An immunomagnetic bead kit for purifying field sponge acid and a preparation method and application thereof
By preparing an immunomagnetic bead kit conjugated with carboxylated agarose magnetic beads and daetian scleroderma monoclonal antibody, the problems of cumbersome detection methods and low recovery rates of daetian scleroderma were solved, achieving efficient, rapid, and accurate detection results.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- QINGDAO PRIBOLAB BIOTECH CO LTD
- Filing Date
- 2026-03-27
- Publication Date
- 2026-05-29
AI Technical Summary
Existing methods for detecting daphne are characterized by long detection cycles, cumbersome operations, a tendency to produce false positives and false negatives, inaccurate quantification, and an inability to distinguish specific toxin components. Traditional pretreatment methods also suffer from low recovery rates and poor stability, making it difficult to achieve high-throughput and high-accuracy detection.
An immunomagnetic bead kit, consisting of carboxylated agarose magnetic beads conjugated with monoclonal antibodies against datian squalene, combined with a buffer preservation solution, was developed to enrich and extract datian squalene using immunomagnetic bead technology. This kit is suitable for purifying datian squalene in aquatic products.
It achieves efficient enrichment and extraction of styrax acid from molluscs, with advantages of high extraction efficiency, simple operation, and rapid detection. It is suitable for accurate detection in different shellfish matrices and a wide range of toxin concentrations, and has wide applicability.
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Figure CN122109525A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomaterials technology, specifically relating to an immunomagnetic bead kit for purifying oolong acid, its preparation method, and its application. Background Technology
[0002] Okadaic acid (OA) is a polyether marine biotoxin with the molecular formula C2. 44 H 68 O 13 Its structural formula is shown in Formula 1. After consuming shellfish contaminated with daphniacin, patients will experience acute poisoning symptoms such as diarrhea and vomiting. Daphniacin is one of the most prevalent marine toxins, and during its transport and metabolism in the bloodstream, it can damage multiple organs, and there is no specific antidote. Therefore, daphniacin is widely recognized as a potential tumor promoter and even a primary carcinogen. Its long-term toxic effects pose a serious threat to the development of shellfish aquaculture and public health.
[0003]
[0004] The structural formula of the large-field soft sponge acid Currently, the detection of diarrhetic shellfish toxins in shellfish uses the national standard "Determination of Diarrhetic Shellfish Toxins in Shellfish" (GB / T5009.212-2016), which uses the mouse method as the standard test method. This method has a long detection cycle, is cumbersome, has large individual animal differences, is prone to false positives and false negatives, and cannot accurately quantify or distinguish specific toxin components. It is no longer able to meet the current requirements for high-throughput and high-accuracy detection. Ordinary liquid-liquid extraction and solid-phase extraction as pretreatment for LC-MS exhibit significant matrix effects and large interference from impurities, resulting in low recovery rates of target toxins, poor stability, and unsatisfactory method reproducibility, making it difficult to achieve accurate and efficient analysis of diarrhetic shellfish toxins in shellfish samples.
[0005] Therefore, there is an urgent need to provide a high-efficiency and stable daetian sponge acid detection kit to enrich, purify and extract daetian sponge acid from samples, thereby achieving accurate detection of daetian sponge acid in the test samples. Summary of the Invention
[0006] This invention addresses the shortcomings of existing technologies by providing an immunomagnetic bead kit for purifying daphnia latifolia, its preparation method, and its applications. The preparation method of the immunomagnetic bead kit for purifying daphnia latifolia provided by this invention is environmentally friendly and the product exhibits high stability. The immunomagnetic bead kit for purifying daphnia latifolia provided by this invention can be used to enrich and extract daphnia latifolia from aquatic products, offering advantages such as high extraction efficiency, simple operation, and intelligent rapid detection.
[0007] To address the above problems, the present invention provides the following technical solution: An immunomagnetic bead kit for purifying daetian sclerosing acid includes: immunomagnetic beads formed by conjugating carboxylated agarose magnetic beads and daetian sclerosing acid monoclonal antibody, and a buffer solution containing preservatives.
[0008] The immunomagnetic bead kit for purifying oolong acid, as described above, uses a buffer solution selected from any one of bicarbonate buffer, citrate buffer, phosphate buffer, or borax buffer.
[0009] The immunomagnetic bead kit for purifying oolong acid, as described above, uses Proclin 300 as the preservative, and its mass fraction in the buffer preservation solution is 0.04-0.06%.
[0010] Based on the same inventive concept, this invention provides a method for preparing an immunomagnetic bead kit for purifying okata sclerosing acid, which involves conjugating carboxylated agarose magnetic beads with okata sclerosing acid monoclonal antibody to form immunomagnetic beads, specifically including the following steps: (1) Synthesis of carboxylated agarose magnetic beads: To prepare an agarose solution, Fe3O4@SiO2 magnetic fluid was added to the agarose solution and mixed evenly to form an aqueous phase. This aqueous phase was then added to the oil phase and stirred to emulsify. After cooling, the aqueous phase was washed with pure water and magnetically separated to obtain uncrosslinked agarose magnetic beads. The uncrosslinked agarose magnetic beads were added to a crosslinking agent and an alkaline solution and reacted at 20-60℃ for 2-6 hours. After washing and magnetic separation, crosslinked agarose magnetic beads were obtained. In a three-necked flask, cross-linked agarose magnetic beads and ammonia were added, and an amination reaction was carried out at 30-40℃ for 3-4 hours. After washing and magnetic separation, the amination-treated agarose magnetic beads were obtained. In a three-necked flask, sodium chloride aqueous solution, amination-modified agarose magnetic beads and acid anhydride were added and reacted at room temperature for 16-24 hours. After the reaction, the mixture was washed, magnetically separated, and then stored in an ethanol aqueous solution to obtain carboxylated agarose magnetic beads. (2) Conjugation of carboxylated agarose magnetic beads and okata sucralose monoclonal antibody: Carboxylated agarose magnetic beads were activated using a carboxyl activating agent; The activated agarose magnetic beads were mixed evenly with Ota Soft Sponge Acid Monoclonal Antibody and coupled at 25-30℃ for 2-4 hours to obtain antibody-coupled magnetic bead microspheres. Magnetic separation was used to remove the supernatant, followed by washing, addition of blocking reagent, and reaction at 25-30℃ for 1-4 hours. The supernatant was removed by magnetic separation, washed, and stored in a buffer solution containing preservatives to obtain the purified Otae sucralose immunomagnetic bead kit.
[0011] As a preferred embodiment, the method for preparing the immunomagnetic beads of the present invention is as follows:
[0012] In the preparation method of the immunomagnetic bead kit for purifying daphne nigra as described above, in step (1), the oil phase contains an organic solvent and an emulsifier; the organic solvent is one or a mixture of any two or more of toluene, cyclohexane, liquid paraffin, and chloroform; the emulsifier is one or a mixture of any two or more of Span-80, Tween-80, and Triton X-100.
[0013] In the preparation method of the immunomagnetic bead kit for purifying daphne as described above, in step (1), the crosslinking agent is any one of epichlorohydrin, 1,4-butanediol glycidyl ether, and ethylene glycol glycidyl ether.
[0014] In the preparation method of the immunomagnetic bead kit for purifying daphniaic acid as described above, in step (1), the acid anhydride is either succinic anhydride or glutaric anhydride.
[0015] In the preparation method of the immunomagnetic bead kit for purifying daphne oleoresin as described above, in step (2), the carboxyl activating agent is one or a mixture of any two or more of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (or simply “EDC” in this invention), N-hydroxysuccinimide (or simply “NHS” in this invention), and N-hydroxysuccinimide sodium sulfonate.
[0016] In the preparation method of the immunomagnetic bead kit for purifying daphne as described above, in step (2), the coupling buffer solution is selected from any one of bicarbonate buffer, citrate buffer, phosphate buffer and borax buffer.
[0017] In the preparation method of the immunomagnetic bead kit for purifying omega-3 spongy acid as described above, in step (2), the blocking reagent is selected from any one or a mixture of two or more of PBST, Tris-HCl, chicken egg albumin, whey protein, casein, ethylamine and ethanolamine.
[0018] Based on the same inventive concept, the present invention provides a method for extracting daphniacin from aquatic products using an immunomagnetic bead kit for purifying daphniacin as described above or an immunomagnetic bead kit for purifying daphniacin prepared by the preparation method described above, and using the immunomagnetic bead kit to extract daphniacin from shellfish samples.
[0019] The shellfish samples described above are mussels, scallops, oysters, and clams.
[0020] Based on the same inventive concept, this invention provides an application of the immunomagnetic bead kit for purifying daetian sclerosing acid prepared by the above-described method or the immunomagnetic bead kit for purifying daetian sclerosing acid prepared by the above-described method in the enrichment, purification and extraction of daetian sclerosing acid.
[0021] Compared with existing technologies, the effects and advantages of this invention are: 1. The immunomagnetic bead kit for purifying daphnia latifolia provided by this invention can be used to enrich and extract daphnia latifolia from aquatic products, and has the advantages of high extraction efficiency, simple operation and rapid detection.
[0022] 2. Experiments of this invention show that, under spiked concentrations of 50 μg / kg and 400 μg / kg, the recoveries of oxytetracycline in various shellfish were: 86.63%–100.26% (mussels), 83.65%–95.34% (scallops), 78.17%–94.52% (oysters), and 82.49%–105.72% (clams). The results indicate that this immunomagnetic bead kit exhibits good recovery rates and accuracy across different shellfish matrices and a wide range of toxin concentrations, demonstrating its excellent extraction efficiency and broad applicability in OA monitoring.
[0023] 3. The immunomagnetic bead kit for purifying oleanolic acid provided by this invention solves the core pain points of low extraction efficiency and cumbersome operation of traditional methods by utilizing antibody-antigen specific recognition in immunomagnetic bead purification technology. The automated platform constructed in conjunction with LC-MS / MS enables high throughput, intelligent operation, precision, and environmental friendliness. Attached Figure Description
[0024] Figure 1 Morphology of the Ota soft sponge acid immunomagnetic beads prepared in Example 1 under a magnetic rack (left) and an optical microscope (right). Figure 2 Magnetization curves of NHS activated agarose magnetic beads and Ota soft sponge acid immunomagnetic beads; Figure 3 A schematic diagram of the preparation method of a fully automated magnetic bead purifier; Figure 4The mass spectrometry elution time of omega-3 sclerosing acid; Figure 5 The mass spectrum of omega-3 sclerosing acid ([MH)) - = 803.52). Detailed Implementation
[0025] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0026] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this specification includes any and all combinations of one or more of the associated listed items.
[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.
[0028] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.
[0029] An immunomagnetic bead kit for purifying daetian sclerosing acid includes: immunomagnetic beads formed by conjugating carboxylated agarose magnetic beads and daetian sclerosing acid monoclonal antibody, and a buffer solution containing preservatives.
[0030] In the immunomagnetic bead kit for purifying oolong acid described above, the buffer solution is selected from any one of bicarbonate buffer, citrate buffer, phosphate buffer, or borax buffer. Preferably, the buffer solution is selected from phosphate buffer.
[0031] The immunomagnetic bead kit for purifying oolong acid, as described above, uses Proclin 300 as the preservative, and its mass fraction in the buffer preservation solution is 0.04-0.06%.
[0032] Based on the same inventive concept, this invention provides a method for preparing an immunomagnetic bead kit for purifying okata sclerosing acid, which involves conjugating carboxylated agarose magnetic beads with okata sclerosing acid monoclonal antibody to form immunomagnetic beads, specifically including the following steps: (1) Synthesis of carboxylated agarose magnetic beads: To prepare an agarose solution, Fe3O4@SiO2 magnetic fluid was added to the agarose solution and mixed evenly to form an aqueous phase. This aqueous phase was then added to the oil phase and stirred to emulsify. After cooling, the aqueous phase was washed with pure water and magnetically separated to obtain uncrosslinked agarose magnetic beads. The uncrosslinked agarose magnetic beads were added to a crosslinking agent and an alkaline solution and reacted at 20-60℃ for 2-6 hours. After washing and magnetic separation, crosslinked agarose magnetic beads were obtained. In a three-necked flask, cross-linked agarose magnetic beads and ammonia were added, and an amination reaction was carried out at 30-40℃ for 3-4 hours. After washing and magnetic separation, the amination-treated agarose magnetic beads were obtained. In a three-necked flask, sodium chloride aqueous solution, amination-modified agarose magnetic beads and acid anhydride were added and reacted at room temperature for 16-24 hours. After the reaction, the mixture was washed, magnetically separated, and then stored in an ethanol aqueous solution to obtain carboxylated agarose magnetic beads. (2) Conjugation of carboxylated agarose magnetic beads and okata sucralose monoclonal antibody: Carboxylated agarose magnetic beads were activated using a carboxyl activating agent; In the conjugation buffer solution, the activated agarose magnetic beads were mixed with the Ota Soft Sponge Acid Monoclonal Antibody and conjugated at 25-30℃ for 2-4 hours to obtain the antibody-conjugated magnetic bead microspheres. Magnetic separation was used to remove the supernatant, followed by washing, addition of blocking reagent, and reaction at 25-30℃ for 1-4 hours. The supernatant was removed by magnetic separation, washed, and stored in a buffer solution containing preservatives to obtain the purified Otae sucralose immunomagnetic bead kit.
[0033] The immunomagnetic bead kit for purifying daphniacin (a type of aquatic fibroblastic acid) provided by this invention is obtained by conjugating carboxylated agarose magnetic beads with a monoclonal antibody against daphniacin. The basic magnetic microspheres are coated with an agarose polymer layer, and the daphniacin monoclonal antibody is obtained through secretion by hybridoma cells. This immunomagnetic bead kit for purifying daphniacin can be used to enrich and extract daphniacin from aquatic products, offering advantages such as high extraction efficiency, simple operation, and intelligent rapid detection.
[0034] As a preferred embodiment, the reaction formula for the preparation method of the immunomagnetic beads of the present invention is as follows:
[0035] In the preparation method of the immunomagnetic bead kit for purifying oolong acid as described above, in step (1), the oil phase comprises an organic solvent and an emulsifier; the organic solvent is one or a mixture of any two or more of toluene, cyclohexane, liquid paraffin, and chloroform; the emulsifier is one or a mixture of any two or more of Span-80, Tween-80, and Triton X-100. Preferably, the organic solvent is toluene; the emulsifier is Span-80.
[0036] In the preparation method of the immunomagnetic bead kit for purifying okara oleic acid as described above, in step (1), the crosslinking agent is any one of epichlorohydrin, 1,4-butanediol glycidyl ether, and ethylene glycol glycidyl ether. Preferably, the crosslinking agent is epichlorohydrin.
[0037] In the preparation method of the immunomagnetic bead kit for purifying daphniaic acid as described above, in step (1), the acid anhydride is either succinic anhydride or glutaric anhydride.
[0038] In the preparation method of the immunomagnetic bead kit for purifying daphne as described above, in step (2), the carboxyl activating agent is any one or a mixture of two or more of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, and N-hydroxysuccinimide sodium sulfonate.
[0039] In the preparation method of the immunomagnetic bead kit for purifying oolong acid as described above, in step (2), the coupling buffer solution is selected from any one of bicarbonate buffer, citrate buffer, phosphate buffer, or borax buffer. The coupling buffer solution is selected from phosphate buffer.
[0040] In the preparation method of the immunomagnetic bead kit for purifying omega-3 sclerosing acid as described above, in step (2), the blocking reagent is selected from any one or a mixture of two or more of PBST, Tris-HCl, chicken ovalbumin, whey protein, casein, ethylamine, and ethanolamine. Preferably, the blocking reagent is selected from PBST blocking solution. Specifically, the PBST blocking solution is prepared as follows: Measure PBS (pH 7.2, 0.01M) solution, add 1 g of BSA and 46 μL of Tween 20, stir evenly, and then adjust the volume to 100 mL with PBS buffer.
[0041] Based on the same inventive concept, the present invention provides a method for extracting daphniacin from aquatic products using an immunomagnetic bead kit for purifying daphniacin as described above or an immunomagnetic bead kit for purifying daphniacin prepared by the preparation method described above, and using the immunomagnetic bead kit to extract daphniacin from shellfish samples.
[0042] The shellfish samples described above are mussels, scallops, oysters, and clams.
[0043] Based on the same inventive concept, this invention provides an application of the immunomagnetic bead kit for purifying daetian sclerosing acid prepared by the above-described method or the immunomagnetic bead kit for purifying daetian sclerosing acid prepared by the above-described method in the enrichment, purification and extraction of daetian sclerosing acid.
[0044] The Fe3O4@SiO2 magnetorheological fluid containing Tween-20 was purchased from Suzhou Beaver Biomedical Engineering Co., Ltd., model: 400nm.
[0045] Example 1 This embodiment provides a method for preparing an immunomagnetic bead kit for purifying oolong acid, including the following steps: (1) Synthesis of carboxylated agarose magnetic beads Weigh 300 g of pure water, add 12 g of agarose and 2 g of sodium chloride, and heat to 95°C. At this point, numerous fine bubbles appear, and the solution becomes transparent. Slowly add 1.5 g of Fe3O4@SiO2 magnetic fluid containing Tween-20, and continue stirring until homogeneous. Heat to 95°C, then cool slightly to obtain the aqueous phase. Weigh 60 g of Span-80 and 1500 mL of toluene into a 2 L three-necked flask, and heat to 70°C, initially maintaining a rotation speed of 500 rpm to obtain the oil phase. Quickly add the aqueous phase to the oil phase, adjust the rotation speed to 1350 rpm, and maintain this temperature for 30 min. Rapidly cool to below 30°C using ice water. Pour off the upper oil phase. The aqueous phase is washed with pure water and magnetically separated to obtain approximately 330 mL of uncrosslinked agarose magnetic beads.
[0046] Measure 300 mL of the uncrosslinked agarose magnetic beads and add 75 g of dimethyl sulfoxide (DMSO) and 300 mL of water. Stir at low speed for 15 min, then add 0.3 g of sodium borohydride, and simultaneously add 60 g of epichlorohydrin and 120 g of a 50% sodium hydroxide aqueous solution. Maintain the temperature at 45 ℃. After the addition is complete, keep the reaction at this temperature for 4 h. Then, wash with pure water and perform magnetic separation to obtain the crosslinked agarose magnetic beads.
[0047] In a 1000 mL three-necked flask, 300 mL of cross-linked agarose magnetic beads and 300 g of ammonia solution (prepared from 30 g of concentrated ammonia and 270 g of pure water) were added. The reaction was maintained at 40 °C and 200 rpm for 4 h. After washing with pure water and magnetic separation, the amination-modified agarose magnetic beads were obtained.
[0048] In a 1000 mL three-necked flask, 300 mL of 0.1 M sodium chloride aqueous solution, 300 mL of amination-modified agarose, and 10 g of succinic anhydride were added. The pH was continuously adjusted to 6 using 0.1 M sodium hydroxide aqueous solution, and the reaction was carried out at room temperature for 16 h. After the reaction, the mixture was washed with pure water, magnetically separated, and then stored in a 20% ethanol aqueous solution to obtain carboxylated agarose magnetic beads. The carboxyl density was determined to be 60 mmol / g by acid-base titration.
[0049] (2) Conjugation of carboxylated agarose magnetic beads and Ota hymenopic acid monoclonal antibody Preparation of MEST solution (100 mM MES, pH 5.0, mass fraction 0.05% Tween 20): Accurately weigh 1.952 g of 2-morpholinoethanesulfonic acid (MES) and dissolve it in ultrapure water. Add 46 μL of Tween 20 and stir well. Then adjust the pH to 5.0 with 0.1 mol / L HCl (NaOH) according to the actual pH value of the solution. Finally, make up to 100 mL to obtain the MEST solution.
[0050] Add 5 mL of carboxylated agarose magnetic beads to a centrifuge tube, magnetically separate to remove the supernatant, and wash three times with 10 mL of MEST solution, then remove the supernatant. Add 5 mL of freshly prepared MEST solution of 10 mg / mL 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 5 mL of MEST solution of 10 mg / mL N-hydroxysuccinimide sodium sulfonate to the centrifuge tube containing the carboxylated agarose magnetic beads, and vortex to fully suspend the magnetic beads. Activate at 25°C for 30 min, using a vertical mixer to maintain the suspension of the magnetic beads during this period.
[0051] After 30 min, the supernatant was removed by magnetic separation. The microspheres were washed three times with PBS buffer, each time with 5 mL of PBS buffer (0.01 M PBS pH=7.2). Then, 11 mL of 6.995 mg / mL OA monoclonal antibody (referred to as "OA antibody") in PBS solution (solvent: 0.01 M PBS pH=7.2) was added, and the mixture was gently mixed. The microspheres were then coupled at 25°C for 2 h to obtain antibody-conjugated magnetic beads. The OA antibody was obtained using the conventional immunization method described in Chinese patent application "CN202111097785.5 A bongkrekic acid complex antigen, bongkrekic acid antibody, and their preparation method and preparation of antibodies in an enzyme-linked immunosorbent assay kit".
[0052] After coupling for 2 hours, the supernatant was removed by magnetic separation. The mixture was washed three times with PBS, and 10 mL of PBST blocking buffer was added for blocking. The mixture was gently mixed and reacted at 25°C with shaking for 1 hour. The PBST blocking buffer was prepared as follows: Measure PBS buffer (0.01M PBS pH=7.2), add 1 g of BSA and 46 μL of Tween 20, stir well, and then bring the volume to 100 mL with PBS buffer.
[0053] After 1 hour, the supernatant was removed by magnetic separation, washed 3 times with PBS, and then PBS preservation solution containing 0.05% Proclin 300 was added. The solution was stored at 4°C to obtain the immunomagnetic bead kit for purified okata sclerosing acid, abbreviated as OA magnetic bead kit.
[0054] The morphology of the prepared Oda soft sponge acid immunomagnetic beads on a magnetic rack and under an optical microscope is shown in the figure. Figure 1 .
[0055] Example 2 The synthesis method of this embodiment is the same as that of Example 1, and the same parts are omitted. The difference from Example 1 is that in this embodiment, the acid anhydride used in step (1) is glutaric anhydride.
[0056] Example 3 The synthesis method of this embodiment is the same as that of Example 1, and the same parts are omitted. The difference from Example 1 is that in this embodiment, in step (2), the carboxyl activating agent is replaced by an equal amount of N-hydroxysuccinimide sodium salt with N-hydroxysuccinimide (NHS).
[0057] Example 4 The synthesis method of this embodiment is the same as that of Example 1, and the same parts are omitted. The difference from Example 1 is that in this embodiment, in step (1), the acid anhydride used is glutaric anhydride; in step (2), the carboxyl activating agent is replaced by an equal amount of N-hydroxysuccinimide sodium salt with N-hydroxysuccinimide (NHS).
[0058] Performance testing I. Characterization of magnetic beads: Magnetization properties of the synthesized Oda soft spongy acid immunomagnetic beads in Example 1 are as follows: Figure 2 As shown, the saturation magnetization of carboxylated agarose beads activated by Sulfo-NHS was approximately 41 emu / g, while that of ohdain leucine immunomagnetic beads decreased to approximately 37 emu / g. This significant decrease in magnetization indicates that the ohdain leucine monoclonal antibody successfully bound to the surface of the NHS-activated agarose beads. The antibody binding reduced the proportion of the magnetic core per unit mass of the beads, which is a direct physicochemical characterization of successful conjugation.
[0059] In addition, according to Figure 2 shown, the hysteresis loops of NHS-activated agarose magnetic beads and okadaic acid immunomagnetic beads are both symmetrically distributed around the origin, and the magnetization curve and demagnetization curve completely overlap; no coercive force and remanent magnetization signals were detected during the experiment, and the above magnetic characteristics all meet the typical judgment criteria of superparamagnetic materials, confirming that the prepared okadaic acid immunomagnetic beads have good superparamagnetic properties.
[0060] II. Experimental method: Detect the capture amount and recovery rate of magnetic beads, and prepare a shellfish sample test specimen: Take fresh shellfish homogenate, and then add a known concentration of okadaic acid (OA) standard to it to obtain a shellfish spiked test specimen; the spiked concentration of okadaic acid in the test specimen is 50 μg / kg and 400 μg / kg, and measure it in parallel twice.
[0061] 1. Use the OA magnetic bead kit of Example 1 to extract OA in the shellfish test specimen and measure the recovery rate. The specific operation steps are as follows: (1) Extraction of OA in shellfish samples Weigh 2.0 g of the edible part of shellfish, place it in a 50 mL centrifuge tube, add 10.0 mL of methanol aqueous solution with a mass fraction of 80%, and perform vortex oscillation for 2 min and ultrasonic extraction for 10 min in sequence. Centrifuge the extract at 4000 rpm for 10 min, collect the supernatant, repeat the above extraction steps for the residue once, and combine the two supernatants. Accurately transfer 10.0 mL of the combined supernatant, add 40.0 mL of PBS buffer (0.01 M, pH 7.0), mix well, which is the shellfish sample extract and serves as the sample solution to be purified.
[0062] (2) Immunomagnetic bead purification In this experiment, a fully automatic magnetic bead purifier is used. The schematic diagram of its operation method is shown in Figure 3 .
[0063] ① Reagent and sample preparation: In the magnetic bead strip of the fully automatic magnetic bead purifier, add the following components in sequence: a) 10.0 mL of the pre-treated and diluted shellfish sample extract; b) 100 μL of the OA magnetic bead kit of Example 1; c) 0.9 mL of PBS buffer (0.01 M, pH 7.0) for diluting the immunomagnetic beads to the working concentration; d) Separate independent wells are pre-loaded with 1.0 mL of washing buffer, and the washing buffer is PBS buffer (0.01 M, pH 7.0); e) 1.0 mL eluent (chromatographic grade methanol).
[0064] ②Immunopurification: After the instrument is started, the magnetic rod enters the magnetic bead solution well under the drive of the magnetic field, adsorbs the immunomagnetic beads, and transfers them to the well containing the sample extract. The instrument is then shaken at 25°C for 50 minutes to allow the OA in the sample to fully bind with the antibodies on the surface of the magnetic beads.
[0065] ③ Washing and removing impurities: The magnetic rod transfers the OA-bound magnetic beads into the wash buffer wells, and the mixture is agitated at 25°C for 2 min to remove non-specific adsorbed impurities. A magnetic field is then applied for 1 min to stabilize the magnetic beads on the magnetic rod surface, and the wash waste liquid is discarded.
[0066] ④ Eluting the target substance: The magnetic rod transfers the magnetic beads into an elution well containing 1.0 mL of chromatographic grade methanol, and the mixture is shaken for 2 min to allow OA to dissociate efficiently from the antibody.
[0067] ⑤ Recover the eluent: After elution is complete, the magnetic rod moves the magnetic beads out of the elution hole and back to the magnetic bead storage hole under the action of the magnetic field.
[0068] Retain 1.0 mL of methanol solution in the elution well; this is the OA test solution after enrichment and purification by immunomagnetic beads.
[0069] The concentration of OA was determined by liquid chromatography-tandem mass spectrometry (LC-MS / MS), and its recovery rate was calculated accordingly. A higher recovery rate indicates better extraction efficiency of the magnetic bead kit. The recovery rate was calculated as follows: Recovery rate = OA detection value / OA spiked concentration × 100%.
[0070] Mass spectrometry analysis revealed a quasi-molecular ion peak [MH]. - = 803.52, consistent with the molecular weight of Datian soft spongy acid, mass spectrum shown in [reference needed]. Figure 4 and Figure 5 .
[0071] The recovery results are shown in Table 1.
[0072] Table 1. Recovery rate of OA extracted from shellfish samples using an OA magnetic bead kit.
[0073] 2. Immunomagnetic bead binding capacity assay Take 10.0 mL of 10% methanol aqueous solution, add 4000 ng of OA standard, mix thoroughly, and prepare a binding capacity detection solution with an OA concentration of 400 ng / mL.
[0074] The same magnetic bead purification procedure as the above shellfish samples was used in steps 1 (1)–1 (2) to process the test reagent on the instrument: the amount of immunomagnetic beads used was 100 μL, and the binding, washing and elution conditions were kept consistent with the above experimental conditions.
[0075] The final eluent was collected to evaluate the binding capacity and enrichment efficiency of immunomagnetic beads for OA at high concentrations. The results of the binding capacity test are shown in Table 2.
[0076] Table 2. Results of binding capacity determination using OA magnetic bead reagent kit.
[0077] 3. Experimental Results and Analysis: Based on the detection results in Table 1, the OA immunomagnetic bead kit combined with liquid chromatography-tandem mass spectrometry (LC-MS / MS) was used to analyze four common shellfish samples: mussels, scallops, oysters, and clams. Under spiked concentrations of 50 μg / kg and 400 μg / kg, the recoveries of okata-sponge acid in each shellfish were: 86.63%–100.26% (mussels), 83.65%–95.34% (scallops), 78.17%–94.52% (oysters), and 82.49%–105.72% (clams), respectively. Combined with volumetric testing (Table 2, spiking amount 4000 ng), the recovery rate was >90%. The results indicate that this immunomagnetic bead kit exhibits good recovery rates and accuracy across different shellfish matrices and a wide range of toxin concentrations, demonstrating excellent extraction efficiency and broad applicability in OA monitoring.
[0078] It is understood that those skilled in the art can make equivalent substitutions based on the technical solutions and concepts of this invention.
Claims
1. An immunomagnetic bead reagent kit for purifying oolong acid, characterized in that, include: Immunomagnetic beads formed by conjugation of carboxylated agarose magnetic beads and Ōta hygroscopic acid monoclonal antibody, and a buffer solution containing preservatives.
2. The immunomagnetic bead kit for purifying oolong acid according to claim 1, characterized in that, The buffer solution is selected from any one of bicarbonate buffer, citrate buffer, phosphate buffer, or borax buffer.
3. A method for preparing an immunomagnetic bead reagent kit for purifying oolong acid, characterized in that, Immunomagnetic beads are formed by conjugating carboxylated agarose magnetic beads with okata hyaluronic acid monoclonal antibody, specifically including the following steps: (1) Synthesis of carboxylated agarose magnetic beads: To prepare an agarose solution, Fe3O4@SiO2 magnetic fluid was added to the agarose solution and mixed evenly to form an aqueous phase. This aqueous phase was then added to the oil phase and stirred to emulsify. After cooling, the aqueous phase was washed with pure water and magnetically separated to obtain uncrosslinked agarose magnetic beads. The uncrosslinked agarose magnetic beads were added to a crosslinking agent and an alkaline solution and reacted at 20-60℃ for 2-6 hours. After washing and magnetic separation, crosslinked agarose magnetic beads were obtained. In a three-necked flask, cross-linked agarose magnetic beads and ammonia were added, and an amination reaction was carried out at 30-40℃ for 3-4 hours. After washing and magnetic separation, the amination-treated agarose magnetic beads were obtained. In a three-necked flask, sodium chloride aqueous solution, amination-modified agarose magnetic beads and acid anhydride were added and reacted at room temperature for 16-24 hours. After the reaction, the mixture was washed, magnetically separated, and then stored in an ethanol aqueous solution to obtain carboxylated agarose magnetic beads. (2) Conjugation of carboxylated agarose magnetic beads and okata sucralose monoclonal antibody: Carboxylated agarose magnetic beads were activated using a carboxyl activating agent; The activated agarose magnetic beads were mixed evenly with Ota Soft Sponge Acid Monoclonal Antibody and coupled at 25-30℃ for 2-4 hours to obtain antibody-coupled magnetic bead microspheres. Magnetic separation was used to remove the supernatant, followed by washing, addition of blocking reagent, and reaction at 25-30℃ for 1-4 hours. The supernatant was removed by magnetic separation, washed, and stored in a buffer solution containing preservatives to obtain the purified Otae sucralose immunomagnetic bead kit.
4. The method for preparing the immunomagnetic bead reagent kit for purifying oolong acid according to claim 3, characterized in that, In step (1), the oil phase comprises an organic solvent and an emulsifier; the organic solvent is one or a mixture of any two or more of toluene, cyclohexane, liquid paraffin, and chloroform; the emulsifier is one or a mixture of any two or more of Span-80, Tween-80, and Triton X-100.
5. The method for preparing the immunomagnetic bead reagent kit for purifying oolong acid according to claim 3, characterized in that, In step (1), the crosslinking agent is any one of epichlorohydrin, 1,4-butanediol glycidyl ether, and ethylene glycol glycidyl ether.
6. The method for preparing the immunomagnetic bead reagent kit for purifying oolong acid according to claim 3, characterized in that, In step (1), the acid anhydride is either succinic anhydride or glutaric anhydride.
7. The method for preparing the immunomagnetic bead reagent kit for purifying oolong acid according to claim 3, characterized in that, In step (2), the carboxyl activating agent is any one or a mixture of two or more of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, and N-hydroxysuccinimide sodium sulfonate.
8. The method for preparing the immunomagnetic bead kit for purifying oolong acid according to claim 3, characterized in that, In step (2), the blocking reagent is selected from any one or any two or more of PBST, Tris-HCl, chicken ovalbumin, whey protein, casein, ethylamine and ethanolamine.
9. A method for extracting daphniacin from aquatic products using an immunomagnetic bead kit for purifying daphniacin as described in any one of claims 1-2 or an immunomagnetic bead kit for purifying daphniacin prepared by any one of claims 3-8, characterized in that, Ota-sponge acid was extracted from shellfish samples using the immunomagnetic bead kit.
10. The application of the immunomagnetic bead kit for purifying daetian sclerosing acid as described in any one of claims 1-2 or the immunomagnetic bead kit for purifying daetian sclerosing acid prepared by the preparation method described in any one of claims 3-8 in the enrichment, purification and extraction of daetian sclerosing acid.