025] Through detection, the performance of the fluorescent microspheres is as follows: the excitation wavelength is 360 to 365 nm, and the emission wavelength is 610 to 620 nm; the dispersion in the aqueous phase is good.

[0026] After the fluorescent microspheres are prepared, pretreatment is required, including washing and activation. In the washing step, a washing solution is prepared, and its formula is: accurately weigh 1.066 g of 2-(N-morpholino)ethanesulfonic acid, dissolve it in 100 mL of ultrapure water, and adjust the pH to 6.0 with 5 M NaOH solution to obtain a washing solution with a final concentration of 50 mM and a pH of 6.0. The washing procedure is as follows: Transfer 100 μL of the fluorescent microspheres into a 2 mL centrifuge tube, add 900 μL of the above washing solution, and mix well by ultrasonic treatment for 10 min. The ultrasonic conditions are: power 300 W, frequency 40 KHz. After completion, centrifuge at 8000 r / min for 20 min to remove the supernatant. Then add 900 μL of the washing solution to the centrifuge tube, mix well by ultrasonic treatment for 5 min. The ultrasonic conditions are: power 300 W, frequency 40 KHz. Subsequently, transfer to the activation step. Prepare an activation solution, and its formula is: Solution A, accurately weigh 10 mg of N-hydroxysuccinimide and dissolve it in 1 mL of ethanol. Solution B, accurately weigh 10 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and dissolve it in 1 mL of ethanol. Add 50 μL of activation solution A and 50 μL of activation solution B to the centrifuge tube obtained in the washing step respectively, vortex and mix well, and place it on a mixer for rotary incubation at room temperature for 40 min. Centrifuge the centrifuge tube containing the activated fluorescent microspheres for 20 min at a speed of 8000 r / min. Remove the supernatant. After repeating the above washing step twice, add 900 μL of the microsphere coupling buffer, and its formula is: accurately weigh 0.213 g of 2-(N-morpholino)ethanesulfonic acid, dissolve it in 100 mL of ultrapure water, and adjust the pH to 6.0 with 5 M NaOH solution to obtain a microsphere coupling buffer with a final concentration of 10 mM and a pH of 6.0. Mix well by ultrasonic treatment for 10 min, then add 0.1 mg of anti-enrofloxacin or 0.1 mg of anti-ciprofloxacin monoclonal antibody, vortex and mix well, and place it on a mixer for rotary incubation at room temperature for 2 h. After completion, centrifuge at 8000 r / min for 20 min to remove the supernatant. Subsequently, add 1 mL of the microsphere blocking solution, and its formula is: accurately weigh 50 mg of BSA and dissolve it in 10 mL of the microsphere coupling buffer. Place it on a mixer for rotary incubation at room temperature for 2 h. After completion, centrifuge at 8000 r / min for 20 min to remove the supernatant. The precipitate is washed once again with the microsphere blocking solution by repeating the above steps. Then, add 1 mL of the microsphere suspension, and its formula is: weigh 0.5 g of trehalose, 0.1 g of BSA, and 0.1 g of polyvinylpyrrolidone (PVP-K30) respectively, dissolve them in 10 mL of the microsphere coupling buffer, then add 10 μL of Tween-20 and 10 μL of ProClin300 preservative, and mix evenly. Disperse by ultrasonic wave (power 300 W, frequency 40 KHz) for 5 min.

[0027] So far, the conjugation of fluorescent microspheres with anti-enrofloxacin monoclonal antibody or anti-ciprofloxacin monoclonal antibody has been completed, and enrofloxacin antibody-fluorescent microsphere conjugate and ciprofloxacin antibody-fluorescent microsphere conjugate have been prepared respectively, which are stored in the dark at 2-8 °C for future use, and the validity period is 12 months.

[0028] Comparative Example 1

[0029] Same as Example 1, except that the particle size of the polystyrene microspheres is 1500 nm.

[0030] Through detection, the performance of the fluorescent microspheres prepared in this comparative example is: excitation wavelength 365 nm, emission wavelength 615 nm; and it has good dispersion in the aqueous phase.

[0031] After the fluorescent microspheres are prepared, cleaning and activation treatments are carried out. 100 μL of the fluorescent microspheres are transferred to a 2 mL centrifuge tube, and 900 μL of the above cleaning solution is added. The mixture is ultrasonically mixed for 10 min under the conditions of 300 W and 40 KHz. After that, the supernatant is removed by centrifugation at a speed of 8000 r / min for 20 min. Then, 900 μL of the cleaning solution is added to the centrifuge tube, and the mixture is ultrasonically mixed for 5 min. After the washing process is completed, activation treatment is carried out, and it is found that the microspheres aggregate and precipitate. It is necessary to vortex, shake and ultrasonically treat again to resuspend the microspheres. The binding ability of the resuspended microspheres with the antibody decreases, and the conjugation effect is poor.

[0032] Comparative Example 2

[0033] Same as Example 1, except that the ultrasonic mixing time is 20 min, and the ultrasonic conditions are: power 300 W, frequency 80 KHz.

[0034] The purpose of ultrasonic treatment is to make the fluorescent microspheres in a uniformly dispersed state, which is the basis for the conjugation effect of monoclonal antibody and fluorescent microspheres. However, in this comparative example, the ultrasonic time is long and the ultrasonic frequency is high, and heat is generated during the ultrasonic process, which destroys the carboxylation modification on the surface of the microspheres, and further separates the europium element embedded in the microspheres from the polystyrene microspheres, resulting in the failure of the experiment.

[0035] Comparative Example 3

[0036] Same as Example 1, except that the amount of antibody used is different. In this comparative example, 1.0 mg of anti-enrofloxacin or 1.0 mg of anti-ciprofloxacin monoclonal antibody is added respectively.

[0037] After the preparation process of this comparative example is completed, it is found that the antibody is in excess. In addition to the binding of the fluorescent microspheres with the antibody, the binding of antibody with antibody also occurs, and precipitation phenomenon appears, resulting in the failure of the experiment.

[0038] The enrofloxacin antibody-fluorescent microsphere conjugate and ciprofloxacin antibody-fluorescent microsphere conjugate prepared in Example 1 were used for the simultaneous detection of the antibiotics enrofloxacin and ciprofloxacin. The specific detection steps are as follows:

[0039] (1)Prepare an immunofluorescence chromatography test card for the simultaneous detection of the antibiotics enrofloxacin and ciprofloxacin, including a bottom plate, a sample pad, a nitrocellulose membrane, and a blotting paper pad arranged on the bottom plate. On the upper surface of the nitrocellulose membrane, three lines are drawn from bottom to top, namely ciprofloxacin-coated antigen (test line T2), enrofloxacin-coated antigen (test line T1), and rabbit anti-chicken IgY (control line C), and the scribing concentrations are 0.8 mg / mL, 0.5 mg / mL, and 1.0 mg / mL respectively. The sample pad and the blotting paper pad are respectively lapped at both ends of the nitrocellulose membrane. Among them, the blotting paper pad is pasted at a position 2 mm away from the edge of the nitrocellulose membrane at one end of the nitrocellulose membrane, and the sample pad is pasted at a position 2 mm away from the edge of the nitrocellulose membrane at the other end of the nitrocellulose membrane. Then it is cut into a test strip with a width of 4 mm and placed in the test strip slot of the lower cover of the test card, and the upper cover of the test card is covered to obtain a complete test card.

[0040] (2)Preparation of three kinds of antibody-fluorescent microsphere working solutions: Dilute the ciprofloxacin antibody-fluorescent nanospheres, enrofloxacin antibody-fluorescent nanospheres, and rabbit anti-chicken IgY polyclonal antibody-fluorescent nanospheres (the preparation method of rabbit anti-chicken IgY polyclonal antibody-fluorescent nanospheres is the same as that of ciprofloxacin antibody-fluorescent nanospheres and enrofloxacin antibody-fluorescent nanospheres, see Example 1, the difference is that 0.1 mg of rabbit anti-chicken IgY polyclonal antibody is added) with PBS solution containing 1 wt% Tween-20 to prepare solutions diluted 1000 times, namely ciprofloxacin antibody-fluorescent nanosphere working solution, enrofloxacin antibody-fluorescent nanosphere working solution, and rabbit anti-chicken IgY polyclonal antibody-fluorescent nanosphere working solution.

[0041] (3)Preparation of mixed microsphere working solutions: Dilute the ciprofloxacin antibody-fluorescent nanospheres and rabbit anti-chicken IgY polyclonal antibody-fluorescent nanospheres with PBS solution containing 1 wt% Tween-20 to prepare a mixed microsphere working solution 1 diluted 1000 times; dilute the enrofloxacin antibody-fluorescent nanospheres and rabbit anti-chicken IgY polyclonal antibody-fluorescent nanospheres with PBS solution containing 1 wt% Tween-20 to prepare a mixed microsphere working solution 2 diluted 1000 times; dilute the ciprofloxacin antibody-fluorescent nanospheres, enrofloxacin antibody-fluorescent nanospheres, and rabbit anti-chicken IgY polyclonal antibody-fluorescent nanospheres with PBS solution containing 1 wt% Tween-20 to prepare a mixed microsphere working solution 3 diluted 1000 times.

[0042] (4)Cross-reactivity of the three antibodies: Take 100 μL of the three microsphere working solutions prepared in (2) above and add them to the test card. Let it stand at room temperature (about 25 °C) for 10 min, and then read the values of the test line T and the quality control C line corresponding to the three antibodies using a time-resolved fluorescence immunoassay analyzer. The measurement results are shown in Table 1. It can be seen from Table 1 that there is no cross-reactivity among the fluorescent microspheres of the three antibodies, indicating that the specificity of the antibodies is good.

[0043] (5)Inhibition rate of ciprofloxacin by ciprofloxacin antibody-fluorescent nanospheres alone: Prepare a series of ciprofloxacin standard concentrations: 0, 0.25, 2.25, 20.25 ng / mL using the mixed microsphere working solution 1 prepared in (3) above. Take 100 μL and add it to the test card. Let it stand at room temperature (about 25 °C) for 10 min, and then read the ratio of the test line T to the quality control C line (T / C) using a time-resolved fluorescence immunoassay analyzer. The measurement results are shown in Table 2.

[0044] (6)Inhibition rate of enrofloxacin by enrofloxacin antibody-fluorescent nanospheres alone: Prepare a series of enrofloxacin standard concentrations: 0, 0.05, 0.45, 4.05 ng / mL using the mixed microsphere working solution 2 prepared in (3) above. Take 100 μL and add it to the test card. Let it stand at room temperature (about 25 °C) for 10 min, and then read the ratio of the test line T to the quality control C line (T / C) using a time-resolved fluorescence immunoassay analyzer. The measurement results are shown in Table 3.

[0045] (7)Inhibition rate when ciprofloxacin antibody-fluorescent nanospheres and enrofloxacin antibody-fluorescent nanospheres are added simultaneously: Prepare a series of enrofloxacin / ciprofloxacin standard concentrations: 0 / 0, 0.05 / 0.25, 0.45 / 2.25, 4.05 / 20.25 ng / mL using the mixed microsphere working solution 3 prepared in (3) above. Take 100 μL and add it to the test card. Let it stand at room temperature (about 25 °C) for 10 min, and then read the ratio of the test line T to the quality control C line (T / C) using a time-resolved fluorescence immunoassay analyzer. The measurement results are shown in Table 4.

[0046] It can be seen from Tables 2 to 4 that adding ciprofloxacin antibody-fluorescent nanospheres, enrofloxacin antibody-fluorescent nanospheres alone and adding the two fluorescent microspheres simultaneously has little effect on the inhibition rate and sensitivity of ciprofloxacin and enrofloxacin, indicating that these two antibiotics can be detected simultaneously.

[0047] Table 1 Cross-reactivity of the fluorescent microspheres of the three antibodies added alone

[0048]

[0049] Table 2 Inhibition rate of ciprofloxacin by ciprofloxacin antibody-fluorescent nanospheres

[0050]

[0051] Table 3 Inhibition rate of enrofloxacin by enrofloxacin antibody-fluorescent nanospheres

[0052]

[0053] Table 4 Inhibition rate when ciprofloxacin antibody-fluorescent nanospheres and enrofloxacin antibody-fluorescent nanospheres are added simultaneously

[0054]

Claims

1. Preparation method of antibody-fluorescent nanospheres conjugate for synchronous detection of antibiotics enrofloxacin and ciprofloxacin, Characterized in that, The steps are as follows: Step 1. Prepare fluorescent microspheres. Modify the surface of polystyrene microspheres with a particle size of 100-500 nm by hydrolysis to carry out carboxylation modification, and then use the embedding method to embed rare earth europium elements inside the microspheres to form a combination of polystyrene microspheres and europium elements to obtain fluorescent microspheres. The specific processes of hydrolysis and embedding are as follows: sequentially add 25 mg / mL of polystyrene microspheres, 7 mg / mL of sodium dodecyl sulfate, and 15 mg / mL of potassium persulfate to a glass container, dissolve with deionized water, mix evenly by ultrasonic wave, with a total volume of 20 mL, then add 1.5 mL of methanol and 1.0 mL of undecylenic acid, and mix well; pass nitrogen, seal, and place in a 70°C constant temperature water bath oscillator for reaction for 4 h. After the reaction, centrifuge the microspheres, wash with alcohol and water three times each, and store in vacuum drying to obtain carboxylated polystyrene microspheres; take 0.1 g of the carboxylated polystyrene microspheres prepared in the above process, add 10 mL of a mixed solution of deionized water and acetone with a volume ratio of 1:1, oscillate at 28°C for 10 h, then add 0.5 mL of 0.25 M europium trichloride-ethanol solution, oscillate and react at 28°C for 20 h, finally remove the organic solvent by rotary evaporation, centrifuge the product at 5000-10000 r / min for 3 min, wash with deionized water and ethanol three times, and finally redissolve with water containing 0.05 wt% sodium azide and store at 4°C to obtain fluorescent microspheres; Step 2. Construct a pretreatment system for fluorescent microspheres, clean and activate the fluorescent microspheres. During cleaning, transfer 100 μL of fluorescent microspheres to a centrifuge tube, add 400-900 μL of cleaning solution, mix evenly by ultrasonic wave at a power of 300 W and a frequency of 40 KHz for 5-10 min. After completion, centrifuge at a speed of 5000-20000 r / min for 10-20 min to remove the supernatant, then add 400-900 μL of cleaning solution to the centrifuge tube, mix evenly by ultrasonic wave at a power of 300 W and a frequency of 40 KHz for 5-8 min, and then carry out activation. Add 20-100 μL of activation solution to the centrifuge tube obtained in the cleaning step, vortex and mix well, place on a mixer and incubate at room temperature with rotation for 20-40 min to obtain a centrifuge tube containing activated fluorescent microspheres; Step 3. Coupling the fluorescent microspheres with the antibody. Centrifuge the centrifuge tube containing the activated fluorescent microspheres at a speed of 5000 - 20000 r / min for 10 - 20 min, remove the supernatant, repeat the above washing steps twice, then add 400 - 900 μL of microsphere coupling buffer, mix well by ultrasonic for 5 - 10 min, then add 0.05 - 0.1 mg of anti-enrofloxacin or anti-ciprofloxacin monoclonal antibody, vortex mix, place it on a mixer and incubate at room temperature with rotation for 1 - 2 h. After completion, centrifuge at a speed of 5000 - 20000 r / min for 10 - 20 min, remove the supernatant, then add 1 mL of microsphere blocking solution, place it on a mixer and incubate at room temperature with rotation for 1 - 2 h. After completion, centrifuge at a speed of 5000 - 20000 r / min for 10 - 20 min, remove the supernatant, repeat the above steps to wash the precipitate with the microsphere blocking solution one more time. Then, add 1 - 2 mL of microsphere suspension, mix evenly, and disperse by ultrasonic for 5 min under the conditions of a power of 300 W and a frequency of 40 KHz. Thus, the coupling of the fluorescent microspheres with the anti-enrofloxacin monoclonal antibody or anti-ciprofloxacin monoclonal antibody is completed, and the enrofloxacin antibody-fluorescent microsphere conjugate and ciprofloxacin antibody-fluorescent microsphere conjugate are respectively prepared; Step 4. The prepared products are stored in the dark at 2 - 8 °C. Among them, when the enrofloxacin antibody-fluorescent microsphere conjugate and ciprofloxacin antibody-fluorescent microsphere conjugate are detected synchronously, there is no cross-reaction.

2. The method for preparing an antibody-fluorescent nanomicrosphere conjugate for simultaneous detection of antibiotics enrofloxacin and ciprofloxacin according to claim 1, characterized in that, the preparation method of the cleaning solution in the second step is as follows: Dissolve 2-(N-morpholino)ethanesulfonic acid in ultrapure water, and the ratio of 2-(N-morpholino)ethanesulfonic acid to ultrapure water is 1.066 g:100 mL, adjust the pH to 6.0 with 5 M NaOH solution to obtain a cleaning solution with a final concentration of 50 mM and a pH of 6.

0.

3. The method for preparing an antibody-fluorescent nanomicrosphere conjugate for simultaneous detection of antibiotics enrofloxacin and ciprofloxacin according to claim 1, characterized in that, the activation solution in the second step includes solution A and solution B. Solution A is an ethanol solution of N-hydroxysuccinimide, and the ratio of N-hydroxysuccinimide to ethanol is 10 mg:1 mL. Solution B is an ethanol solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and the ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to ethanol is 10 mg:1 mL.

4. The method for preparing an antibody-fluorescent nanomicrosphere conjugate for simultaneous detection of antibiotics enrofloxacin and ciprofloxacin according to claim 1, characterized in that, Add 10 - 50 μL of activation solution A and 10 - 50 μL of activation solution B to the centrifuge tubes obtained in the washing step respectively, and vortex mix.

5. The preparation method of the antibody-fluorescent nanospheres conjugate for simultaneous detection of antibiotics enrofloxacin and ciprofloxacin according to claim 1, characterized in that, the preparation method of the microsphere coupling buffer in step 3 is as follows: dissolve 2-(N-morpholino)ethanesulfonic acid in ultrapure water, and the ratio of 2-(N-morpholino)ethanesulfonic acid to ultrapure water is 0.213 g:100 mL, adjust the pH to 6.0 with 5 M NaOH solution to obtain a microsphere coupling buffer with a final concentration of 10 mM and a pH of 6.0; the preparation method of the microsphere blocking solution is as follows: dissolve BSA in the microsphere coupling buffer, and the ratio of BSA to the microsphere coupling buffer is 50 mg:10 mL; the preparation method of the microsphere suspension is as follows: respectively dissolve trehalose, BSA and polyvinylpyrrolidone PVP-K30 in the microsphere coupling buffer, then add Tween-20 and ProClin300 preservatives and mix well, wherein the ratio of trehalose, BSA, polyvinylpyrrolidone PVP-K30, microsphere coupling buffer, Tween-20 and ProClin300 preservative is 0.5~1 g:0.1~0.5 g:0.1~0.5 g:10 mL:10~50 μL:10 μL.

6. The antibody-fluorescent nanospheres conjugate for simultaneous detection of antibiotics enrofloxacin and ciprofloxacin prepared by the method according to claim 1.

7. The application of the antibody-fluorescent nanospheres conjugate according to claim 6 in the simultaneous detection of antibiotics enrofloxacin and ciprofloxacin.

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