Magnetic particle chemical luminous kit for detecting clenbuterol and application thereof

[0036] Example 1 Preparation of specific components of the kit

[0037] 1. Preparation of Luminescent Labels

[0038] a) Synthesis of Clenbuterol Hapten

[0039] Clenbuterol is acylated with succinic anhydride to acylate the alcohol hydroxyl group on the molecular structure of clenbuterol into a clenbuterol hapten containing a 4-carbon carboxyl indirect arm.

[0040] b) Preparation of Luminescent Labels

[0041] Take 4.5mmol/L ABEI, dissolve it in 4ml distilled water, dissolve 5.0mmol/L N-hydroxysuccinimide in 0.5ml N,N-dimethylformamide, mix the two thoroughly and react at room temperature for 3-4h . Take 15 mg of the clenbuterol hapten prepared above, adjust the volume to 1.5 ml with pH 7.4 PBS, then add the above activated ABEI solution, mix well, react overnight at room temperature, and purify through G-25 gel column.

[0042] 2. Preparation of fluorescent markers

[0043] a) Preparation of immunogens:

[0044] The immunogen was obtained by coupling the clenbuterol hapten and ovalbumin by the mixed acid anhydride method.

[0045] b) Preparation of clenbuterol monoclonal antibody

[0046]Animal immunization: Balb/c mice were immunized with immunogen at a dose of 150 μg/mice to produce antiserum.

[0047] Cell fusion and cloning: The spleen cells of immunized Balb/c mice were taken and fused with SP2/0 myeloma cells in a ratio of 9:1 to obtain a hybridoma cell line of monoclonal antibody.

[0048] Cell cryopreservation and recovery: make 1×10 hybridoma cells with cryopreservation solution 6 cells/ml, and stored in liquid nitrogen for a long time. When resuscitated, take out the cryopreservation tube, immediately put it into a 37°C water bath to thaw quickly, remove the cryopreservation solution by centrifugation, and transfer it to a culture bottle for culture.

[0049] Preparation and purification of monoclonal antibodies: Balb/c mice were intraperitoneally injected with sterilized paraffin oil 0.5ml/mouse, and 5×10 hybridoma cells were intraperitoneally injected 7 days later. 5 Ascites was collected after 7 days. It was purified by the octanoic acid-saturated ammonium sulfate method, and the purified ascites was stored at -20°C.

[0050] C) Fluorescent Label Preparation

[0051] Dilute the clenbuterol monoclonal antibody to a concentration of 1% by mass with 0.025mol/L carbonate buffer pH 9.0; add 0.01mg FITC per mg of immunoglobulin according to the total amount of protein to be labeled, Accurately weigh the desired FITC powder with an analytical balance. Use the same buffer to prepare a 0.1 mg/ml solution of FITC, mix 3-5 times the volume of the above antibody solution into the FITC dilution solution; magnetically stir and label for 30-48 hours at 4℃ in the dark; 3000 r/min, centrifuge at room temperature for 20 min, remove a small amount of sediment, put it into a dialysis bag, and dialyze the PBS buffer with pH 7.4 for 2-3 days, during which the dialysate is replaced at least 3 times; take the marker dialyzed overnight, Free fluorescein was separated by Sephadex G-25 or G-50 column, and the labeled fluorescent marker was collected for identification, aliquoted, and stored in a refrigerator at 4°C.

[0052] 3. Preparation of separation reagents

[0053] a) Magnetic bead activation

[0054] Magnetic beads with surface -COOH group (purchased from DYNAL, particle size is 2.8μm), its content is 0.15eq/g; take 100μl magnetic beads, wash with 100μl 25mmol/L, pH5.0, 0.05% Tween-20MES solution Twice, the supernatant was removed after magnetic separation; before use, 50 mmol/L EDC and NHS solutions were prepared with 25 mmol/L MES solution stored at 4 °C; 50 μl of newly prepared EDC and NHS solutions were added to the magnetic beads. The centrifuge tube was placed in a centrifuge tube, vortexed and mixed, and activated at room temperature for 30 minutes; the centrifuge tube was placed on a magnetic separation rack for magnetic separation for 4 minutes, the supernatant was removed, and 100 μl, 25 mmol/L, pH 5.0 was added, and MES was washed 2-3 times. Surface carboxyl-activated magnetic beads are available.

[0055] b) Magnetic beads conjugated goat anti-FITC monoclonal antibody

[0056] Dissolve 50-100 μg goat anti-FITC monoclonal antibody into 60 μl, 25 mmol/L, pH 5.0 MES, adjust the total volume to 100 μl with the MES solution, and mix the magnetic beads and the antibody gently; at least 30min or 30 minutes of coupling at room temperature. Coupling at 4°C for 2h, during this period, the magnetic beads can be kept mixed by a vortex; the centrifuge tube is placed on a magnetic separation rack for magnetic separation for 3-5min, and the supernatant is removed; in order to quench unreacted -COOH, you can Add 100μl, pH7.4, TRIS reaction for 15min or 100μl, pH8.0, PBS containing 50mmol/L ethanolamine to block magnetic beads; wash the blocked magnetic beads with 100μl, 0.1-0.3%BSA, 0.1%Tween-20 PBS or TRIS Magnetic beads 3-5 times; finally magnetic beads were reconstituted with 0.1-0.5% BSA, 0.01-0.1% Tween-20, 0.02% NaN 3 Store in PBS or TRIS buffer at 2-8°C.

[0057] The formation of the second test kit

[0058] A magnetic particle chemiluminescence detection kit for clenbuterol was constructed to contain the following components:

[0059] Fluorescent label for FITC-labeled clenbuterol monoclonal antibody

[0060] Luminescent marker of ABEI-labeled clenbuterol hapten

[0061] Separation Reagent for Paramagnetic Nanospheres Coated with Goat Anti-FITC Monoclonal Antibody

[0062] Clenbuterol standard solution (0ng/ml, 0.01ng/ml, 0.03ng/ml, 0.09ng/ml, 0.27ng/ml, 0.81ng/ml), the standard dilution is pH7.4, containing 0.03% NaN 3 , 0.05mol/L TRIS buffer. The percentage content is the mass percentage content.

[0063] The concentration of Clenbuterol quality control solution is 0.02ng/ml and 0.5ng/ml, respectively, and the quality control solution is pH7.4, containing 0.03% NaN 3 , 0.05mol/L TRIS buffer. The percentage content is the mass percentage content.

[0064] Concentrated wash solution pH 7.6, 0.4% Tween-20, 0.02% NaN 3 , 0.1mol/L PBS buffer. The percentage content is the mass percentage content.

[0065] Example 3 Detection of Clenbuterol in Actual Samples

[0066] 1. Sample pretreatment

[0067] (1) Urine

[0068] Take 20μl of clear urine sample for direct determination (if the urine sample is cloudy, it must be filtered or more than 3000g, centrifuged at 15°C for 10min until clear), the sample not used temporarily should be frozen and stored, the sample dilution ratio: 1

[0069] (2) Tissues with low-fat meat, liver and other tissues

[0070] Weigh 2.0±0.05g of homogenized sample into a 50ml polystyrene centrifuge tube; add 6ml of 4% NaCl-0.1M HCl-methanol mixture, shake with a shaker until uniform; let stand for 10 minutes, centrifuge above 3000g for 5 minutes ; Liver samples: Take 1ml of supernatant and add 20μl of 1M sodium hydroxide solution and mix well (after mixing, measure the pH value, it is about 8); Muscle samples: Take 1ml of supernatant and add 30μl of 1M sodium hydroxide solution and mix well (mixed After homogenization, measure the pH value, about 8); take 20 μl for analysis, sample dilution factor: 1

[0071] (3) Feed

[0072] Grind the feed sample with a mortar, weigh 2.0 ± 0.05g of the ground sample into a 50ml polystyrene centrifuge tube, add 2ml of 1M hydrochloric acid solution, add 16ml of deionized water to homogenize with a homogenizer; use a vortexer Vortex for 3min, put it on the shaker for 15min; over 3000g, centrifuge for 20min, transfer all the supernatant to a 10ml polystyrene centrifuge tube, add 2ml 1M sodium hydroxide solution and mix well, check whether the pH value is between 6.5- Between 7.5; (adjust the pH value with hydrochloric acid or sodium hydroxide solution); over 3000g, centrifuge for 20min, take 100μl supernatant to a 2ml clean polystyrene centrifuge tube, add 900μl deionized water and mix well; take 20μl for For analysis, sample dilution: 100

[0073] (4) Milk

[0074] Take 100 μl of milk sample, add 400 μl of 4% sodium chloride solution, vortex and mix, take 20 μl for analysis, sample dilution factor: 5.

[0075] 2. Detection and result analysis with the kit

[0076] Pipette 20μl-100μl standard or sample respectively, then add 20μl-100μl luminescent marker, add 20μl-100μl fluorescein marker, mix well, incubate at 37°C for 15min; add goat anti-FITC monoclonal antibody coated 80-150μl of paramagnetic nano-beads, mixed well and incubated at 37°C for 5min; separated with a magnetic separation rack for 5min, discarded the supernatant and rinsed the complex precipitate with 300-500μl of washing solution; the obtained separated complex was directly put into Enter the measurement dark box, add excitation substrate 1 and excitation substrate 2, and detect the relative light intensity (RLU) emitted after a delay of 3-5s. The content of clenbuterol in the sample is proportional to the RLU, which can be combined with the standard by RLU. The concentration of clenbuterol was calculated by the curve method.

[0077] Excitation substrate 1 is NaOH, excitation substrate 2 is H 2 O 2. Before loading the substrate, rinse the substrate pump 10-20 times with distilled water, and after emptying the remaining water traces in the pipeline, put the corresponding substrate directly into the instrument, and insert the pump tube into the substrate bottle; Flush the tube 5 times and measure the RLU value of the substrate. Under normal circumstances, the RLU value of the substrate should not exceed 1200. If it exceeds 1200, it is necessary to re-clean the tubing and substrate pump with distilled water more times until the blank value falls within a reasonable range. If no experiment is performed for more than three days, unload the substrate bottle and close the lid to prevent evaporation. The pipes are then rinsed with distilled water and emptied to avoid corrosion of the substrate pump by the strong alkaline solution.

[0078] The present invention uses 6 clenbuterol standard substances (0ng/ml, 0.01ng/ml, 0.03ng/ml, 0.09ng/ml, 0.27ng/ml, 0.81ng/ml) for curve mapping. The instrument detects and obtains a calibration curve related to the RLU value and the concentration of clenbuterol according to the method. In the subsequent measurement, the concentration of clenbuterol in each sample is compared with the standard curve to obtain the clenbuterol in the sample. content.