Kit for detecting contents of fentanyl and analogues thereof and application of kit

Abstract

The invention discloses a kit for detecting the content of fentanyl and analogues thereof and application of the kit. The kit contains an anti-fentanyl monoclonal antibody and a fentanyl complete antigen. The enzyme linked immunosorbent assay kit and the test strip can be used for rapidly detecting fentanyl and analogues thereof in urine and hair samples so as to evaluate the content of fentanyl in the urine and hair samples and judge whether to suck fentanyl or not, and have the advantages of rapidness, simplicity, convenience, portability and the like. The enzyme linked immunosorbent assay kit and the test strip for rapid detection of fentanyl and analogues thereof, disclosed by the invention, have good accuracy and precision, can be used for broad-spectrum detection of 33 kinds of fentanyl, and provide a means suitable for on-site rapid detection for public security departments.

Description

Technical Field

[0001] The invention relates to a kit for detecting the content of fentanyl and its analogs and application thereof, belonging to the field of biomedicine. Background Art

[0002] Fentanyl is a very effective opioid analgesic, approximately 50-100 times more potent than morphine. Although fentanyl is commonly used for intraoperative analgesia, its abuse can turn it into a deadly poison. An overdose of fentanyl can cause drowsiness, confusion, and nausea, followed by addiction, hypotension, and ultimately respiratory depression and death. Fentanyl has a variety of analogs, such as carfentanil, sufentanil, and remifentanil, which have similar pharmacological activities. Recognizing the hazards of fentanyl and its analogs, the Chinese government classified the entire fentanyl class as non-medical narcotic drugs and psychotropic drugs in 2019. However, due to the regulation of the entire fentanyl class, there is an urgent need to develop a rapid detection method for the entire fentanyl class.

[0003] Although patents (2023110962083, 2023114143279) disclose different fentanyl compounds and fentanyl-specific antibodies thereto, different fentanyl antibodies have different recognition abilities, the fentanyl analogs that can be detected are limited, and the detection effect of fentanyl analogs in hair is not disclosed. Summary of the invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide a kit for detecting the content of fentanyl or its analogs in biological samples with high sensitivity and its application.

[0005] Technical solution: To solve the above technical problems, the present invention provides a kit for detecting the content of fentanyl or its analogs in biological samples, which contains an anti-fentanyl monoclonal antibody and a fentanyl complete antigen, wherein the anti-fentanyl monoclonal antibody comprises a light chain and a heavy chain; the nucleotide sequence of the variable region of the light chain is shown in SEQ ID NO.4; the nucleotide sequence of the variable region of the heavy chain is shown in SEQ ID NO.5; the fentanyl complete antigen is shown in Formula I:

[0006]

[0007]

[0008] Wherein, the protein is BSA or OVA; the analogs include acetylfentanyl, benzylfentanyl, benzoylfentanyl, thiofentanyl, β-hydroxyfentanyl, N-methylfentanyl, benzylfuranfentanyl, 3'-methylfentanyl, 2'-methylfentanyl, 4'-methylfentanyl, norfentanyl, butyrylfentanyl, isobutyrylfentanyl, acryloylfentanyl, crotonylfentanyl, tetrahydrofuran Fentanyl, ciprofentanyl, methoxyacetylfentanyl, 4-fluoroisobutyrylfentanyl, para-fluorofentanyl, o-fluorofentanyl, m-fluorofentanyl, p-methoxyfentanyl, m-methylfentanyl, o-methylfentanyl, p-methylfentanyl, valerylfentanyl, 4-methoxyfuranylfentanyl, 2-methylfentanyl, 3-furanylfentanyl, furanylfentanyl, or carfentanyl; biological specimens include hair, urine, serum, or saliva.

[0009] Wherein, the anti-fentanyl monoclonal antibody is obtained by co-culturing and purification of the full-length expression plasmid of the light chain of 9D5 and the full-length expression plasmid of the heavy chain of 9D5; the full-length expression plasmid of the light chain of 9D5 contains a signal peptide + a light chain variable region + a mouse kappa constant region; the full-length expression plasmid of the heavy chain of 9D5 contains a signal peptide + a heavy chain variable region + a mouse IgG1 constant region; the nucleotide sequence of the signal peptide is shown in SEQ ID NO.1, the nucleotide sequence of the light chain variable region is shown in SEQ ID NO.4, and the nucleotide sequence of the mouse kappa constant region is shown in SEQ ID NO.6; the nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO.5, and the nucleotide sequence of the mouse IgG1 constant region is shown in SEQ ID NO.7.

[0010] The present invention also provides a plasmid vector, which contains the nucleotide sequence shown as SEQ ID NO.6 or the nucleotide sequence shown as SEQ ID NO.7.

[0011] The present invention also provides a method for constructing the plasmid vector, comprising the following steps: using pcDNA3.4 as an expression vector, using the nucleotide sequence shown in SEQ ID NO.1 as a light chain signal peptide, using the nucleotide sequence shown in SEQ ID NO.3 as a light chain variable region, and using the nucleotide sequence shown in SEQ ID NO.6 as a mouse kappa light chain constant region to construct a light chain plasmid.

[0012] The present invention also provides a method for constructing the plasmid vector, comprising the following steps: using pcDNA3.4 as an expression vector, using the nucleotide sequence shown in SEQ ID NO.1 as a heavy chain signal peptide, using the nucleotide sequence shown in SEQ ID NO.5 as a heavy chain variable region, and using the nucleotide sequence shown in SEQ ID NO.7 as a mouse IgG1 heavy chain constant region to construct a heavy chain plasmid.

[0013] Wherein, the complete fentanyl antigen is prepared by coupling fentanyl hapten with a carrier protein.

[0014] Wherein, the structural formula of the fentanyl hapten is as follows:

[0015]

[0016] Wherein, the method for preparing the fentanyl hapten comprises the following steps:

[0017] (1) Using (2-bromoethyl)benzoic acid as the starting material, the carboxyl group is reduced to an alcoholic hydroxyl group under the action of borane to generate (2-bromoethyl)benzyl alcohol;

[0018] (2) Under the action of pyridinium chlorochromate, the (2-bromoethyl)benzyl alcohol described in step (1) is oxidized to generate (2-bromoethyl)benzaldehyde; a substitution reaction is carried out with N-phenyl-N-(4-piperidinyl)piperidinamide, and the mixture is extracted with ethyl acetate and separated by column chromatography and distilled under reduced pressure to obtain fentanyl hapten.

[0019] The present invention also provides the use of fentanyl hapten, fentanyl complete antigen or anti-fentanyl monoclonal antibody in preparing a reagent for detecting the fentanyl content in a biological sample; the biological sample includes hair, urine, serum or saliva.

[0020] The present invention also provides the use of fentanyl hapten, fentanyl complete antigen or anti-fentanyl monoclonal antibody in detecting the fentanyl content in biological samples; the biological samples include hair, urine, serum or saliva.

[0021] Among them, ELISA or colloidal gold immunochromatography is used to detect the content of fentanyl or its analogs in biological samples.

[0022] Among them, when the ELISA method was used to detect the fentanyl content in hair, the detection limit was 0.0044ng / mg, and the detection range was 0.011-0.24ng / mg; when the ELISA method was used to detect the fentanyl content in urine, the detection limit was 0.16ng / mL, and the detection range was 0.41-8.97ng / mL.

[0023] Among them, when the colloidal gold immunochromatography method was used to detect the fentanyl content in hair, the detection limit was 0.0010ng / mg, and the detection range was 0.0033-0.24ng / mg; when the colloidal gold immunochromatography method was used to detect the fentanyl content in urine, the detection limit was 0.15ng / mL, and the detection range was 0.38-8.97ng / mL.

[0024] The present invention also provides the use of fentanyl hapten, fentanyl complete antigen or anti-fentanyl monoclonal antibody in preparing an enzyme-linked immunosorbent assay kit for fentanyl detection.

[0025] The present invention targets the variable region amino acid sequence of the fentanyl-specific antibody, constructs a mouse antibody expression plasmid vector, and transfects it in the eukaryotic system HEK 293F cells, expresses and purifies it in large quantities. The purified antibody is used to establish an immunoassay method to achieve rapid detection of the fentanyl content in samples such as urine and hair.

[0026] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The fentanyl-specific antibody sequence provided by the present invention increases the diversity of antibody sequences, and the constructed expression plasmid can be successfully expressed in a eukaryotic system and applied to establish a highly sensitive rapid immune detection method; 2. The immunoassay method established based on the prepared fentanyl-specific antibody can simultaneously determine 33 types of fentanyl and its analogs, IC 50 Between 0.068-88.53ng / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Fentanyl hapten Fent-1 1 H-NMR spectrum;

[0028] Figure 2 Fentanyl hapten Fent-1 13 C-NMR spectrum;

[0029] Figure 3 This is the standard curve for ELISA determination of fentanyl content in hair (B) and urine (A);

[0030] Figure 4 This is the standard curve for determining fentanyl in hair and urine by colloidal gold immunochromatography: A and C are colloidal gold test strip graphs of urine and hair standard curves, respectively; B and D are colloidal gold test strip concentration and T / C curves of urine and hair standard curves, respectively. DETAILED DESCRIPTION

[0031] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0032] Example 1 Fentanyl Complete Antigen Protein-Fentanyl

[0033] 1. Synthesis and characterization of fentanyl hapten Fent-1

[0034] Synthesis route:

[0035]

[0036] 4-(2-bromoethyl)benzoic acid (500 mg, 1 eq) was dissolved in 5 mL tetrahydrofuran (THF), stirred under ice bath conditions, and borane tetrahydrofuran solution (563 mg, 3 eq) was drawn into the reaction solution with a syringe, and stirred until the raw materials reacted completely. Under the action of borane, the carboxyl group was reduced to an alcohol hydroxyl group to generate 4-(2-bromoethyl)benzyl alcohol, which is intermediate 2; intermediate 2 (500 mg, 1 eq) was dissolved in 10 mL dichloromethane solvent, and under the action of (1.002 g, 2 eq) pyridinium chlorochromate (PCC), 4-(2-bromoethyl)benzyl alcohol was oxidized to generate 4-(2-bromoethyl)benzaldehyde, which is intermediate 3; intermediate 3 (200 mg, 1 eq), N-phenyl-N-(4-piperidinyl)piperidinamide (219 mg, 1 eq), and K2CO3 (389 mg, 3 eq) were added to 5 mL Substitution reaction takes place in DMF solvent to generate intermediate 4; finally, the aldehyde group on the benzene ring of intermediate 4 (200 mg, 1 eq) and carboxymethyloxyamine hemihydrochloride (180 mg, 3 eq) are dehydrated and condensed in pyridine (130 mg, 3 eq) with methanol as solvent, extracted with ethyl acetate and separated by column chromatography to obtain hapten Fent-1. The hydrogen and carbon spectra of the prepared fentanyl hapten Fent-1 are shown in Figures 1-2 shown. 1 H NMR (500MHz, CDCl3) δ8.14(s,1H),7.48-7.42(m,3H),7.39(d,J=8.1Hz,2H),7.10(dd,J=7.9,1.7Hz,2H),7.05(d,J=8.2Hz,2H),4.82(dt,J= 11.9,7.3Hz,1H),4.51(s,2H),3.64(d,J=11.7Hz,2H),3.04-2.91(m,4H),2.67(t,J=11.5Hz,2H),2.01-1.85(m,6H),1.04(t,J=7.4Hz,3H). 13 C NMR (126MHz, CDCl3) δ174.74,174.08,148.95,138.73,138.02,130.89,129.95,129.80,129.06 ,128.95,127.49,72.09,51.96,50.35,30.78,29.73,28.46,27.96,9.53.HRMS(ESI):calcd.for C 25 H 31 N3O4[M+H] + 438.2393, found 438.2365.

[0037] 2. Synthesis of complete fentanyl antigen:

[0038] Weigh 5 mg of hapten Fent-1, weigh EDCI (6 eq), NHS (6 eq), Et3N (6 eq) and dissolve in 500 μL DMF, stir at room temperature for 6 hours until the hapten reaction is complete, and obtain a hapten mixed solution. Weigh 10 mg of protein, dissolve the protein in 2 mL of PBS solution to prepare a 5 mg / mL solution, add it to the above hapten mixed solution, stir overnight at 4°C, take it out the next day, put it in a MW 3000 dialysis bag, and dialyze it with 500 mL of pH 7.4 PBS each time, dialyze it 4 times in total, and obtain the complete antigen Protein-Fentanyl, which is then divided and frozen at -80°C for use. When the protein is bovine serum albumin (BSA, Biosharp), the complete antigen obtained is the coating antigen BSA-Fentanyl; when the protein is chicken ovalbumin (OVA, ovalbumin), the complete antigen obtained is the coating antigen OVA-Fentanyl. The structural formula of complete antigen Protein-Fentanyl is as follows:

[0039]

[0040] Herein, Protein represents BSA or OVA.

[0041] Example 2 Preparation of fentanyl antibodies

[0042] 1. Construction of fentanyl antibody expression plasmid

[0043] The antibody subtype of 9D5 hybridoma cell is IgG1, and the light chain is kappa (Xiao-Yi Chen, Li Wang, Xiao Ma, Fan Yang, Xiao Wang, Peng Xu*, Li-Li Xu*, Bin Di*. Development of fentanyl-specific monoclonal antibody (mAb) to antagonize the pharmacological effects of fentanyl. Toxicology and Applied Pharmacology. 2024, (486) 116918.). The nucleotide sequences of the light and heavy chain variable regions were obtained after sequencing the antibody variable regions. The construction of mouse recombinant antibody light and heavy chain plasmids was completed by Detai Biotechnology (Nanjing) Technology Co., Ltd. The light chain plasmid construction: signal peptide + light chain variable region + mouse kappa constant region, finally connected to the pcDNA3.4 vector; heavy chain plasmid: signal peptide + heavy chain variable region + mouse IgG1 constant region, finally connected to the pcDNA3.4 vector.

[0044] The nucleotide sequence of the signal peptide is shown in SEQ ID NO.1: ATGGGCTGGAGCTGCATCAT CCTGTTCCTCGTGGCTACAGCTACCGGAGTGCACAGC. The amino acid sequence of the light chain variable region of 9D5 is shown in SEQ ID NO.2: DIVMTQSQRFMSTSVGDRVTVTCKASQNVDTDVVWYQ QKPGRSPKALIYSTSYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNSNPF TFGSGTKLEIK; the nucleotide sequence of the light chain variable region of 9D5 is shown in SEQ ID NO.3: GACATTGT GATGACCCAGTCTCAAAGATTCATGTCCACATCAGTAGGAGACAGGGTCACCGTCACCTGCAAGGCCAGTCAGAATGTGGATACTGATGTAGTCTGGTATCAACAGAAACCAGGGCGATCTCCTAAAGCACTGATTTACTCGACATCCTATCGGTACAGTGGAGTC CCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAATGTGCAGTCTGAAGACTTGGCAGAATATTTCTGTCAACAATATAACAGCAATCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA; the amino acid sequence of the heavy chain variable region of 9D5 is as SEQ ID NO.4 shows: QVQLQQSGAELARPGASLKMSCKASGYTFTTFTIYWIKQRPG LGLEWIGYINPGSAYTNYNQKFKNKATLTADKSSSTAYMQLTSLTSEDSAVYYCSIEVR RGVYVMDYWGQGTSVTVSS; the nucleotide sequence of the heavy chain variable region of 9D5 is as shown in SEQ ID NO.As shown in Figure 5: The nucleotide sequence of the mouse kappa light chain constant region is as shown in SEQ ID NO. 6: AGGGCCGACGCAGCTCCTACCGTGTCTATCTTCCCCCCTAGCAGC GAGCAGCTGACATCAGGAGGAGCTAGCGTGGTCTGCTTCCTGAACAACTTCTACCCCAAGGACATCAACGTCAAGTGGAAGATCGACGGCAGCGAGAGGCAGAACGGCGTGCTGAACTCTTGGACCGACCAGGATAGCAAGGACAGCACCTACAGCATGAGCAGCACCCTGACCCTGACCAAGGACGAGTACGAGCGGCACAACAGCTACACCTGCGAGGCTACACACAAGACCAGCACCAGCCCCATCGTGAAGAGCTTCAACCGGAACGAGTGC; The nucleotide sequence of the mouse IgG1 heavy chain constant region is as shown in SEQ ID NO.As shown in Figure 7: GCTAAGACAACC CCTCCTTCTGTGTACCCTCTGGCTCCAGGAAGCGCAGCTCAGACCAACAGCATGGTGACACTGGGCTGCCTGGTGAAGGGATACTTCCCCGAGCCAGTGACCGTGACTTGGAACTCAGGCAGCCTGAGCAGCGGAGTGCACACATTTCCAGCCGTGCTGCAGAGCGACCTGTACACACTGAGCAGCAGCGTGACCGTGCCTTCTAGCACTTGGCCTAGCGAGACCGTGACTTGCAACGTGGCTCACCCAGCCAGCAGCACCAAGGTGGACAAGAAGATCGTGCCCAGGGATTGCGGTTGCAAGCCTTGCATCTGTACCGTGCCCGAAGTGTCCAGCGTGTTCATCTTCCCCCCCAAGCCCAAGGACGTGCTGACCATCACCCTGACCCCCAAAGTGACTTGCGTGGTGGTGGACATCAGCAAGGACGACCCCGAGGTGCAGTTCTCTTGGTTCGTGGACGACGTGGAGGTGCACACAGCTCAGACACAGCCTAGGGAGGAGCAGTTCAACAGCACCTTCCGGAGCGTGTCCGAACTGCCCATCATGCACCAGGATTGGCTGAACGGCAAGGAGTTCAAGTGTCGCGTGAACAGCGCCGCTTTCCCAGCCCCCATCGAGAAGACCATCAGCAAGACCAAGGGCAGGCCTAAGGCTCCTCAGGTGTACACAATCCCTCCTCCCAAGGAGCAGATGGCCAAGGACAAGGTGTCCCTGACTTGCATGATCACCGACTTCTTCCCCGAGGACATCACCGTCGAGTGGCAGTGGAACGGACAGCCAGCCGAGAACTACAAGAACACCCAGCCCATCATGGACACCGACGGCAGCTACTTCGTGTACAGCAAACTGAACGTGCAGAAGAGCAATTGGGAGGCCGGCAACACCTTCACTTGCAGCGTGCTGCACGAGGGACTGCACAACCACCACACCGAGAAGAGCCTGAGCCACAGCCCAGGAAAG。.

[0045] 2. Expression and purification of recombinant fentanyl antibodies

[0046] Take out the frozen HEK 293F cells (frozen in the laboratory) from the -80℃ refrigerator, quickly put them into a preheated 37℃ water bath, shake them back and forth until there are no ice crystals in the cryotube, centrifuge at 1000rpm for 5min, discard the supernatant, add 1mL OPM-293CD05 Medium medium to resuspend the cells, transfer them to a shaker containing OPM-293CD05 Medium medium (Shanghai Aopuma Biotech Co., Ltd.), shake at 120rpm, set the temperature to 37℃, and culture overnight with 5% CO2. When the HEK 293F cells grow to 3.0×10 6 When the density reached 1.5447 W / v, transfection was started by adding 150 μg of each of the constructed 9D5 light and heavy chain full-length expression plasmids into 10 mL of blank culture medium, slowly dropping 1.2 mg of 1 mg / mL PEI transfection reagent (PolySciences), vortexing to mix, letting stand for 15 min, and slowly adding to 300 mL of cell culture medium. Five days after transfection, the cell supernatant was collected for purification. After filtration, Protein A (Changzhou Tiandi Renhe Biotechnology Co., Ltd.) was used for purification. The collected supernatant was loaded onto the Protein A affinity column, repeated 3 times, and then 5 column volumes of equilibrium washing solution (0.15M NaCl, 20mM Na2HPO4, pH7.0) were used to elute the impurities from the affinity column, and then 2mL of elution solution (0.1M glycine, pH 2.7) was added each time to elute the antibody from the affinity column. 200μL of neutralizing solution (1M Tris-HCl, pH8.5) was added to each collection tube in advance to adjust to neutrality to obtain the antibody solution. The antibody solution was concentrated by a 50kD ultrafiltration tube (Millipore) and stored in a PBS solution. The concentration of the antibody solution was measured by NanoDrop to be 17.559mg / mL, and the mouse fentanyl-specific antibody 9D5 was obtained, which was packaged and frozen for use.

[0047] Example 3 Competitive ELISA method to determine the affinity of purified antibodies to fentanyl and its analogs

[0048] The specific operation steps of the competitive ELISA method include:

[0049] 1. Coating: Dilute the coating antigen BSA-Fentanyl to 0.025μg / mL with coating solution, add 100μL / well to a 96-well ELISA plate, seal the plate with a sealing film and incubate overnight in a 4°C refrigerator; the coating solution formula is as follows: KCl 0.2g, KH2PO4 0.24g, Na2HPO4.12H2O 3.63g, NaCl 8g, add ddH2O to make up to 1000mL (pH 7.4);

[0050] 2. Blocking: Add 100 μL / well of blocking solution and incubate at 37°C for 1.5 h. The formula of the blocking solution is as follows: KCl 0.2 g, KH2PO4 0.24 g, Na2HPO4.12H2O 3.63 g, NaCl 8 g, 50 g skim milk powder, add ddH2O to make up to 1000 mL (pH 7.4).

[0051] 3. Primary antibody incubation: After blocking, wash the plate 5 times with washing solution, add 50 μL and 10 ng / mL of the purified fentanyl-specific antibody 9D5 obtained in the previous step and 50 μL of PBS solution containing different concentrations of fentanyl (0, 0.12, 0.49, 1.95, 7.81, 31.25, 125, 500 ng / mL) to the wells, and incubate at 37°C for 1 h; the washing solution formula is as follows: KCl 0.2 g, KH2PO4 0.24 g, Na2HPO4.12H2O 3.63 g, NaCl 8 g, 0.5 mL Tween 20, add ddH2O to make up to 1000 mL (pH 7.4);

[0052] 4. Incubate with secondary antibody: add 100 μL / well of freshly diluted goat anti-mouse HRP enzyme-labeled secondary antibody (Jackson ImmunoResearch, 115-035-003) (PBST: goat anti-mouse HRP enzyme-labeled secondary antibody = 1:5000, v / v), and incubate at 37°C for 30 min;

[0053] 5. Color development: Add 50 μL / well of TMB color development solution (Biyuntian, P0209 product) and incubate for 10 minutes in the dark;

[0054] 6. Termination: Add 2M H2SO4 (55mL concentrated H2SO4 plus dd H2O to 500mL) at 50μL / well to terminate the color reaction, and detect the absorbance at 450nm. The results of the competitive ELISA of 9D5 against fentanyl and its structural analogs are shown in Table 1.

[0055] Table 1 IC of fentanyl antibodies and fentanyl and its analogs 50 value

[0056]

[0057]

[0058]

[0059] The results showed that 9D5 had a high affinity for most fentanyl and its analogs (calculated IC 50The smaller the value, the higher the affinity), but it has almost no affinity for sufentanil, alfentanil, and remifentanil. IC 50 The value was 1.046 ng / mL, which was comparable to the IC 50 Between 0.068-88.53ng / mL.

[0060] Example 4 Competitive ELISA for detecting fentanyl in urine and hair samples

[0061] The operation steps are the same as those for determining specificity by competitive ELISA, except that the fentanyl PBS solution is replaced with a standard curve prepared by diluting fentanyl with urine and hair sample solutions during the primary antibody incubation step. The results are shown in Figure 2. Figure 3 shown.

[0062] Coating: dilute the complete antigen BSA-Fentanyl to 0.025 μg / mL with coating solution, add 100 μl / well to 96-well ELISA plate, seal the plate with sealing film and incubate in a 4°C refrigerator overnight; wherein, the coating solution formula is as follows: KCl 0.2g, KH2PO4 0.24g, Na2HPO4.12H2O 3.63g, NaCl 8g, add ddH2O to make up to 1000mL (pH7.4);

[0063] Blocking: 100 μl / well of blocking solution was added, and incubated at 37°C for 1.5 h. The blocking solution had the following formula: KCl 0.2 g, KH2PO4 0.24 g, Na2HPO4.12H2O 3.63 g, NaCl 8 g, 50 g skim milk powder, and ddH2O was added to make up to 1000 mL (pH 7.4).

[0064] Primary antibody incubation: After blocking, wash the plate 5 times with washing solution, add 50 μL and 10 ng / mL of fentanyl-specific antibody 9D5 and 50 μL of sample solutions containing different concentrations of fentanyl (urine and hair samples) into the wells, respectively. The fentanyl concentrations in urine samples are 0, 0.2, 0.5, 1, 2, 5, 10, 20 ng / mL; the fentanyl concentrations in hair samples are 0, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0 ng / mg, and incubate at 37°C for 1 h. The washing solution has the following formula: KCl 0.2 g, KH2PO4 0.24 g, Na2HPO4.12H2O 3.63 g, NaCl 8 g, 0.5 mL Tween 20, and add ddH2O to make up to 1000 mL (pH 7.4).

[0065] Incubate with secondary antibody: add 100 μL / well of freshly diluted goat anti-mouse HRP enzyme-labeled secondary antibody (PBST: goat anti-mouse HRP enzyme-labeled secondary antibody = 1:5000, v / v), and incubate at 37°C for 30 min;

[0066] Color development: 50 μL / well of TMB color development solution (Biyuntian, P0209 product) was added and incubated for 10 min in the dark;

[0067] Termination: Add 2M H2SO4 (55 mL concentrated H2SO4 plus dd H2O to 500 mL) at 50 μL / well to terminate the color reaction and detect the absorbance at 450 nm.

[0068] Among them, the urine pretreatment steps are: blank urine (provided by laboratory staff) is adjusted to pH 7.4 using 1M NaOH solution, with or without fentanyl added, added to an ELISA 96-well ELISA plate containing fentanyl-specific antibody 9D5, and incubated for detection.

[0069] Hair pretreatment steps: Weigh 20 mg of washed and chopped blank hair (provided by laboratory staff) and add it to a grinding tube containing 0.8 mL of hair lysis solution (Zhejiang Noga Biotechnology Co., Ltd.) and grinding beads, add / not add fentanyl, grind it in a hair grinder for 10 minutes, centrifuge and take the supernatant, add it to an ELISA 96-well enzyme-labeled plate containing 9D5, and incubate for detection.

[0070] The results of the standard curve competition ELISA of urine and hair samples (fentanyl-specific antibody 9D5 and sample solutions containing different concentrations of fentanyl were added to the wells, respectively) are as follows: Figure 3 As shown, A and B are the competitive ELISA standard curve results of urine and hair, respectively, the horizontal axis is the fentanyl concentration, and the vertical axis is the OD450 value. Using the competitive ELISA method, the detection limit (LOD) of fentanyl content in urine samples is 0.16ng / mL, and the LOD in hair samples is 0.0044ng / mg.

[0071] Example 5 Determination of fentanyl content in urine and hair samples using colloidal gold immunochromatographic test strips

[0072] Step 1: Use a gold spray film streaking instrument to streak 0.6 mg / mL of BSA-Fentanyl coated antigen and 0.6 mg / mL of goat anti-mouse secondary antibody (Jackson ImmunoResearch, 205-035-108) on the NC membrane at 1 μL / cm and dry at 37°C to serve as the detection line T and quality control line C, respectively;

[0073] Step 2: Assemble the sample pad, NC membrane, and absorbent pad on the PVC base plate in order, and use a chopper to cut them into 3 mm wide test strips;

[0074] Step 3: 50 μL of urine and hair sample solutions containing different concentrations of fentanyl and 50 μL of colloidal gold solution modified with antibody 9D5 (2 μL of gold-labeled antibody stock solution mixed with 48 μL of PBS) were mixed and incubated in a 96-well plate for 2 minutes, and the test strip was inserted into the microwell. The mixed solution moved along the chromatography direction under the action of capillary siphon. When it reached the test line T and the quality control line C, the ratio of the test line to the quality control line was read by the colloidal gold reader TSR-100 (Hangzhou Aosheng Instrument Co., Ltd.). According to the different fentanyl concentrations, the different values ​​read by the test line T and the quality control line C, the ratio T / C of the test line and the quality control line was obtained. The standard curve was made by using the ratio and the corresponding fentanyl concentration to achieve the detection of fentanyl. Among them, the fentanyl concentrations in urine samples were 0, 0.2, 0.5, 1, 2, 5, 10, and 20 ng / mL; the fentanyl concentrations in hair samples were 0, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, and 2.0 ng / mg.

[0075] Among them, the preparation of the gold-labeled antibody mother solution: 2mL of 1% trisodium citrate aqueous solution was quickly added to the boiling 100mL of 0.01% HAuCl4 aqueous solution, and the solution was stirred and boiled for 15min. The colloidal gold solution was then cooled to room temperature and stored at 4°C. The fentanyl-specific antibody 9D5 was attached to the colloidal gold by electrostatic adsorption. First, 1mL of colloidal gold solution was drawn into a 1.5mL EP tube, 5μL of 0.2mol / L K2CO3 was added to adjust the pH of the colloidal gold solution, and then 4μg of fentanyl-specific antibody 9D5 was added and incubated at room temperature for 1h. Bovine serum albumin (BSA) dissolved in 0.01M boric acid buffer (BB, pH 8.2) at a final concentration of 10mg / mL was added to the above mixture and incubated for another 1 hour. The mixture was centrifuged at 10000 rpm / min for 1 hour, the supernatant was discarded, and the precipitate was resuspended in 100 μL BB solution containing 2% bovine serum albumin and 3% sucrose to obtain the gold-labeled antibody stock solution.

[0076] Colloidal gold immunochromatography was used to establish a standard curve for fentanyl detection in urine and hair. The pretreatment steps of urine and hair samples were the same as those in Example 4. The results are shown in Table 1. Figure 4 As shown, A and C are colloidal gold test strips of urine and hair respectively, B and D are colloidal gold test strips of urine and hair respectively, the horizontal axis is concentration, and the vertical axis is T / C. With the increase of fentanyl concentration, the T / C value gradually decreases, and the established colloidal gold test strip immunochromatography method can detect the fentanyl content in urine and hair.

Claims

1. A reagent or kit for detecting the content of fentanyl or its analogs in a biological sample, characterized in that: It contains an anti-fentanyl monoclonal antibody and a complete fentanyl antigen, wherein the anti-fentanyl monoclonal antibody comprises a light chain and a heavy chain; the nucleotide sequence of the variable region of the light chain is shown in SEQ ID NO.4; the nucleotide sequence of the variable region of the heavy chain is shown in SEQ ID NO.5; and the complete fentanyl antigen is shown in Formula I: Wherein, Protein is BSA or OVA; The analogs include acetylfentanyl, benzylfentanyl, benzoylfentanyl, thiofentanyl, β-hydroxyfentanyl, N-methylfentanyl, benzylfuranfentanyl, 3'-methylfentanyl, 2'-methylfentanyl, 4'-methylfentanyl, norfentanyl, butyrylfentanyl, isobutyrylfentanyl, acryloylfentanyl, crotonylfentanyl, tetrahydrofuranfentanyl, ciprofentanyl, methoxyacetylfentanyl, 4-fluoroisobutyrylfentanyl, para-fluorofentanyl, ortho-fluorofentanyl, meta-fluorofentanyl, para-methoxyfentanyl, meta-methylfentanyl, ortho-methylfentanyl, para-methylfentanyl, valerylfentanyl, 4-methoxyfuranfentanyl, 2-methylfentanyl, 3-furanfentanyl, furanfentanyl or carfentanyl; and the biological sample includes hair, urine, serum or saliva.

2. The reagent or kit according to claim 1, characterized in that The anti-fentanyl monoclonal antibody is obtained by co-transfection, expression and purification of the full-length expression plasmid of the light chain of 9D5 and the full-length expression plasmid of the heavy chain of 9D5 in a HEK 293F expression system; the full-length expression plasmid of the light chain of 9D5 contains a signal peptide + a light chain variable region + a mouse kappa constant region; the full-length expression plasmid of the heavy chain of 9D5 contains a signal peptide + a heavy chain variable region + a mouse IgG1 constant region; the nucleotide sequence of the signal peptide is shown in SEQ ID NO.1, the nucleotide sequence of the light chain variable region is shown in SEQ ID NO.4, and the nucleotide sequence of the mouse kappa constant region is shown in SEQ ID NO.6; the nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO.5, and the nucleotide sequence of the mouse IgG1 constant region is shown in SEQ ID NO.

7.

3. Use of anti-fentanyl monoclonal antibodies, fentanyl complete antigens or fentanyl haptens in the preparation of reagents or kits for detecting the content of fentanyl or its analogs in biological samples; the biological samples include hair, urine, serum or saliva; the analogs include acetylfentanyl, benzylfentanyl, benzoylfentanyl, thiofentanyl, β-hydroxyfentanyl, N-methylfentanyl, benzylfuranfentanyl, 3'-methylfentanyl, 2'-methylfentanyl, 4'-methylfentanyl, norfentanyl, butanol The fentanyl hapten is fentanyl, fentanyl acet ...

4. Use of an anti-fentanyl monoclonal antibody or a full-length expression plasmid of the light chain of 9D5 and a full-length expression plasmid of the heavy chain of 9D5 in the preparation of a reagent or a kit for detecting the content of fentanyl in a biological sample; the biological sample includes hair, urine, serum or saliva.

5. Use of an anti-fentanyl monoclonal antibody, a fentanyl complete antigen, a fentanyl hapten, or the reagent or kit according to claim 1 or 2 in detecting the content of fentanyl or its analogs in a biological sample; the biological sample comprises hair, urine, serum or saliva; the analogs comprise acetylfentanyl, benzylfentanyl, benzoylfentanyl, thiofentanyl, β-hydroxyfentanyl, N-methylfentanyl, benzylfuranfentanyl, 3'-methylfentanyl, 2'-methylfentanyl, 4' -Methylfentanyl, norfentanyl, butyrylfentanyl, isobutyrylfentanyl, acryloylfentanyl, crotonylfentanyl, tetrahydrofuranylfentanyl, ciprofentanyl, methoxyacetylfentanyl, 4-fluoroisobutyrylfentanyl, para-fluorofentanyl, ortho-fluorofentanyl, meta-fluorofentanyl, para-methoxyfentanyl, meta-methylfentanyl, ortho-methylfentanyl, para-methylfentanyl, valerylfentanyl, 4-methoxyfuranylfentanyl, 2-methylfentanyl, 3-furanylfentanyl, furanylfentanyl, or carfentanyl.

6. The use according to claim 5, characterized in that: Competitive ELISA or colloidal gold immunochromatography is used to detect the content of fentanyl or its analogs in biological samples.

7. The use according to claim 5, characterized in that: When the ELISA method was used to detect the fentanyl content in hair, the detection limit of fentanyl was 0.0044 ng / mg, and the detection range was 0.011-0.24 ng / mg; when the ELISA method was used to detect the fentanyl content in urine, the detection limit of fentanyl was 0.16 ng / mL, and the detection range was 0.41-8.97 ng / mL.

8. The use according to claim 5, characterized in that: When the colloidal gold immunochromatography method was used to detect the fentanyl content in hair, the detection limit was 0.0010 ng / mg and the detection range was 0.0033-0.24 ng / mg; when the colloidal gold immunochromatography method was used to detect the fentanyl content in urine, the detection limit was 0.15 ng / mL and the detection range was 0.38-8.97 ng / mL.

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