A method for recovering trace enrofloxacin antibiotic in aquaculture water by using magnetic microspheres coupled with antibody specificity
By using a method of conjugating antibodies to magnetic microspheres, the problem of separating and enriching trace amounts of enrofloxacin in water has been solved, achieving efficient and specific adsorption and recovery, and reducing the risk of enrofloxacin residue in water.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- FRESHWATER FISHERIES RES CENT OF CHINESE ACAD OF FISHERY SCI
- Filing Date
- 2024-04-24
- Publication Date
- 2026-06-09
AI Technical Summary
Existing technologies involve cumbersome procedures and cannot accurately and specifically separate and enrich trace amounts of enrofloxacin antibiotics in water, leading to an increased risk of antibiotic-resistant pollution by microorganisms in water bodies.
The method of using magnetic microspheres to conjugate antibodies involves activating magnetic microspheres, biotinylated enrofloxacin monoclonal antibody, preparing magnetic microsphere-conjugated antibodies, and then enriching and eluting enrofloxacin under specific conditions to achieve specific adsorption and recovery.
It achieved a highly efficient and specific adsorption rate of 97.68% and an elution recovery rate of over 83% for trace enrofloxacin in water, simplifying the operation process and reducing the risk of enrofloxacin residue in water.
Smart Images
Figure SMS_1 
Figure SMS_2
Abstract
Description
Technical Field
[0001] This invention relates to the field of aquatic environment remediation technology, specifically to a method for the specific recovery of trace amounts of enrofloxacin antibiotics from aquaculture water using magnetic microspheres coupled with antibodies. Background Technology
[0002] The presence of trace amounts of antibiotics in water bodies can lead to selective burden on aquatic microorganisms, causing antibiotic-resistant pollution. Long-term exposure to trace amounts of antibiotics can accelerate the horizontal transfer of antibiotic resistance genes, which may also be transferred to bacteria belonging to the human gut microbiota, posing a risk to global ecosystems and human health.
[0003] Enrofloxacin (ENR) is an important veterinary antibiotic used in aquaculture. Its risks are higher than other antibiotics due to its lipophilic nature, slow degradation in aquatic environments, long half-life, and wide tissue distribution. It easily accumulates in animal-derived foods, posing a potential health risk to consumers. Long-term presence of trace amounts of enrofloxacin in water bodies can harm human health through the food chain.
[0004] However, traditional antibiotic separation methods such as solid phase extraction, immunoaffinity chromatography, molecular imprinting, supercritical fluid extraction, and accelerated solvent extraction are cumbersome and cannot achieve precise and specific separation and enrichment of trace amounts of antibiotics in water. Summary of the Invention
[0005] Technical problem solved: In view of the problems in existing technologies, such as cumbersome operation procedures and inability to accurately and specifically separate and enrich trace amounts of enrofloxacin antibiotics in water, this invention proposes a method for specifically recovering trace amounts of enrofloxacin antibiotics in aquaculture water using magnetic microspheres coupled with antibodies. The method is easy to operate and can specifically adsorb trace amounts (a few μg / L) of enrofloxacin in water.
[0006] Technical solution: A method for the specific recovery of trace amounts of enrofloxacin antibiotics from aquaculture water using magnetic microspheres coupled with antibodies, comprising the following steps:
[0007] Step 1: Activate the magnetic microspheres. Take streptavidin magnetic microspheres into a centrifuge tube, vortex to mix, then mix on a roller mixer, remove the supernatant by magnetic separation, add PBST Buffer, mix on a roller mixer at room temperature, remove the supernatant after magnetic separation, repeat the washing process with PBST Buffer several times, remove the supernatant and set aside for later use.
[0008] Step 2: Biotinylated enrofloxacin monoclonal antibody. Biotin was added to the enrofloxacin monoclonal antibody and reacted at room temperature in the dark on a mixer. After the reaction was completed, blocking buffer was added and the mixture was blocked at room temperature. Then, it was purified by PD10 chromatography column to obtain biotinylated enrofloxacin monoclonal antibody.
[0009] Step 3: Prepare magnetic microsphere-conjugated antibody. Add biotinylated enrofloxacin monoclonal antibody to the activated magnetic microspheres, add PBS buffer, and then vortex mix. Then place it in a roller mixer for incubation at a temperature of 25-37°C. At this point, the conjugation is complete. The conjugate is represented as MNP@Ab, where MNP represents magnetic microspheres, Ab represents antibody, and @ represents conjugation.
[0010] Step 4: Enrofloxacin enrichment and elution. The conjugate MNP@Ab was directly added to the water sample containing trace amounts of enrofloxacin for extraction. The extract was incubated in a mixer set at 25-37°C. After incubation, the supernatant was removed by magnetic separation to obtain the enrofloxacin-bound MNP@Ab.
[0011] Step 5: Elution and recovery of enrofloxacin. Add the enrofloxacin-bound MNP@Ab to the eluent and incubate at 65°C for no more than 2 minutes. After incubation, vortex to mix and then use magnetic separation technology to obtain the supernatant. At this time, enrofloxacin is eluted into the supernatant. The eluent is a mixture of 95wt% formamide and 20mM EDTA at a volume ratio of 1:1.
[0012] Preferably, the streptavidin magnetic microspheres in step one have a particle size of 3-5 μm.
[0013] Preferably, in step one, streptavidin magnetic microspheres are placed in a centrifuge tube, vortexed for 10 seconds, then mixed on a roller mixer for 15 minutes, magnetically separated on a magnetic separator for 1 minute, the supernatant is removed, PBST Buffer is added, and the mixture is mixed on a roller mixer at room temperature for 5 minutes. After magnetic separation, the supernatant is removed, and the washing is repeated twice with PBST Buffer. The supernatant is then removed for later use, thus completing the activation process.
[0014] Preferably, the PBST Buffer is PBS with 0.05 wt% Tween 20 added and the pH adjusted to 7.4.
[0015] Preferably, in step two, the molar ratio of enrofloxacin monoclonal antibody to biotin is 1:30.
[0016] Preferably, in step two, biotin is pretreated before addition. After equilibration at room temperature, it is dissolved in DMSO. The enrofloxacin monoclonal antibody is diluted with labeling buffer, and then the dissolved biotin is added. The mixture is wrapped in aluminum foil to protect it from light and placed on a mixer to react at room temperature for 3 hours. After the reaction is complete, blocking buffer is added, and the mixture is placed on a mixer to block at room temperature for 30 minutes. Then, it is purified using a PD10 chromatography column to obtain the biotinylated enrofloxacin monoclonal antibody.
[0017] Preferably, the concentration of streptavidin magnetic microspheres in step one is 10 mg / mL, and the ratio of biotinylated enrofloxacin monoclonal antibody to streptavidin magnetic microspheres in step three is 10-20 μg: 100 μL.
[0018] Preferably, in step three, the biotinylated enrofloxacin monoclonal antibody is added to the activated magnetic microspheres, and PBS buffer is added to make the total volume 100 μL. Then, the mixture is vortexed for 10 seconds and then placed in a roller mixer for 60 minutes at a temperature of 37°C. At this point, the coupling is complete.
[0019] Preferably, in step four, the conjugate MNP@Ab is directly added to the water sample containing trace amounts of enrofloxacin for extraction. The extract is then placed in a mixer with the temperature set at 37°C and incubated for 30 minutes. After incubation, the supernatant is removed by magnetic separation, which specifically involves placing the sample on a magnetic rack and letting it stand for 3 minutes.
[0020] Preferably, in step five, the enrofloxacin-bound MNP@Ab is added to the elution buffer and incubated at 65°C for no more than 2 minutes. After incubation, the mixture is vortexed for 30 seconds, and then the supernatant is obtained using magnetic separation technology, at which point enrofloxacin is eluted into the supernatant.
[0021] Beneficial Effects: This invention utilizes magnetic microspheres coupled with antibodies to construct a magnetic immunoadsorption method, and specifically developed and optimized elution conditions to achieve the specific recovery of trace enrofloxacin in aquaculture water. This invention is simple to operate and can specifically adsorb trace amounts (a few μg / L) of enrofloxacin in water, achieving an adsorption rate as high as 97.68% and an elution recovery rate exceeding 83%. Detailed Implementation
[0022] The present invention will be further described below with reference to specific embodiments.
[0023] Unless otherwise specified, all raw materials and equipment used in the embodiments of this specification are from commercially available products.
[0024] A method for the specific recovery of trace amounts of enrofloxacin antibiotics from aquaculture water using magnetic microspheres coupled with antibodies comprises the following steps:
[0025] Step 1: Activate the magnetic microspheres. Take streptavidin magnetic microspheres into a centrifuge tube, vortex to mix, then mix on a roller mixer, remove the supernatant by magnetic separation, add PBST Buffer, mix on a roller mixer at room temperature, remove the supernatant after magnetic separation, repeat the washing process with PBST Buffer several times, remove the supernatant and set aside for later use.
[0026] Step 2: Biotinylated enrofloxacin monoclonal antibody. Biotin was added to the enrofloxacin monoclonal antibody and reacted at room temperature in the dark on a mixer. After the reaction was completed, blocking buffer was added and the mixture was blocked at room temperature. Then, it was purified by PD10 chromatography column to obtain biotinylated enrofloxacin monoclonal antibody.
[0027] Step 3: Prepare magnetic microsphere-conjugated antibody. Add biotinylated enrofloxacin monoclonal antibody to the activated magnetic microspheres, add PBS buffer, and then vortex mix. Then place it in a roller mixer for incubation at a temperature of 25-37°C. At this point, the conjugation is complete. The conjugate is represented as MNP@Ab, where MNP represents magnetic microspheres, Ab represents antibody, and @ represents conjugation.
[0028] Step 4: Enrofloxacin enrichment and elution. The conjugate MNP@Ab was directly added to the water sample containing trace amounts of enrofloxacin for extraction. The extract was incubated in a mixer set at 25-37°C. After incubation, the supernatant was removed by magnetic separation to obtain the enrofloxacin-bound MNP@Ab.
[0029] Step 5: Elution and recovery of enrofloxacin. Add the enrofloxacin-bound MNP@Ab to the eluent and incubate at 65°C for no more than 2 minutes. After incubation, vortex to mix and then use magnetic separation technology to obtain the supernatant. At this time, enrofloxacin is eluted into the supernatant. The eluent is a mixture of 95wt% formamide and 20mM EDTA at a volume ratio of 1:1.
[0030] As a further preferred embodiment of the present invention, the particle size of the streptavidin magnetic microspheres in step one is 3-5 μm.
[0031] As a further preferred embodiment of the present invention, in step one, streptavidin magnetic microspheres are placed in a centrifuge tube, vortexed for 10 seconds, then mixed on a roller mixer for 15 minutes, magnetically separated on a magnetic separator for 1 minute, the supernatant is removed, PBST Buffer is added, and the mixture is mixed on a roller mixer at room temperature for 5 minutes. After magnetic separation, the supernatant is removed, and the washing is repeated twice with PBST Buffer. The supernatant is then removed for later use, thus completing the activation.
[0032] As a further preferred embodiment of the present invention, the PBST Buffer is PBS with 0.05 wt% Tween 20 added and the pH adjusted to 7.4.
[0033] As a further preferred embodiment of the present invention, the molar ratio of enrofloxacin monoclonal antibody to biotin in step two is 1:30.
[0034] As a further preferred embodiment of the present invention, in step two, biotin is pretreated before addition, dissolved in DMSO after equilibration at room temperature, enrofloxacin monoclonal antibody is diluted with labeling buffer, then the dissolved biotin is added, wrapped in aluminum foil to protect from light, and placed on a mixer to react at room temperature for 3 hours. After the reaction is completed, blocking solution is added, and the mixture is placed on a mixer to block at room temperature for 30 minutes. Then it is purified by PD10 chromatography column to obtain biotinylated enrofloxacin monoclonal antibody.
[0035] As a further preferred embodiment of the present invention, the concentration of streptavidin magnetic microspheres in step one is 10 mg / mL, and the ratio of biotinylated enrofloxacin monoclonal antibody to streptavidin magnetic microspheres in step one is 10-20 μg: 100 μL in step three.
[0036] As a further preferred embodiment of the present invention, in step three, biotinylated enrofloxacin monoclonal antibody is added to activated magnetic microspheres, PBS buffer is added to make a total volume of 100 μL, and then vortexed for 10 s. Subsequently, it is placed in a roller mixer and incubated for 60 min at a temperature of 37 °C. Thus, the coupling is completed.
[0037] As a further preferred embodiment of the present invention, in step four, the conjugate MNP@Ab is directly added to the water sample containing trace amounts of enrofloxacin for extraction. The extract is placed in a mixer with the temperature set at 37°C and incubated for 30 minutes. After incubation, the supernatant is removed by magnetic separation. Specifically, the supernatant is placed on a magnetic rack and left to stand for 3 minutes.
[0038] As a further preferred embodiment of the present invention, in step five, the MNP@Ab bound to enrofloxacin is added to the elution buffer and incubated at 65°C for no more than 2 minutes. After incubation, the mixture is vortexed for 30 seconds, and then the supernatant is obtained by magnetic separation technology. At this time, enrofloxacin is eluted into the supernatant.
[0039] Example 1
[0040] This embodiment provides a method for the specific recovery of trace amounts of enrofloxacin antibiotics from aquaculture water using magnetic microspheres coupled with antibodies. The steps are as follows:
[0041] (1) Selection and activation of magnetic microspheres. Streptavidin magnetic microspheres (purchased from Suzhou Weidu Biotechnology Co., Ltd.) with a particle size of 3-5 μm were selected. 100 μL (concentration 10 mg / mL) of streptavidin magnetic microspheres was added to a 1.5 mL centrifuge tube, vortexed for 10 s, mixed on a roller mixer for 15 min, and then magnetically separated on a magnetic separator for 1 min. The supernatant was removed. 400 μL of PBST Buffer (PBS with 0.05 wt% Tween 20, pH adjusted to 7.4) was added; the mixture was mixed on a roller mixer for 5 min at room temperature (20-25℃), magnetically separated, and the supernatant was removed. The microspheres were washed twice with PBST Buffer, and the supernatant was then removed for later use. Activation was complete.
[0042] (2) Biotinylation of enrofloxacin monoclonal antibody. After equilibration at room temperature (20-25℃), an appropriate amount of biotin was weighed and dissolved in DMSO to a concentration of 5 mg / mL. 1 mg of enrofloxacin monoclonal antibody (purchased from an antibody company, not manufactured in-house) was diluted with labeling buffer, and then 22.5 μL of dissolved biotin was added (antibody:biotin = 1:30, molar ratio). The mixture was wrapped in aluminum foil to protect it from light and placed on a mixer for incubation at room temperature for 3 h. After the reaction, 100 μL of blocking buffer was added, and the mixture was placed on a mixer for incubation at room temperature for 30 min. The product was purified using a PD10 chromatography column. The A280 value of the collected product was measured using a UV spectrophotometer, and the concentration C of the biotinylated antibody was calculated using the formula C = A280 × 0.7.
[0043] (3) Evaluation of the conjugation effect of the magnetic microspheres to the antibody. Add 10 μg of the biotinylated enrofloxacin monoclonal antibody purified and collected in step (2) to the centrifuge tube from step (1). Controlling for a single variable, add PBS buffer to make a total volume of 100 μL, vortex for 10 s, and then incubate in a roller mixer for 60 min at 37°C. The conjugation is now complete. The conjugate is denoted as MNP@Ab, where MNP represents magnetic microspheres, Ab represents antibody, and @ represents conjugation.
[0044] After coupling in step (3), place the centrifuge tubes on a magnetic rack for 3 minutes and collect the supernatant. There are three ways to evaluate the coupling effect: ① Use the BCA method to determine the concentration of monoclonal antibody in the supernatant. Perform three replicates of each supernatant, take the average value, and obtain the remaining antibody amount in the supernatant. Then calculate the specific amount of antibody coupled using the difference method. ② Use UV-Vis absorption spectroscopy to determine the UV-Vis absorption spectrum of the magnetic microspheres before and after coupling. The change in the maximum absorption peak can determine the antibody coupling effect. ③ Use a Zeta potential analyzer to determine the potential and particle size changes of the magnetic microspheres before and after coupling.
[0045] In this embodiment, the first method was selected to evaluate the conjugation effect. The conjugation rate was calculated by measuring the amount of antibody added to the supernatant before and after conjugation and the amount of remaining antibody. The final conjugation rate was 90.4%.
[0046] (4) The MNP@Ab conjugate, evaluated for its coupling effect, was directly added to a water sample containing 4 μg / L enrofloxacin for extraction. To ensure thorough mixing and reaction, an appropriate amount of PBS buffer was added to maintain a suitable reaction environment. The mixture was then placed in a roller mixer set to 37°C and incubated for 30 minutes to promote the binding of MNP@Ab with the target analyte, enrofloxacin. After incubation, the centrifuge tubes were placed on a magnetic rack and allowed to stand for 3 minutes. During this time, MNP@Ab aggregated on one side of the centrifuge tube due to its magnetism, while simultaneously binding tightly to the target analyte. Thus, using magnetic separation technology, the enrofloxacin-bound MNP@Ab could be easily separated from the supernatant.
[0047] Enrofloxacin in the supernatant was filtered through a 0.22 μm filter membrane, and its concentration was then determined using a triple quadrupole liquid chromatography-mass spectrometry (LC-MS / MS). The binding rate (%) was calculated based on the change in enrofloxacin concentration before and after the experiment using the following formula.
[0048]
[0049] In this embodiment, the original amount of enrofloxacin was 2 μg, and the remaining amount of enrofloxacin in the supernatant was 0.06 μg.
[0050] The binding rate reflects the efficiency of MNP@Ab adsorption of the target analyte enrofloxacin. The adsorption rate tested using this method reached 97.68%.
[0051] (5) Elution and recovery of enrofloxacin from the conjugate. First, a suitable eluent was selected, formulated as a mixture of 95 wt% formamide and 20 mM EDTA at a volume ratio of 1:1. 500 μL of the eluent was incubated at a specific temperature (65 °C) and time (2 min) to dissociate the target analyte from the MNP@Ab. After incubation, the mixture was vortexed for 30 seconds to ensure thorough dispersion of the complex in the eluent. Magnetic separation was then used to easily separate the eluent from the MNP@Ab. At this point, the target analyte, enrofloxacin, had been eluted into the supernatant with an elution rate of 83%. The specific value of the target analyte, enrofloxacin, in this step was 1.6 μg.
[0052] The formula for calculating the elution rate is as follows:
[0053]
[0054] Comparative Example 1
[0055] Similar to Example 1, except that the magnetic microspheres selected in step (1) of this comparative example have a particle size of 600 nm. 100 μL (concentration 10 mg / mL) of the magnetic microspheres was transferred to a 1.5 mL centrifuge tube, vortexed for 10 s, mixed on a roller mixer for 15 min, and then magnetically separated on a magnetic separator for 1 min. The supernatant was removed. 900 μL of PBST Buffer (PBS with 0.05 wt% Tween 20 added, pH adjusted to 7.4) was added, and the mixture was mixed on a roller mixer for 5 min at room temperature (20-25℃). After magnetic separation, the supernatant was removed, and the mixture was washed twice with PBST Buffer, then the supernatant was removed and the mixture was ready for use. Activation was complete.
[0056] After activation, it is conjugated with the biotinylated enrofloxacin monoclonal antibody described in step (2). The conjugation results are shown in the table below. Due to the small particle size of the microspheres, the conjugation effect is poor.
[0057] Microsphere particle size 600nm 3μm Coupling rate / % 72 90.4
[0058] Comparative Example 2
[0059] Similar to Example 1, except that in step (3) of this comparative example, 5 μg, 15 μg and 20 μg of the biotinylated enrofloxacin monoclonal antibody purified and collected in step (2) were respectively taken into the centrifuge tube of step (1).
[0060] The coupling rate was calculated, and the data are shown in the table below:
[0061] Antibody dosage / μg 5 10 15 20 Coupling rate / % 97.21 90.4 81.64 79.77
[0062] As can be seen from the table, antibody conjugation is basically saturated at 10 μg. Considering the cost, 10 μg of antibody was selected for conjugation with 100 μL of magnetic microspheres.
[0063] Comparative Example 3
[0064] Similar to Example 1, the difference is that in step (4) of this comparative example, the water sample containing 4 μg / L enrofloxacin was replaced with a water sample containing 4 μg / L ciprofloxacin. The test showed that the adsorption rate was 0%, indicating that the conjugate prepared by the present invention has strong specificity for the adsorption of enrofloxacin. Even though ciprofloxacin and enrofloxacin have very similar structures, ciprofloxacin cannot be adsorbed.
[0065] Comparative Example 4
[0066] Similar to Example 1, except that in step (4) of this comparative example, the mixture is placed in a roller mixer set to 25°C and incubated for 30 minutes to promote the full binding of MNP@Ab with the target analyte enrofloxacin.
[0067] The coupling rate measured in this comparative example was 57.78%, which is far less effective than the coupling effect at 37℃.
[0068] Comparative Example 5
[0069] Similar to Example 1, except that in step (5) of this comparative example, the eluent is replaced with a weak acid solution of pH=4, methanol or 0.1% SDS. Compared with the 95wt% formamide and 20mM EDTA mixture with a volume ratio of 1:1 in Example 1, the elution effect of the first three is worse, at 17.84%, 35% and 46% respectively.
[0070] Comparative Example 6
[0071] Similar to Example 1, the difference is that in step (5) of this comparative example, the elution incubation temperature is controlled at 90°C, and ENR is not detected in the elution solution, which cannot be compared with the elution effect at 65°C.
[0072] Comparative Example 7
[0073] Similar to Example 1, the difference is that in step (5) of this comparative example, the elution incubation time was extended from 2 min to 60 min, and the elution rate was reduced from 83% to 35%. The reason is that enrofloxacin produced other degradation products.
[0074] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.
Claims
1. A method for the specific recovery of trace amounts of enrofloxacin antibiotics from aquaculture water using magnetic microspheres coupled with antibodies, characterized in that, The steps are as follows: Step 1: Activate the magnetic microspheres. Take streptavidin magnetic microspheres into a centrifuge tube, vortex to mix, then mix on a roller mixer, remove the supernatant by magnetic separation, add PBST Buffer, mix on a roller mixer at room temperature, remove the supernatant after magnetic separation, repeat the washing process several times with PBST Buffer, remove the supernatant and set aside. The activation is complete. The particle size of the streptavidin magnetic microspheres is 3-5 μm. Step 2: Biotinylate enrofloxacin monoclonal antibody. Add biotin to the enrofloxacin monoclonal antibody, place it in a mixer in the dark and react at room temperature. After the reaction is complete, add blocking buffer and block at room temperature. Then purify with a PD10 chromatography column to obtain biotinylated enrofloxacin monoclonal antibody. The molar ratio of enrofloxacin monoclonal antibody to biotin is 1:
30. Step 3: Prepare magnetic microsphere-conjugated antibody. Add biotinylated enrofloxacin monoclonal antibody to the activated magnetic microspheres, add PBS buffer, and then vortex mix. Then place it in a roller mixer for incubation at a temperature of 25-37°C. At this point, the conjugation is complete. The conjugate is represented as MNP@Ab, where MNP represents magnetic microspheres, Ab represents antibody, and @ represents conjugation. Step 4: Enrofloxacin enrichment and elution. The conjugate MNP @Ab was directly added to the water sample containing trace amounts of enrofloxacin for extraction. The extract was incubated in a mixer set at 37°C for 30 min. After incubation, the supernatant was removed by magnetic separation. Specifically, the supernatant was placed on a magnetic rack and left to stand for 3 min to obtain the MNP @Ab bound to enrofloxacin. Step 5: Elution and recovery of enrofloxacin. Add the enrofloxacin-bound MNP @Ab to the eluent and incubate at 65°C for no more than 2 minutes. After incubation, vortex to mix and then use magnetic separation technology to obtain the supernatant. At this time, enrofloxacin is eluted into the supernatant. The eluent is a mixture of 95 wt% formamide and 20 mM EDTA at a volume ratio of 1:
1.
2. The method for specifically recovering trace amounts of enrofloxacin antibiotics from aquaculture water using magnetic microspheres coupled with antibodies, as described in claim 1, is characterized in that... In step one, streptavidin magnetic microspheres are placed in a centrifuge tube, vortexed for 10 seconds, then mixed on a roller mixer for 15 minutes, magnetically separated on a magnetic separator for 1 minute, the supernatant is removed, PBST Buffer is added, and the mixture is mixed on a roller mixer at room temperature for 5 minutes. After magnetic separation, the supernatant is removed, and the washing is repeated twice with PBST Buffer. The supernatant is then removed and the product is ready for use, thus completing the activation process.
3. A method for the specific recovery of trace amounts of enrofloxacin antibiotics from aquaculture water using magnetic microspheres coupled with antibodies, as described in claim 1 or 2, characterized in that... The PBST Buffer is prepared by adding 0.05 wt% Tween 20 to PBS and adjusting the pH to 7.
4.
4. The method for specifically recovering trace amounts of enrofloxacin antibiotics from aquaculture water using magnetic microspheres coupled with antibodies, as described in claim 1, is characterized in that... In step two, biotin is pretreated before addition. After equilibration at room temperature, it is dissolved in DMSO. The enrofloxacin monoclonal antibody is diluted with labeling buffer, and then the dissolved biotin is added. The mixture is wrapped in aluminum foil to protect it from light and placed on a mixer to react at room temperature for 3 h. After the reaction is complete, blocking buffer is added, and the mixture is placed on a mixer to block at room temperature for 30 min. Then, it is purified using a PD10 chromatography column to obtain the biotinylated enrofloxacin monoclonal antibody.
5. The method for specifically recovering trace amounts of enrofloxacin antibiotics from aquaculture water using magnetic microspheres coupled with antibodies, as described in claim 1, is characterized in that... In step one, the concentration of streptavidin magnetic microspheres is 10 mg / mL, and in step three, the ratio of biotinylated enrofloxacin monoclonal antibody to streptavidin magnetic microspheres in step one is 10~20 μg:100 μL.
6. The method for specifically recovering trace amounts of enrofloxacin antibiotics from aquaculture water using magnetic microspheres coupled with antibodies, as described in claim 1, is characterized in that... In step three, biotinylated enrofloxacin monoclonal antibody is added to the activated magnetic microspheres, and PBS buffer is added to make the total volume 100 μL. The mixture is then vortexed for 10 s and incubated in a roller mixer for 60 min at a temperature of 37°C. The coupling is now complete.
7. The method for specifically recovering trace amounts of enrofloxacin antibiotics from aquaculture water using magnetic microspheres coupled with antibodies, as described in claim 1, is characterized in that... In step five, the enrofloxacin-bound MNP @Ab is added to the elution buffer and incubated at 65°C for no more than 2 minutes. After incubation, the mixture is vortexed for 30 seconds, and then the supernatant is obtained using magnetic separation technology. At this time, enrofloxacin is eluted into the supernatant.