A method for preparing a fentanyl antigen and a method and kit for preparing a fentanyl antibody

Abstract

The application belongs to the field of biology and particularly relates to a fentanyl antigen preparation method, a fentanyl antibody preparation method and a kit. The fentanyl antibody preparation method comprises the following steps: immunizing 9-week-old, 25-35 g female Balb / c mice with a fentanyl antigen, immunizing the mice at a dose of 100 ug / time, immunizing the mice once a month, a total of 4 times, obtaining peripheral blood of the mice after each immunization to detect and screen, fusing positive B cells with bone marrow hybridoma cells SP2 / 0, and obtaining fentanyl antibodies through a dilution method after 1 week. The kit is used for detecting fentanyl, the kit is provided with a colloidal gold combination pad and a detection line, colloidal gold particles in the colloidal gold combination pad are used for marking fentanyl antibodies, and the detection line is coated with a fentanyl antigen. The application has the following beneficial effects: 1. The preparation method is convenient and low in cost; 2. The kit is convenient and fast to use, low in cost and capable of rapidly detecting fentanyl.

Description

Technical Field

[0001] This invention belongs to the field of biology, specifically relating to a method for preparing fentanyl antigen and a method and kit for preparing fentanyl antibody. Background Technology

[0002] Fentanyl, chemical name: N-[l-(2-phenylethyl)-4-piperidinyl]-N-phenylpropionamide, molecular formula is C 22 H 28 N2 has a molecular weight of 336.48.

[0003] Fentanyl is a potent opioid analgesic suitable for various types of pain and for pain relief during and after surgical procedures, including gynecological and surgical procedures. It is also used to prevent or reduce postoperative delirium and can be used in combination with anesthetics as an adjunct to anesthesia. When combined with droperidol, it is formulated as a "tranquilizing analgesic" for pain relief during extensive dressing changes and minor surgeries.

[0007] Fentanyl is a deadly poison. Its pharmacological characteristics are the main reason for its fatal adverse reactions and toxicity. This is primarily manifested in its potent analgesic effect and high potential for abuse. It can rapidly cross the cell membrane and blood-brain barrier to reach the brain, quickly reaching a peak blood concentration, and easily leading to tolerance and dependence. Although fentanyl is safe for clinical use, its abuse or recreational use is extremely dangerous, easily resulting in acute poisoning and death due to respiratory depression.

[0008] Various analytical methods are available for the preliminary screening of fentanyl and its derivatives in biological samples, such as immunoassay, gas chromatography-mass spectrometry (GC-MS), and liquid chromatography-mass spectrometry (HPLC-MS / MS). Immunoassay utilizes the specific binding of antigens (targets) and antibodies for determination; however, different immunoassays have limitations in cross-reactivity with fentanyl analogues, and some cross-reactivity is unknown. GC-MS cannot directly determine non-volatile, polar, or thermally unstable substances, requiring derivatization of the target analyte, which is inconsistent with the future trend of rapid and efficient analytical chemistry. HPLC-MS, as the most commonly used detection method, offers higher sensitivity, greater stability, and a wider range of applications compared to GC-MS. However, both HPLC-MS and GC-MS require specialized equipment and skilled personnel, making them unsuitable for rapid on-site detection. Summary of the Invention

[0009] Purpose of the invention: The present invention addresses the problems existing in the prior art by disclosing a method for preparing fentanyl antigen and a method and kit for preparing fentanyl antibody.

[0010] This invention utilizes the fundamental principles of immunology and immunochemistry, along with residue analysis techniques, to design and synthesize small-molecule target analyte haptens, which are then conjugated with carrier proteins to prepare effective artificial fentanyl antigens and antibodies. The preparation method of this invention is simple, feasible, and low-cost, with a high hapten yield. The fentanyl antigen of this invention, when used to immunize animals, can generate specific fentanyl antibodies against fentanyl, enabling rapid detection of fentanyl residues.

[0011] The structure of fentanyl is as follows: .

[0012] The structure of the fentanyl hapten is as follows: .

[0013] The synthetic route for fentanyl hapten is as follows: .

[0014] FEN-BSA synthesis (one of the fentanyl antigens) .

[0015] FEN-KLH Synthesis (Fentanyl Antigen II) .

[0016] Technical solution: A method for preparing fentanyl antigen, comprising the following steps: (1) The synthesis of fentanyl hapten, by mass, is as follows: (11) Take one copy of the structural formula as Compound 1 was dissolved in 50-100 parts of dichloromethane. 1-1.5 parts of triethylamine were added under stirring, and the mixture was stirred in an ice bath. 0.5-1 parts of chloroacetyl were added dropwise, and the reaction was allowed to proceed overnight at room temperature to obtain a reaction solution. The reaction solution was then washed with water, and the organic phase was collected. The organic phase was dried and evaporated to dryness to obtain a solid product. This solid product was then purified by column chromatography to obtain the structural formula […]. Compound 2; (12) Dissolve compound 2 obtained in step (11) in 5-10 parts of dichloromethane, then add 7-10 parts of trifluoroacetic acid, stir at room temperature for at least 3 hours, evaporate the solvent to obtain the residue, add 20-40 parts of ethyl acetate to 20-50 parts of sodium carbonate aqueous solution with a mass concentration of 1%-5% and stir evenly to obtain a second washing solution, add the residue to the second washing solution and wash with water at least once, take the organic phase, then dry the organic phase to obtain a solid product, then purify the solid product by column chromatography to obtain the structure with the following formula: Compound 3; (13) Dissolve compound 3 obtained in step (12) in 10-20 parts of ethanol, then add 5-10 parts of triethylamine and 5-10 parts of ethyl bromobutyrate to it and reflux overnight to obtain a reaction solution. Add 20-40 parts of ethyl acetate to 20-50 parts of water and stir evenly to obtain a third washing solution. Place the reaction solution in the third washing solution and wash with water at least once. Take the organic phase, dry and evaporate the organic phase to obtain a solid product. Then purify the solid product by column chromatography to obtain the structure with the following formula: Compound 4; (14) Dissolve compound 4 obtained in step (13) in 8-15 parts of an ethanol-water mixture, then add 0.4-1 parts of sodium hydroxide and hydrolyze for at least 3 hours. After the reaction is complete, adjust the pH of the reaction solution to 5 with dilute hydrochloric acid, then extract with ethyl acetate, take the organic phase, dry and evaporate the organic phase to obtain a solid product, and then purify the solid product by column chromatography to obtain the structure with the following formula: Fentanyl hapten; (2) The preparation of fentanyl antigen, by weight, is as follows: (21) Take 1 part of the fentanyl hapten obtained in step (1), 1-1.5 parts of N-hydroxysuccinimide (NHS), 1.5-2 parts of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), and 3-5 parts of N,N-dimethylformamide and stir rapidly at room temperature overnight. After the reaction is completed, an activation solution is obtained. (22) Stir 35-50 parts of PBS solution of carrier protein with a concentration of 8-12 mg / mL under ice water bath cooling, then slowly add the activation solution obtained in step (21) dropwise, react at room temperature overnight, and obtain the reaction solution after the coupling reaction is completed. (23) Transfer the reaction solution obtained in step (22) to PBS dialysate and dialyze at 2-8℃ for more than 7 times, with an interval of not less than 2 hours between two adjacent dialyses; (24) Transfer the dialyzed solution into a centrifuge tube and centrifuge at 6000-7000 rpm for 5-6 minutes at 2-8℃. Take the supernatant to obtain fentanyl antigen.

[0017] Furthermore, in step (14), the volume ratio of ethanol to water in the ethanol-water mixture is (4-6):1.

[0018] Furthermore, the carrier protein mentioned in step (22) is one of BSA and KLH.

[0019] A method for preparing fentanyl antibodies, comprising the following steps: Nine-week-old, female Balb / c mice weighing 25–35 g were immunized with the fentanyl antigen at a dose of 100 μg per dose, once a month for a total of four immunizations. Peripheral blood was collected from the mice after each immunization for screening. Positive B cells were fused with bone marrow hybridoma cells SP2 / 0, and fentanyl antibodies were obtained one week later by dilution.

[0020] A kit for detecting fentanyl, the kit comprising a colloidal gold conjugate pad and a detection line, wherein colloidal gold particles in the colloidal gold conjugate pad are labeled with the aforementioned fentanyl antibody, and the detection line is coated with the aforementioned fentanyl antigen.

[0021] Principle of fentanyl colloidal gold detection: Colloidal gold particles are labeled with fentanyl antibodies, while the test line is coated with fentanyl antigen and the control line is coated with anti-mouse IgG.

[0022] During testing, the fentanyl test antigen in the sample first binds to the gold-labeled antibody. As it ascends to the test band, if the fentanyl test antigen has completely saturated a certain amount of antibody, the gold-labeled antibody will not be able to bind to the antigen in the test band, and no visible red band will appear, indicating a positive result. If the sample does not contain the test antigen, the gold-labeled antibody will bind to the antigen in the test band, forming a red band, indicating a negative result.

[0023] For the detection of fentanyl small molecule antigens, the competitive inhibition method yields good results. Because small molecules have only one antigenic site and cannot bind to two antibodies simultaneously, the double-antibody sandwich method cannot be used for detection.

[0024] Beneficial effects: The fentanyl antigen preparation method and fentanyl antibody preparation method and kit disclosed in this invention have the following beneficial effects: 1. The methods for preparing fentanyl antigen and fentanyl antibody are convenient and low in cost; 2. The reagent kit is easy and quick to use, low in cost, and has a wide range of applications. It can quickly detect fentanyl and is of great significance. Attached Figure Description

[0025] Figure 1 This is a flowchart of a fentanyl antigen preparation method disclosed in this invention.

[0026] Figure 2 This is a schematic diagram of the test results for Test Example 1. Detailed Implementation

[0027] The specific embodiments of the present invention are described in detail below.

[0028] The "range" disclosed in this invention is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of a particular range. Ranges defined in this way can include or exclude endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range. For example, if a range of 10–50 is listed for a specific parameter, it is also expected that ranges of 10–40 and 20–50 are also included. Furthermore, if the minimum range values ​​are 1 and 2, and the maximum range values ​​are 3, 4, and 5, then the following ranges are all expected: 1–3, 1–4, 1–5, 2–3, 2–4, and 2–5. In this application, unless otherwise stated, the numerical range "a–b" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers. For example, the numerical range "0–5" means that all real numbers between "0–5" have been listed herein; "0–5" is merely a shortened representation of these numerical combinations.

[0029] Unless otherwise specified, all embodiments and optional embodiments of this application can be combined to form new technical solutions.

[0030] Unless otherwise specified, all technical features and optional technical features of this application may be combined to form new technical solutions.

[0031] Unless otherwise specified, all steps in this application may be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), indicating that the method may include steps (a) and (b) performed sequentially, or it may include steps (b) and (a) performed sequentially. For example, the mention that the method may also include step (c) indicates that step (c) may be added to the method in any order. For example, the method may include steps (a), (b), and (c), or it may include steps (a), (c), and (b), or it may include steps (c), (a), and (b), etc.

[0032] Unless otherwise specified, the terms "comprising" and "including" as used in this application can be open-ended or closed-ended. For example, "comprising" and "including" can mean that other components not listed may also be included, or that only the listed components may be included.

[0033] Unless otherwise specified, the reaction will proceed under normal temperature and pressure conditions.

[0034] Unless otherwise specified, all parts or percentages are by weight or by weight percentage.

[0035] In this invention, all the substances used are known substances that can be purchased or synthesized by known methods.

[0036] In this invention, all the devices or equipment used are conventional devices or equipment known in the art and are readily available.

[0037] Example 1 A method for preparing fentanyl antigen, comprising the following steps: (1) The synthesis of fentanyl hapten, by mass, is as follows: (11) Take one copy of the structural formula as Compound 1 was dissolved in 80 parts of dichloromethane. 1.2 parts of triethylamine were added under stirring, and the mixture was stirred in an ice bath. 0.8 parts of chloroacetyl were then added dropwise, and the reaction was allowed to proceed overnight at room temperature. The reaction solution was then washed with water, and the organic phase was collected. The organic phase was dried and evaporated to dryness to obtain a solid product. This solid product was then purified by column chromatography to obtain the structural formula […]. Compound 2; (12) Dissolve compound 2 obtained in step (11) in 8 parts of dichloromethane, then add 9 parts of trifluoroacetic acid, stir at room temperature for 5 hours, evaporate the solvent to obtain the residue, add 30 parts of ethyl acetate to 40 parts of 3% sodium carbonate aqueous solution and stir evenly to obtain the second washing solution, add the residue to the second washing solution and wash twice with water, take the organic phase, then dry the organic phase to obtain the solid product, then purify the solid product by column chromatography to obtain the structure with the following formula: Compound 3; (13) Dissolve compound 3 obtained in step (12) in 15 parts of ethanol, then add 8 parts of triethylamine and 8 parts of ethyl bromobutyrate to it and reflux overnight to obtain a reaction solution. Add 30 parts of ethyl acetate to 35 parts of water and stir evenly to obtain a third washing solution. Place the reaction solution in the third washing solution and wash twice with water. Take the organic phase, dry and evaporate the organic phase to obtain a solid product. Then purify the solid product by column chromatography to obtain the structure with the following formula: Compound 4; (14) Dissolve compound 4 obtained in step (13) in 10 parts of an ethanol-water mixture, then add 0.8 parts of sodium hydroxide and hydrolyze for 5 hours. After the reaction is complete, adjust the pH of the reaction solution to 5 with dilute hydrochloric acid, then extract with ethyl acetate, take the organic phase, dry the organic phase to obtain a solid product, and then purify the solid product by column chromatography to obtain the structure of [structure not specified]. Fentanyl hapten; (2) The preparation of fentanyl antigen, by weight, is as follows: (21) Take 1 part of the fentanyl hapten obtained in step (1), 1.2 parts of N-hydroxysuccinimide (NHS), 1.8 parts of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), and 4 parts of N,N-dimethylformamide and stir rapidly at room temperature overnight. After the reaction is completed, an activation solution is obtained. (22) Stir 40 portions of PBS solution containing 10 mg / mL carrier protein under ice-water bath cooling, then slowly add the activation solution obtained in step (21) dropwise, react at room temperature overnight, and obtain the reaction solution after the coupling reaction is completed. (23) Transfer the reaction solution obtained in step (22) to PBS dialysate and dialyze 10 times at 4°C, with a time interval of 3 hours between two adjacent dialyses; (24) Transfer the dialyzed solution into a centrifuge tube and centrifuge at 6500 rpm for 5.5 minutes at 4°C. Take the supernatant to obtain fentanyl antigen.

[0038] Furthermore, in step (14), the volume ratio of ethanol to water in the ethanol-water mixture is 5:1.

[0039] Furthermore, the carrier protein mentioned in step (22) is BSA.

[0040] A method for preparing fentanyl antibodies, comprising the following steps: Nine-week-old, 30g female Balb / c mice were immunized with the fentanyl antigen at a dose of 100μg per dose, once a month for a total of four immunizations. Peripheral blood was collected from the mice after each immunization for screening. Positive B cells were fused with bone marrow hybridoma cells SP2 / 0, and fentanyl antibodies were obtained one week later by dilution.

[0041] A kit for detecting fentanyl, the kit comprising a colloidal gold conjugate pad and a detection line, wherein colloidal gold particles in the colloidal gold conjugate pad are labeled with the aforementioned fentanyl antibody, and the detection line is coated with the aforementioned fentanyl antigen.

[0042] Example 2 A method for preparing fentanyl antigen, comprising the following steps: (1) The synthesis of fentanyl hapten, by mass, is as follows: (11) Take one copy of the structural formula as Compound 1 was dissolved in 50 parts of dichloromethane. 1 part of triethylamine was added under stirring, and the mixture was stirred in an ice bath. 0.5 parts of chloroacetyl were then added dropwise, and the reaction was allowed to proceed overnight at room temperature. The reaction solution was then washed with water, and the organic phase was collected. The organic phase was dried and evaporated to dryness to obtain a solid product. This solid product was then purified by column chromatography to obtain the structural formula […]. Compound 2; (12) Dissolve compound 2 obtained in step (11) in 5 parts of dichloromethane, then add 7 parts of trifluoroacetic acid, stir at room temperature for 3 hours, evaporate the solvent to obtain the residue, add 20 parts of ethyl acetate to 20 parts of 1% sodium carbonate aqueous solution and stir evenly to obtain the second washing solution, add the residue to the second washing solution and wash once with water, take the organic phase, then dry the organic phase to obtain the solid product, then purify the solid product by column chromatography to obtain the structure with the following formula: Compound 3; (13) Dissolve compound 3 obtained in step (12) in 10 parts of ethanol, then add 5 parts of triethylamine and 5 parts of ethyl bromobutyrate to it and reflux overnight to obtain a reaction solution. Add 20 parts of ethyl acetate to 20 parts of water and stir evenly to obtain a third washing solution. Place the reaction solution in the third washing solution and wash once with water. Take the organic phase, dry and evaporate the organic phase to obtain a solid product. Then purify the solid product by column chromatography to obtain the structure with the following formula: Compound 4; (14) Dissolve compound 4 obtained in step (13) in 8 parts of an ethanol-water mixture, then add 0.4 parts of sodium hydroxide and hydrolyze for 3 hours. After the reaction is complete, adjust the pH of the reaction solution to 5 with dilute hydrochloric acid, then extract with ethyl acetate, take the organic phase, dry the organic phase to obtain a solid product, and then purify the solid product by column chromatography to obtain the structure of [structure not specified]. Fentanyl hapten; (2) The preparation of fentanyl antigen, by weight, is as follows: (21) Take 1 part of the fentanyl hapten obtained in step (1), 1 part of N-hydroxysuccinimide (NHS), 1.5 parts of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), and 3 parts of N,N-dimethylformamide and stir rapidly at room temperature overnight. After the reaction is completed, an activation solution is obtained. (22) Stir 35 portions of PBS solution containing 8 mg / mL carrier protein under ice-water bath cooling, then slowly add the activation solution obtained in step (21) to it, and react at room temperature overnight to complete the coupling reaction and obtain the reaction solution. (23) Transfer the reaction solution obtained in step (22) to PBS dialysate and dialyze 7 times at 2°C, with a time interval of 2 hours between two adjacent dialyses; (24) Transfer the dialyzed solution into a centrifuge tube and centrifuge at 6000 rpm for 6 minutes at 2°C. Take the supernatant to obtain fentanyl antigen.

[0043] Furthermore, in step (14), the volume ratio of ethanol to water in the ethanol-water mixture is 4:1.

[0044] Furthermore, the carrier protein mentioned in step (22) is KLH.

[0045] A method for preparing fentanyl antibodies, comprising the following steps: Nine-week-old, 25g female Balb / c mice were immunized with the fentanyl antigen at a dose of 100μg per dose, once a month for a total of four immunizations. Peripheral blood was collected from the mice after each immunization for screening. Positive B cells were fused with bone marrow hybridoma cells SP2 / 0, and fentanyl antibodies were obtained one week later by dilution.

[0046] A kit for detecting fentanyl, the kit comprising a colloidal gold conjugate pad and a detection line, wherein colloidal gold particles in the colloidal gold conjugate pad are labeled with the aforementioned fentanyl antibody, and the detection line is coated with the aforementioned fentanyl antigen.

[0047] Example 3 A method for preparing fentanyl antigen, comprising the following steps: (1) The synthesis of fentanyl hapten, by mass, is as follows: (11) Take one copy of the structural formula as Compound 1 was dissolved in 100 parts of dichloromethane. 1.5 parts of triethylamine were added under stirring, and the mixture was stirred in an ice bath. 1 part of chloroacetyl was then added dropwise, and the reaction was allowed to proceed overnight at room temperature. The reaction solution was then washed with water, and the organic phase was collected. The organic phase was dried and evaporated to dryness to obtain a solid product. This solid product was then purified by column chromatography to obtain the structural formula […]. Compound 2; (12) Dissolve compound 2 obtained in step (11) in 10 parts of dichloromethane, then add 10 parts of trifluoroacetic acid, stir at room temperature for 6 hours, evaporate the solvent to obtain the residue, add 20-40 parts of ethyl acetate to 50 parts of 5% sodium carbonate aqueous solution and stir evenly to obtain the second washing solution, add the residue to the second washing solution and wash with water 3 times, take the organic phase, then dry the organic phase to obtain the solid product, then purify the solid product by column chromatography to obtain the structure with the following formula: Compound 3; (13) Dissolve compound 3 obtained in step (12) in 20 parts of ethanol, then add 10 parts of triethylamine and 10 parts of ethyl bromobutyrate to it and reflux overnight to obtain a reaction solution. Add 40 parts of ethyl acetate to 20-50 parts of water and stir evenly to obtain a third washing solution. Place the reaction solution in the third washing solution and wash it three times with water. Take the organic phase, dry and evaporate it to obtain a solid product. Then purify the solid product by column chromatography to obtain the structure with the following formula: Compound 4; (14) Dissolve compound 4 obtained in step (13) in a mixture of 15 parts ethanol and water, then add 1 part sodium hydroxide and hydrolyze for 6 hours. After the reaction is complete, adjust the pH of the reaction solution to 5 with dilute hydrochloric acid, then extract with ethyl acetate, take the organic phase, dry the organic phase to obtain a solid product, and then purify the solid product by column chromatography to obtain the structure of [structure not specified]. Fentanyl hapten; (2) The preparation of fentanyl antigen, by weight, is as follows: (21) Take 1 part of the fentanyl hapten obtained in step (1), 1.5 parts of N-hydroxysuccinimide (NHS), 2 parts of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), and 5 parts of N,N-dimethylformamide and stir rapidly at room temperature overnight. After the reaction is completed, an activation solution is obtained. (22) Stir 50 portions of PBS solution containing 12 mg / mL carrier protein under ice-water bath cooling, then slowly add the activation solution obtained in step (21) dropwise, react at room temperature overnight, and obtain the reaction solution after the coupling reaction is completed. (23) Transfer the reaction solution obtained in step (22) to PBS dialysate and dialyze 10 times at 8°C, with a time interval of 3 hours between two adjacent dialyses; (24) Transfer the dialyzed solution into a centrifuge tube and centrifuge at 7000 rpm for 5 minutes at 8°C. Take the supernatant to obtain fentanyl antigen.

[0048] Furthermore, in step (14), the volume ratio of ethanol to water in the ethanol-water mixture is 6:1.

[0049] Furthermore, the carrier protein mentioned in step (22) is BSA.

[0050] A method for preparing fentanyl antibodies, comprising the following steps: Nine-week-old, 35g female Balb / c mice were immunized with the fentanyl antigen at a dose of 100μg per dose, once a month for a total of four immunizations. Peripheral blood was collected from the mice after each immunization for screening. Positive B cells were fused with bone marrow hybridoma cells SP2 / 0, and fentanyl antibodies were obtained one week later by dilution.

[0051] A kit for detecting fentanyl, the kit comprising a colloidal gold conjugate pad and a detection line, wherein colloidal gold particles in the colloidal gold conjugate pad are labeled with the aforementioned fentanyl antibody, and the detection line is coated with the aforementioned fentanyl antigen.

[0052] Test Example 1: 1. The labeling of the fentanyl antibody prepared in Example 1 is as follows: (11) Take six clean 2ml PE tubes and mark them as 1, 2, 3, 4, 5, 6. Clean the PE tubes with DD water and add 1ml of gold solution to each tube. (12) Add 0.1M potassium carbonate to the six groups of PE pipes respectively, with addition amounts of 2ul, 4ul, 6ul, 8ul, 10ul, and 12ul. (13) Take 60ug of the antibody prepared in Example 1, add it according to the labeling amount, vortex and let stand for 10min; (14) Take 10% BSA, add 60 μL of each, vortex and let stand for 5 min; (15) Take 10% PEG, add 60ul to each, vortex to obtain a mixture; (16) The mixture obtained in step (5) is purified by centrifugation using a high-speed refrigerated centrifuge, and the steps are as follows: (161) Centrifuge at 10000 RPM, 4℃, for 5-8 min; (161) Suspend the precipitate, discard the supernatant, and then add 1 ml of the reconstituted solution and mix well; 2. Detection of gold-labeled antibodies using a spectrophotometer, including: Use the diluent as a blank control; The absorbance of the gold-labeled antibody was scanned between 500 and 550 nm, and the peak value changed by 3-5 nm after antibody labeling. Note: Labeling solutions should be used immediately after labeling and should not be stored for long periods.

[0053] 3. Pretreatment of colloidal gold pads and sample pads (31) Determine the quantity to be processed, A4 paper (or cut into other sizes), stack 10 sheets at a time.

[0054] (32) Prepare the buffer solution (approximately 30 ml / A4 paper size). Due to slight differences in material thickness and performance, the volume of the treatment solution needs to be adjusted flexibly. Please prepare the treatment solution formula according to the specific product. Basic elements: buffer + NaCl + others.

[0055] (33) Wearing gloves, immerse the material completely in the buffer solution for 10 minutes, then remove it and dehydrate for 3 minutes, and place it flat on a sieve. (34) Place it in a 37℃ drying oven (humidity less than 20%) and dry it until the humidity is constant at around 20%.

[0056] (35) After drying, pack the material into aluminum foil bags or plastic bags and seal them for storage. Label the bags and record the batch number. Note that the treated glass fiber should not be exposed to a high humidity environment for a long time.

[0057] 4. Spotting (41) Colloidal gold sputtering point to colloidal gold pad (411) Dilute the labeled colloidal gold to 5 OD / ul (the concentration can be adjusted by yourself), and add (final concentration): 10-20% sucrose, 1-3% BSA, 0.5-1% NaCl, and 0.5-1% surfactant. (412) Under the conditions of 20-40% humidity and room temperature, the solution was sprayed onto the colloidal gold pad using the AIRJET nozzle of the BIODOT instrument, with the pressure set to 10 PSI and the spray point parameters being 2-6 ul / cm.

[0058] (413) After the sprayed colloidal gold strip is dried at 37°C and the humidity is kept constant at 20%, it is collected and sealed for storage.

[0059] (414) When using, simply cut the entire strip. Note that the edges at both ends of the gold strip should be discarded due to edge effect.

[0060] (42) Antigen / antibody spraying onto NC membrane (421) Remove the NC membrane and equilibrate it for 1 hour at room temperature and normal humidity.

[0061] (422) Dilute the antigen / antibody to a suitable concentration (0.1-5 mg / ml) with a buffer solution. Buffer systems may include phosphate, Tris, or carbonate; pH range: 6.0-9.0 (depending on the isoelectric point). (423) Using the BIODOT instrument, set the spray volume to 0.5-1ul / cm and spot the sample.

[0062] (424) Dry in an oven at 37 degrees Celsius for 2 hours until the humidity is below 20%, then seal and package for later use.

[0063] Note: No treatment is required before spotting the NC membrane.

[0064] For testing, fentanyl standards of different concentrations were diluted from 1 mg / ml to 100 ng / ml, 10 ng / ml, 5 ng / ml, and 2 ng / ml. Results are shown in [the table below]. Figure 2 .

[0065] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention.

Claims

1. A method for preparing fentanyl antigen, characterized in that, The steps are as follows: (1) The synthesis of fentanyl hapten, by mass, is as follows: (11) Take one copy of the structural formula as Compound 1 was dissolved in 50-100 parts of dichloromethane. 1-1.5 parts of triethylamine were added under stirring, and the mixture was stirred in an ice bath. 0.5-1 parts of chloroacetyl were added dropwise, and the reaction was allowed to proceed overnight at room temperature to obtain a reaction solution. The reaction solution was then washed with water, and the organic phase was collected. The organic phase was dried and evaporated to dryness to obtain a solid product. This solid product was then purified by column chromatography to obtain the structural formula […]. Compound 2; (12) Dissolve compound 2 obtained in step (11) in 5-10 parts of dichloromethane, then add 7-10 parts of trifluoroacetic acid, stir at room temperature for at least 3 hours, evaporate the solvent to obtain the residue, add 20-40 parts of ethyl acetate to 20-50 parts of sodium carbonate aqueous solution with a mass concentration of 1%-5% and stir evenly to obtain a second washing solution, add the residue to the second washing solution and wash with water at least once, take the organic phase, then dry the organic phase to obtain a solid product, then purify the solid product by column chromatography to obtain the structure with the following formula: Compound 3; (13) Dissolve compound 3 obtained in step (12) in 10-20 parts of ethanol, then add 5-10 parts of triethylamine and 5-10 parts of ethyl bromobutyrate to it and reflux overnight to obtain a reaction solution. Add 20-40 parts of ethyl acetate to 20-50 parts of water and stir evenly to obtain a third washing solution. Place the reaction solution in the third washing solution and wash with water at least once. Take the organic phase, dry and evaporate the organic phase to obtain a solid product. Then purify the solid product by column chromatography to obtain the structure with the following formula: Compound 4; (14) Dissolve compound 4 obtained in step (13) in 8-15 parts of an ethanol-water mixture, then add 0.4-1 parts of sodium hydroxide and hydrolyze for at least 3 hours. After the reaction is complete, adjust the pH of the reaction solution to 5 with dilute hydrochloric acid, then extract with ethyl acetate, take the organic phase, dry and evaporate the organic phase to obtain a solid product, and then purify the solid product by column chromatography to obtain the structure with the following formula: Fentanyl hapten; (2) Preparation of fentanyl antigen, by weight, the specific steps are as follows: (21) Take 1 part of the fentanyl hapten obtained in step (1), 1-1.5 parts of N-hydroxysuccinimide, 1.5-2 parts of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and 3-5 parts of N,N-dimethylformamide and stir rapidly at room temperature overnight. After the reaction is completed, an activation solution is obtained. (22) Stir 35-50 parts of PBS solution of carrier protein with a concentration of 8-12 mg / mL under ice water bath cooling, then slowly add the activation solution obtained in step (21) dropwise, react at room temperature overnight, and obtain the reaction solution after the coupling reaction is completed. (23) Transfer the reaction solution obtained in step (22) to PBS dialysate and dialyze at 2-8℃ for more than 7 times, with an interval of not less than 2 hours between two adjacent dialyses; (24) Transfer the dialyzed solution into a centrifuge tube and centrifuge at 6000-7000 rpm for 5-6 minutes at 2-8℃. Take the supernatant to obtain fentanyl antigen.

2. The method for preparing fentanyl antigen as described in claim 1, characterized in that, The volume ratio of ethanol to water in the ethanol-water mixture in step (14) is (4-6):

1.

3. The method for preparing fentanyl antigen as described in claim 1, characterized in that, The carrier protein mentioned in step (22) is one of BSA or KLH.

4. A method for preparing fentanyl antibodies, characterized in that, The steps are as follows: Nine-week-old female Balb / c mice weighing 25-35g were immunized with the fentanyl antigen described in any one of claims 1-3 at a dose of 100μg per dose. The mice were immunized once a month for a total of four immunizations. Peripheral blood of the mice was collected after each immunization for screening. Positive B cells were fused with bone marrow hybridoma cells SP2 / 0. Fentanyl antibodies were obtained one week later by dilution.

5. A reagent kit, characterized in that, For the detection of fentanyl, the kit includes a colloidal gold conjugate pad and a test line, wherein the colloidal gold particles in the colloidal gold conjugate pad are labeled with the fentanyl antibody of claim 4, and the test line is coated with the fentanyl antigen of any one of claims 1-3.

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