Application of protein coupling antigen in preparation of anti-fentanyl monoclonal antibody

Abstract

The invention discloses application of a protein coupling antigen with a structure shown as a formula (I) in preparation of an anti-fentanyl monoclonal antibody, and the prepared anti-fentanyl monoclonal antibody has extremely high affinity to fentanyl and can well recognize most fentanyl analogues; meanwhile, an enzyme-linked immunosorbent assay and a colloidal gold immunochromatography method established on the basis of the antibody can be used for preparing kits with extremely high detection sensitivity, and qualitative or quantitative detection of fentanyl and analogues thereof in biological samples such as hair and urine is realized.

Description

Technical Field

[0001] This invention relates to the field of antibodies, and more particularly to the application of a protein-coupled antigen in the preparation of anti-fentanyl monoclonal antibodies. Background Technology

[0002] Novel synthetic opioids (NSOs) are a new class of drugs belonging to the novel psychoactive substances (NPS) category. They mainly include fentanyl and non-fentanyl analogues. These molecules possess pharmacological properties different from traditional opioids. Overdose can rapidly cause respiratory depression and may be life-threatening; long-term use may lead to neurotoxicity. Developing simple, rapid, and low-cost methods for the rapid detection of fentanyl and its analogues is of great significance for combating drug crimes and maintaining public safety.

[0003] Rapid detection methods based on immunoassay technology have become important tools for on-site screening due to their convenience, speed, and high specificity. In law enforcement or emergency medical situations, these methods can quickly provide a preliminary assessment of the presence of fentanyl-related substances. However, because these methods rely on the specific recognition of the target by antibodies, they have significant limitations. The primary problem is insufficient broad-spectrum recognition. Antibodies are typically designed to target specific structural sites of fentanyl; when illicit molecules modify key sites such as the phenethyl, piperidine ring, or amide side chain, the original antibodies may fail to recognize them, leading to false negatives. Furthermore, the complex mixtures in illicit drugs can also interfere with the detection process.

[0004] Although patent CN202210146811.7 discloses a fentanyl hapten modified with a propionyl site, it only clarifies that the hapten can be used to prepare drugs for treating opioid abuse and opioid use disorder, and does not involve its related applications in the preparation of anti-fentanyl monoclonal antibodies and detection reagents. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide a protein-coupled antigen for the preparation of a high-affinity anti-fentanyl monoclonal antibody and the application of the antibody or a kit containing the antibody in the qualitative or quantitative detection of fentanyl and its analogues in biological samples.

[0006] Technical solution: The application of the protein-coupled antigen with the structure shown in formula (I) of this invention in the preparation of anti-fentanyl monoclonal antibodies. Formula (I), The protein is selected from any one of bovine serum albumin, keyhole hemocyanin, and ovalbumin.

[0007] Preferably, the application steps include: (1) Immunize animals with protein-coupled antigens and collect spleen cells after immunization; (2) The spleen cells obtained in step 1 were fused with myeloma cells and cultured to obtain hybridoma cells; (3) The hybridoma cells obtained in step 2 were used to prepare anti-fentanyl monoclonal antibodies by in vivo induction method.

[0008] Preferably, step 1 specifically involves: immunizing the animal for the first time using Freund's complete adjuvant mixed with the protein-coupled antigen; subsequently immunizing the animal multiple times using Freund's incomplete adjuvant mixed with the protein-coupled antigen; and finally immunizing the animal with the protein-coupled antigen, followed by collection of spleen cells.

[0009] Preferably, the animal is a mouse; more preferably, when immunizing with Freund's complete adjuvant mixed with the protein-coupled antigen, the dose of the protein-coupled antigen is 50-100 μg / mouse; when immunizing with Freund's incomplete adjuvant mixed with the protein-coupled antigen, the dose of the protein-coupled antigen is 50-100 μg / mouse; when immunizing with the protein-coupled antigen alone, the dose is 100-200 μg / mouse.

[0010] The anti-fentanyl monoclonal antibody described in this invention is obtained from hybridoma cells prepared after immunizing animals with a protein-coupled antigen as shown in formula (I).

[0011] The kit or test reagent of the present invention contains the aforementioned anti-fentanyl monoclonal antibody; more preferably, the kit or test reagent also contains a protein-coupled antigen with the structure shown in formula (I).

[0012] The application of the anti-fentanyl monoclonal antibody, kit, or detection reagent described in this invention in detecting fentanyl and its analogues in samples.

[0013] Preferably, the application is for the qualitative or quantitative detection of fentanyl and its analogues in a sample; more preferably, the detection method is enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay (CLIA), immunochromatography (such as test strips), radioimmunoassay (RIA), or immunofluorescence assay.

[0014] Preferably, the sample includes biological samples or non-biological samples; more preferably, the biological sample includes hair, urine, serum, saliva or sweat; the non-biological sample includes environmental swabs, environmental water samples, powder or food.

[0015] Preferably, the fentanyl analogues include acetylfentanyl, benzylfentanyl, benzoylfentanyl, thiofentanyl, β-hydroxyfentanyl, N-methylfentanyl, benzylfuran fentanyl, 3'-methylfentanyl, 2'-methylfentanyl, 4'-methylfentanyl, norfentanyl, butyryl fentanyl, isobutyryl fentanyl, acryl fentanyl, butenoyl fentanyl, tetrahydrofuran fentanyl, cyclopropanyl fentanyl, methoxyacetylfentanyl, 4-fluoroisobutyryl fentanyl, p-fluorofentanyl, o-fluorofentanyl, m-fluorofentanyl, p-methoxyfentanyl, m-methylfentanyl, o-methylfentanyl, p-methylfentanyl, valerate fentanyl, 4-methoxyfuran fentanyl, 2-methylfentanyl, 3-furan fentanyl, or furanyl fentanyl.

[0016] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The anti-fentanyl monoclonal antibody prepared by protein-coupled antigen with the structure shown in formula (I) has extremely high affinity for fentanyl and can well recognize most fentanyl analogues. The enzyme-linked immunosorbent assay and colloidal gold immunochromatography method established based on the anti-fentanyl monoclonal antibody can be used to prepare kits with extremely high detection sensitivity to achieve qualitative or quantitative detection of fentanyl and its analogues in biological samples such as hair and urine. Attached Figure Description

[0017] Figure 1 Hapten, a fentanyl hapten compound Fent of 1 H NMR spectrum; Figure 2 Hapten, a fentanyl hapten compound Fent of 13 C NMR spectrum; Figure 3 The graph shows the antibody titer and affinity assay results for mice immunized with protein-conjugated antigens at 21 and 35 days. Figure 4 This is a standard curve for the determination of fentanyl solution using a competitive ELISA method based on the anti-fentanyl monoclonal antibody AA8. Figure 5 Standard curves for determining fentanyl levels in urine (A) and hair (B) using a competitive ELISA method based on the anti-fentanyl monoclonal antibody AA8; Figure 6 A schematic diagram (top) and a standard curve (bottom) of a test strip for the determination of fentanyl solution based on the AA8 anti-fentanyl monoclonal antibody colloidal gold immunochromatographic assay. Figure 7 The results of determining fentanyl content in urine (A) and hair (B) using colloidal gold immunochromatography based on anti-fentanyl monoclonal antibody AA8 are shown in the figure. Figure 8The image shows the results of cross-reactivity testing of fentanyl, fentanyl analogues and common drugs at a concentration of 1 μg / mL using colloidal gold immunochromatography based on the anti-fentanyl monoclonal antibody AA8. Figure 9 The image shows the results of cross-reactivity testing of fentanyl, fentanyl analogues, and common drugs at a concentration of 100 ng / mL using colloidal gold immunochromatography based on the anti-fentanyl monoclonal antibody AA8. Detailed Implementation

[0018] The technical solution of the present invention will be further described below.

[0019] The fentanyl and its analogues, as well as common narcotics, used in the following examples were provided by the Joint Laboratory for Key Technologies of Narcotics Control of the National Narcotics Control Commission Office and China Pharmaceutical University; all other materials and reagents used, unless otherwise specified, were commercially available. Experimental methods not specifically described in the examples were generally performed under standard conditions or as recommended by the manufacturer.

[0020] Example 1: Preparation of protein-coupled antigens 1. Synthesis of haptens

[0021] Weigh N-phenylethyl-4-piperidinone (200 mg, 0.98 mmol), aniline (91 mg, 0.98 mmol), and glacial acetic acid (59 mg, 0.98 mmol) into a reaction flask. Add 20 mL of CH2Cl2, stir at room temperature, and add sodium triacetoxyborohydride (209 mg, 0.98 mmol). Monitor the reaction by thin-layer chromatography (TLC) until complete. Wash the reaction solution successively with saturated sodium bicarbonate solution and saturated brine. Collect the organic phase and purify by column chromatography to obtain intermediate 1.

[0022] Weigh intermediate 1 (100 mg, 0.36 mmol), add 10 mL of CH2Cl2, add two drops of glacial acetic acid, add glutaric anhydride (123 mg, 1.08 mmol), and react at room temperature to generate intermediate 2.

[0023] Weigh intermediate 2 (205 mg, 0.52 mmol), EDCI (151 mg, 0.79 mmol), and HOBt (106 mg, 0.79 mmol) into a reaction flask. Add 5 mL of DMF dissolved in the solution and stir in an ice bath for 0.5 h. Then add methyl 4-aminobutyrate hydrochloride (97 mg, 0.63 mmol) and DIPEA (203 mg, 1.57 mmol). Monitor the reaction by TLC until it is complete. After post-processing and column chromatography, intermediate 3 is obtained.

[0024] Intermediate 3 was dissolved in 5 mL of methanol, and saturated potassium hydroxide solution was added. The reaction was monitored by TLC until the reactants reacted completely. Residual methanol was removed using a rotary evaporator, and the solid was dissolved in an appropriate amount of pure water. The pH of the reaction solution was adjusted to 4-5 with 1 M HCl, at which point a large amount of solid precipitated. After filtration and drying, the solid was obtained as the hapten product. Fent .

[0025] Prepared Hapten Fent The proton and carbon spectra are as follows: 1 H NMR (500 MHz, DMSO- d 6) δ 12.08(s, 1H), 7.71 (t, J = 5.1 Hz, 1H), 7.50 (dq, J = 14.2, 7.2 Hz, 3H), 7.35 (t, J =7.4 Hz, 2H), 7.26 (d, J = 7.5 Hz, 5H), 4.76 (t, J = 11.7 Hz, 1H), 3.58 (d, J = 11.2Hz, 2H), 3.19 (d, J = 39.0 Hz, 4H), 2.96 (dq, J = 24.5, 7.7, 7.1 Hz, 4H), 2.18(t, J = 7.4 Hz, 2H), 2.02 – 1.92 (m, 4H), 1.85 (t, J = 7.3 Hz, 2H), 1.67 (p, J =7.1 Hz, 2H), 1.55 (dq, J = 19.4, 12.0, 9.4 Hz, 4H). 13 C NMR (126 MHz, DMSO- d 6) δ174.68, 172.04, 171.62, 138.56, 137.39, 130.88, 130.00, 129.16, 129.05,127.35, 57.01, 51.69, 49.30, 38.28, 34.96, 34.10, 31.60, 30.12, 25.15, 21.47.HRMS (ESI) m / z: [M+H] +calculated=480.2862; found=480.2797.

[0026] 2. Synthesis of protein-coupled antigens Weigh out 5 mg of the hapten compound Hapten Fent 12 mg EDCI and 13.6 mg NHS were dissolved in 500 μL DMF, and 8.68 μL Et3N was added. The mixture was stirred at room temperature for 6 h until the hapten reaction was complete. Then, 2 mL of 5 mg / mL keyhole hemocyanin (Solarbio, catalog number K8160)-PBS solution was added, and the mixture was stirred overnight at 4°C. The solution was then transferred to a dialysis bag with a molecular weight cutoff of 3000 Da and dialyzed with 500 mL PBS (pH 7.4) for 4-6 h each time, for a total of 4 dialysis cycles, to obtain the keyhole hemocyanin-conjugated antigen KLH-Hapten. Fent The structure is shown in equation (II).

[0027] Equation (II).

[0028] Based on the above method, 2 mL of bovine serum albumin-PBS solution (5 mg / mL) or 2 mL of ovalbumin-PBS solution (5 mg / mL) was added for reaction, and the bovine serum albumin-conjugated antigen BSA-Hapten was finally obtained. Fent The structure is shown in formula (III); and the ovalbumin-conjugated antigen OVA-Hapten Fent The structure is shown in equation (IV).

[0029] Formula (III);

[0030] Formula (IV).

[0031] Example 2: Preparation of anti-fentanyl monoclonal antibody 1. Mouse immunization KLH-Hapten was injected subcutaneously at three points on the back of 6-week-old female Balb / c mice. Fent To administer immunizations.

[0032] Freund's complete adjuvant (purchased from Sigma-Aldrich Trading Ltd., catalog number F5581) was used with 100 μg KLH-Hapten Fent After emulsification to a constant volume, the first immunization was performed; two weeks later, Freund's incomplete adjuvant (purchased from Sigma-Aldrich Trading Ltd., catalog number F5506) and 100 μg KLH-Hapten were administered. FentEmulsify to an equal volume and administer a second booster immunization; administer a third booster immunization two weeks after the second booster immunization, using the same method as the second booster immunization.

[0033] Blood was collected from the orbital region of mice on days 21 and 35 after the initial immunization. The serum was collected by centrifugation at 6000 rpm and 4 ℃, then aliquoted and frozen at -80℃ for later use.

[0034] 2. Evaluation of immune response 2.1 Serum titer determination Coating: Dilute the BSA-Hapten prepared in Example 1 with coating solution Fent Add 100 μL of the solution to 1 μg / mL in a 96-well microplate, seal with a sealing film, and incubate overnight at 4°C.

[0035] The coating solution was formulated as follows: KCl 0.2 g, KH2PO4 0.24 g, Na2HPO4·12H2O 3.63 g, NaCl 8 g, and ddH2O was added to bring the volume to 1 L. The pH was 7.4. Blocking: After washing the plate three times with washing solution, add 100 μL / well of blocking solution and block at 37℃ for 1 h. The washing solution is a coating solution containing 0.5% Tween-20 and the blocking solution is a coating solution containing 5% (w / v) skim milk powder. Primary antibody incubation: After blocking, wash the plate 5 times with washing buffer, and add 100 μL / well of serum from immunized mice or unimmunized mice obtained on day 21 or day 35 after the initial immunization obtained in step 1. The dilution gradients include 1:1000, 3000, 9000, 27000, 81000, 243000, 729000, and 2187000. Incubate at 37 °C for 1 h. Secondary antibody incubation: After primary antibody incubation, wash the plate 5 times with washing buffer, add 100 μL / well of HRP-labeled goat anti-mouse secondary antibody diluted 1:5000, and incubate at 37℃ for 30 min; Color development: After the secondary antibody incubation, wash the plate 5 times with washing buffer, add 50 μL / well of TMB color development solution (purchased from Beyotime Biotechnology Co., Ltd., catalog number P0209), and incubate at 37°C for 10 min in the dark. Termination: Add 50 μL / well of 2 M H2SO4 to terminate the colorimetric reaction. Measure the absorbance (OD) at 450 nm using a microplate reader. 450 ).

[0036] 2.2 Fentanyl Affinity Determination The competitive affinity assay was performed based on the aforementioned method for serum titer determination, with the difference being that the primary antibody incubation step was replaced by adding 50 μL OD to each well. 450Diluted serum with a concentration in the range of 1 to 1.5 was incubated with 50 μL of serially diluted fentanyl solution at fentanyl concentration gradients of 0, 0.05, 0.20, 0.78, 3.12, 12.50, 50, and 200 μM.

[0037] As shown in Figure 3, the serum antibody titers produced after the second immunization (day 21) and the third immunization (day 35) both reached approximately 10. 6 The above indicates that the antigens have good immunization effects and can be used to produce monoclonal antibodies; the affinity of the serum antibodies produced by this group of antigens on day 35 for fentanyl, i.e., IC50, is... 50 The value ranges from 168 to 585 nM, indicating that this group of antigens can produce high-affinity monoclonal antibodies.

[0038] 3. Preparation of anti-fentanyl monoclonal antibodies 3.1 Preparation, screening, and monoclonalization of hybridomas Selecting serum antibodies and fentanyl IC 50 The mouse with the best IC value (IC 50 =168 nM), and a final pulse immunization was performed 3 days before fusion: an intraperitoneal injection of 150 μg of the KLH-Hapten immunogen without adjuvant. Fent Three days later, the mice were sacrificed, disinfected by soaking in 75% alcohol, and the spleens were removed under aseptic conditions. The spleen cells were then squeezed, ground, and counted.

[0039] SP2 / 0 myeloma cells in logarithmic growth phase were fused with spleen cells at a cell ratio of 1:5. The steps were as follows: After mixing spleen cells with SP2 / 0 myeloma cells, the mixture was centrifuged at 1000 rpm for 10 min. The cell mixture pellet was placed in a 37°C water bath and 1 mL of PEG1450 was added dropwise over 30 s. After the addition was complete, the mixture was allowed to stand for 30 s. Then, 1 mL of 37°C preheated 1640 medium was added dropwise at a rate of 1 mL per min, 2 mL per min, 3 mL per min, and 4 mL per min. The mixture was then allowed to stand for 10 min, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and HAT complete medium was added. The cell suspension was seeded at 200 μL / well into 96-well cell culture plates for culture.

[0040] On day 3 after fusion, a partial medium change was performed using HT complete medium; a complete medium change was performed on day 7. On the second day, a small amount of cell culture supernatant was taken, diluted, and measured according to the method described in step 2.1. The difference was that the primary antibody incubation step was changed to adding 50 μL of 16-fold diluted cell supernatant and 50 μL of 400 nM fentanyl solution to each well for incubation. Cell wells that showed an inhibition rate of 90% at a concentration of 400 nM fentanyl were selected, and monoclonalization was performed on these wells using limiting dilution until a monoclonal cell line stably expressing fentanyl-specific antibody was obtained.

[0041] 3.2 Preparation of anti-fentanyl monoclonal antibodies by in vivo induction method Female Balb / c mice aged 8-9 weeks were selected, and 0.5 mL of sterile liquid paraffin was injected intraperitoneally. One week later, 600 μL of a 10% concentration was added. 6 Hybridoma cells obtained from the aforementioned screening at a rate of [number] cells / mL were injected into the peritoneal cavity of mice to induce peritoneal tumors and ascites. One week later, mice were sacrificed and the ascites was collected. After centrifugation to remove the supernatant, the supernatant was discarded, and the supernatant was collected. The supernatant was diluted 3-fold with equilibration washing buffer (0.15 M NaCl, 20 mM Na2HPO4, pH=7.0) and loaded onto a Protein A affinity column (purchased from Changzhou Tiandi Renhe Biotechnology Co., Ltd., catalog number SA012005). This was repeated 5 times, with the column allowed to stand for 2 h after loading. Then, 5 column volumes of equilibration washing buffer were used to elute contaminating proteins from the affinity column. 2 mL of elution buffer (0.1 M glycine, pH=2.7) was added to elute the antibody from the affinity column. 200 μL of neutralization buffer (1 M Tris-HCl, pH=8.5) was added to each collection tube beforehand to adjust the solution to neutral, yielding an anti-fentanyl monoclonal antibody AA8 solution. This solution was then subjected to 50 [units of measurement - missing from original text]. kD ultrafiltration tubes (purchased from Millipore, catalog number UFC905096) were stored in PBS solution after ultrafiltration concentration, and the concentration of anti-fentanyl monoclonal antibody AA8 in the solution was determined using NanoDrop.

[0042] Example 3: Establishment of a competitive ELISA method based on anti-fentanyl monoclonal antibody The specific operational steps of the competitive ELISA method include: Coating: Dilute the BSA-Hapten prepared in Example 1 with coating solution Fent Add 100 μL of the solution to a 96-well microplate to a concentration of 0.062 μg / mL, seal with a sealing film, and incubate overnight at 4°C.

[0043] Blocking: After washing the plate three times with washing solution, add 100 μL / well of blocking solution and block at 37℃ for 1.5 h; Primary antibody incubation: After blocking, wash the plate 5 times with washing buffer, and add 50 μL / well of the anti-fentanyl monoclonal antibody AA8 solution with a concentration of 31 ng / mL obtained in Example 2, and 50 μL / well of fentanyl solution with concentrations of 0, 0.020, 0.039, 0.078, 0.16, 0.31, 0.62, 1.25, 2.5, 5, 10, and 20 ng / mL or 50 μL / well of the sample solution to be tested, respectively, and incubate at 37 ℃ for 1 h; Secondary antibody incubation: After primary antibody incubation, wash the plate 5 times with washing buffer, add 100 μL / well of 1:5000 diluted HRP-labeled goat anti-mouse secondary antibody, and incubate at 37℃ for 30 min; Color development: After the secondary antibody incubation is complete, wash the plate 5 times with washing buffer, add 50 μL / well of TMB color development solution, and incubate at 37°C for 10 min in the dark; Termination: Add 50 μL / well of 2 M H2SO4 to terminate the colorimetric reaction, and use a microplate reader to detect the absorbance value at a wavelength of 450 nm.

[0044] The sample solution to be tested is either a urine sample solution or a hair sample solution.

[0045] The urine sample solution was prepared as follows: the actual urine sample (provided by the volunteer) was adjusted to pH 7.4 with 1 M NaOH solution, and then fentanyl was added at final concentrations of 0, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, and 10 ng / mL before use.

[0046] The hair sample solution was prepared as follows: 20 mg of cleaned and shredded blank hair (provided by laboratory personnel) was weighed and added to a grinding tube (purchased from Zhejiang Nuojia Biotechnology Co., Ltd.) containing 0.8 mL of hair lysis buffer and grinding beads. Fentanyl was added at final concentrations of 0, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, and 1 ng / mL. The sample was then ground in a hair grinding instrument for 10 min, centrifuged, and the supernatant was collected for use.

[0047] The results of the competitive ELISA detection of fentanyl solution are as follows: Figure 4 As shown, the standard curve IC based on AA8 monoclonal antibody 50 The effective concentration was 0.22 ng / mL, the LOD was 0.021 ng / mL, and the linear range was 0.051~0.99 ng / mL.

[0048] Competitive ELISA results for urine and hair samples are as follows: Figure 5 As shown, the standard curve IC50 based on AA8 monoclonal antibody in urine samples 50The effective concentration was 0.388 ng / mL, the LOD was 0.046 ng / mL, and the linear range was 0.101–1.484 ng / mL; the standard curve IC50 based on AA8 monoclonal antibody in hair samples was [data missing]. 50 The effective dose was 0.0077 ng / mg, the LOD was 0.001 ng / mg, and the linear range was 0.002~0.023 ng / mg.

[0049] Example 4: Establishment of a colloidal gold immunochromatographic method based on anti-fentanyl monoclonal antibody Step 1: Coating antigen (BSA-Hapten) Fent 0.8 mg / mL) and goat anti-mouse secondary antibody (purchased from Jackson Immuno Research Inc., catalog number 205-035-108) were applied to a nitrocellulose membrane (NC membrane) at a rate of 1 μL / cm using a gold-spraying membrane scribing instrument and dried at 37 °C, serving as the test line T and control line C, respectively. Step 2: Assemble the sample pad, NC membrane, and absorbent pad onto the PVC base plate in sequence, and cut them into 3 mm wide test strips; Step 3: Mix 50 μL of colloidal gold solution modified with anti-fentanyl monoclonal antibody AA8 and 50 μL of fentanyl solution with concentrations of 0, 0.05, 0.1, 0.2, 0.5, 1, 2, and 5 ng / mL or 50 μL of the sample solution to be tested in a 96-well plate and incubate for 2 min. Insert the test strip obtained in Step 2 into the microwell. Under the action of capillary siphon, the mixed solution moves along the chromatography direction. When it reaches the detection line T and the control line C, read the values ​​of the detection line and the control line using a colloidal gold reader TSR-100 (Hangzhou Aosheng Instrument Co., Ltd.). Calculate the ratio of the detection line to the control line T / C. Plot a standard curve using this ratio and the corresponding fentanyl concentration for detection.

[0050] The preparation method of the colloidal gold solution modified with anti-fentanyl monoclonal antibody AA8 is as follows: 2 mL of 1% trisodium citrate aqueous solution is rapidly added to 100 mL of boiling 0.01% HAuCl4 aqueous solution, and the mixture is stirred and boiled for 15 min to obtain a colloidal gold solution. The solution is then cooled to room temperature and stored at 4 °C. 1 mL of the colloidal gold solution is taken, and 5 μL of 0.1 mol / L K2CO3 solution and 4 μg of the anti-fentanyl monoclonal antibody AA8 obtained in Example 2 are added. The solution is then incubated at room temperature for 1 h. Bovine serum albumin dissolved in 0.01 M borate buffer (pH=8.2) to a final concentration of 10 mg / mL was added to the above mixture and incubated for 1 h. After centrifugation at 10000 rpm for 15 min, the precipitate was resuspended in 100 µL borate buffer containing 2% bovine serum albumin and 3% sucrose to obtain the gold-labeled antibody stock solution. The gold-labeled antibody stock solution was mixed with PBS at a ratio of 3:47 (v:v) to obtain a colloidal gold solution modified with anti-fentanyl monoclonal antibody AA8.

[0051] The sample solution to be tested is either a urine sample solution or a hair sample solution. The urine sample solution is prepared as follows: the pH of the actual urine sample is adjusted to 7.4 using 1 M NaOH solution, and fentanyl is added at final concentrations of 0, 0.05, 0.1, 0.2, 0.5, 1, 2, and 5 ng / mL before use. The hair sample solution is prepared as follows: 20 mg of washed and shredded blank hair is weighed and added to a grinding tube containing 0.8 mL of hair lysis buffer and grinding beads. Fentanyl is added at final concentrations of 0, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, and 0.5 ng / mL, and then ground using a hair grinding instrument for 10 min. The supernatant is collected after centrifugation and used.

[0052] The results of colloidal gold immunochromatographic assay of fentanyl solution are as follows: Figure 6 As shown, the upper figure is a schematic diagram of the colloidal gold test strip after detection, and the lower figure is a standard curve of concentration-T / C value; the standard curve IC based on AA8. 50 The effective concentration was 0.39 ng / mL, the LOD was 0.073 ng / mL, and the linear range was 0.14~1.12 ng / mL.

[0053] Results of colloidal gold immunochromatographic assay of urine and hair are as follows Figure 7 As shown, the T / C value gradually decreases with increasing fentanyl concentration. The established colloidal gold test strip immunochromatographic assay can quantify fentanyl in urine and hair; the IC50 value of fentanyl in urine samples based on the AA8 standard curve... 50 The concentration was 0.14 ng / mL, the LOD was 0.016 ng / mL, and the linear range was 0.036–0.57 ng / mL; the standard curve IC50 based on AA8 monoclonal antibody in hair samples was [data missing].50 The effective dose was 0.012 ng / mg, the LOD was 0.0019 ng / mg, and the linear range was 0.0037~0.038 ng / mg.

[0054] Example 5: Cross-reactivity test 1. Cross-reactivity test based on competitive ELISA method The experimental method was the same as in Example 3, evaluating cross-reactivity with 36 fentanyl and its analogues and 6 common drugs (heroin, methamphetamine, phenamine, ketamine, JWH-018, and MDMB-FUBICA). The concentration gradients of the 36 fentanyl and 6 common drugs were set to 0, 0.68, 2.06, 6.17, 18.5, 55.6, 167, and 500 ng / mL.

[0055] Anti-fentanyl monoclonal antibody AA8 and its IC50 values ​​for 36 fentanyl and its analogues, as well as 6 common drugs. 50 The values ​​are shown in Table 1. AA8 has IC values ​​for remifentanil, alfentanil, sufentanil, carfentanil, and six other common drugs. 50 The values ​​were all greater than 1 μg / mL, indicating that it did not cross-react with the six common drugs and could identify 32 fentanyl-related substances other than remifentanil, alfentanil, sufentanil, and carfentanil.

[0056] Table 1. IC50 of anti-fentanyl monoclonal antibody AA8 against fentanyl and its analogues as well as common drugs. 50

[0057] Continued from the previous table

[0058] 2. Cross-reactivity test based on colloidal gold immunochromatography The experimental method is the same as in Example 9, except that the concentrations of 36 fentanyl and its analogues and 6 common drugs are 1 μg / mL or 100 ng / mL.

[0059] The results are shown in Figures 8 and 9. The colloidal gold immunochromatography method also showed that AA8 did not cross-react with the six common drugs (CR < 1%). The test strip based on AA8 could basically achieve the elimination line (i.e., inhibition rate > 50%) for 30 fentanyl-related substances, excluding p-methoxyfentanyl, p-methylfentanyl, remifentanyl, sufentanyl, 4-methoxyfuranfentanyl, and carfentanyl, at a competitive concentration of 1 μg / mL. The test strip based on monoclonal antibody AA8 could basically achieve the elimination line (i.e., inhibition rate > 50%) for 27 fentanyl-related substances, excluding 4'-methylfentanyl, nofentanyl, p-methoxyfentanyl, p-methylfentanyl, remifentanyl, alfentanyl, sufentanyl, 4-methoxyfuranfentanyl, and carfentanyl, at a competitive concentration of 100 ng / mL.

Claims

1. The application of a protein-coupled antigen with a structure as shown in formula (I) in the preparation of anti-fentanyl monoclonal antibodies. Equation (I), in, The protein is selected from any one of bovine serum albumin, keyhole hemocyanin, and ovalbumin.

2. The application according to claim 1, characterized in that, step include: (1) Immunize animals with protein-coupled antigens and collect spleen cells after immunization; (2) The spleen cells obtained in step 1 were fused with myeloma cells and cultured to obtain hybridoma cells; (3) The hybridoma cells obtained in step 2 were used to prepare anti-fentanyl monoclonal antibodies by in vivo induction method.

3. The application according to claim 2, characterized in that, Step 1 specifically involves: administering a first immunization to the animal using Freund's complete adjuvant mixed with the protein-coupled antigen; subsequently administering multiple immunizations to the animal using Freund's incomplete adjuvant mixed with the protein-coupled antigen; and finally immunizing the animal with the protein-coupled antigen, followed by the collection of spleen cells.

4. The application according to claim 2 or 3, characterized in that, The animal in question is a mouse.

5. The application according to claim 4, characterized in that, When immunizing with a mixture of Freund's complete adjuvant and protein-coupled antigen, the dose of protein-coupled antigen is 50-100 μg / animal; when immunizing with a mixture of Freund's incomplete adjuvant and protein-coupled antigen, the dose of protein-coupled antigen is 50-100 μg / animal; when immunizing with a mixture of protein-coupled antigen and protein-coupled antigen, the dose is 100-200 μg / animal.

6. An anti-fentanyl monoclonal antibody, characterized in that, The antibody is obtained from hybridoma cells prepared after immunizing animals with a protein-coupled antigen as shown in formula (I).

7. A reagent kit or detection reagent, characterized in that, The kit or test reagent contains the anti-fentanyl monoclonal antibody as described in claim 6.

8. The kit or detection reagent according to claim 7, characterized in that, The kit or test reagent also contains a protein-coupled antigen with a structure as shown in formula (I).

9. The use of the anti-fentanyl monoclonal antibody of claim 6 or the kit or detection reagent of any one of claims 7-8 in the detection of fentanyl and its analogues in a sample.

10. The application according to claim 9, wherein the fentanyl analogue comprises acetylfentanyl, benzylfentanyl, benzoylfentanyl, thiofentanyl, β-hydroxyfentanyl, N-methylfentanyl, benzylfuranfentanyl, 3'-methylfentanyl, 2'-methylfentanyl, 4'-methylfentanyl, norfentanyl, butyroylfentanyl, isobutyroylfentanyl, acryloylfentanyl, butenoylfentanyl, tetrahydrofuranfentanyl, cyclopropanylfentanyl, methoxyacetylfentanyl, 4-fluoroisobutyroylfentanyl, p-fluorofentanyl, o-fluorofentanyl, m-fluorofentanyl, p-methoxyfentanyl, m-methylfentanyl, o-methylfentanyl, p-methylfentanyl, valerate fentanyl, 4-methoxyfuranfentanyl, 2-methylfentanyl, 3-furanfentanyl, and furanylfentanyl.

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