Amitriptyline antibody and kit

Abstract

The invention belongs to the technical field of biochemical engineering, and particularly relates to an amitriptyline antibody and a kit. The amitriptyline hapten has a structural formula shown as a formula (I) or a formula (II). The amitriptyline antibody prepared in the invention has the advantages of high titer, strong specificity and high affinity, the LOD of amitriptyline by using the Elisa method established by the amitriptyline antibody is 0.013 ng / mL, the IC50 is 0.865 ng / mL, the quantitative detection range is 0.07-470.3 ng / mL, the detection sensitivity is high, and the linear range is wide.

Description

[0001] This application is a divisional application of Chinese patent application No. 2025118120330, filed on December 4, 2025, entitled "An amitriptyline hapten and its preparation method, amitriptyline antigen and its preparation method, antibody, reagent kit and application". Technical Field

[0002] This invention belongs to the field of biochemical technology, specifically relating to an amitriptyline antibody and a reagent kit. Background Technology

[0003] Amitriptyline, also known as N,N-dimethyl-3-[10,11-dihydro-5H-dibenzo[a,d]cycloheptatrien-5-ylidene]-1-propylamine, has the molecular formula C2. 20 H 23 N. The structural formula is as follows: .

[0004] Amitriptyline is a tricyclic antidepressant used to treat various types of depression or depressive states. It is particularly effective for endogenous depression and menopausal depression, and also effective for reactive depression and depressive states associated with neurosis. For patients with both anxiety and depressive symptoms, amitriptyline is more effective than imipramine. When used in combination with electroconvulsive therapy (ECT) for major depressive disorder, it can reduce the frequency of ECT. It is also used to relieve chronic pain and treat childhood enuresis and ADHD.

[0005] However, amitriptyline has a narrow therapeutic window and significant inter-individual pharmacokinetic variability. Its effective blood concentration range is close to its potential toxic concentration, and its metabolism is easily affected by various factors such as age, liver and kidney function, concomitant medications, and genetic polymorphism. Therefore, establishing an immunoassay method for detecting amitriptyline levels in serum is particularly urgent. The key to establishing such an immunoassay method lies in designing a suitable artificial antigen for amitriptyline and obtaining antibodies with high sensitivity and specificity. However, no reports on amitriptyline antibodies have been found to date. Summary of the Invention

[0006] The purpose of this invention is to provide an amitriptyline antibody and kit to overcome the problem of low affinity of existing amitriptyline antibodies.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: An amitriptyline hapten, the structural formula of which is shown in formula (I) or formula (II): .

[0008] The amitriptyline hapten shown in formula (I) is named systematically as: 4-((3-(10,11-dihydro-5H-dibenzo[a,d][7]annulen-5-ylidene)propyl)(methyl)amino)butanoic acid, i.e., 4-((3-(10,11-dihydro-5H-dibenzo[a,d][7]benzocyclohepten-5-ylmethylene)propyl)(methyl)amino)butanoic acid.

[0009] The amitriptyline hapten shown in formula (II) is named systematically as: 6-((3-(10,11-dihydro-5H-dibenzo[a,d][7]annulen-5-ylidene)propyl)(methyl)amino)hexanoic acid, i.e., 6-((3-(10,11-dihydro-5H-dibenzo[a,d][7]cyclononen-5-ylidene)propyl)(methyl)amino)hexanoic acid.

[0010] According to embodiments of the present invention, the present invention can be further optimized, and the optimized technical solution is as follows:

[0011] Based on the same inventive concept, this invention also claims protection for a method for preparing the amitriptyline hapten, comprising the following steps:

[0012] After dissolving 3-(10,11-dihydro-5H-dibenzo[a,d]cycloheptene-5-ylidene)-N-methyl-1-propanamine hydrochloride, a weak base and a bromide ester were added, and the mixture was stirred at 50-70°C. The resulting product was separated, purified, dried, dissolved, and then reacted with trifluoroacetic acid to obtain the amitriptyline hapten.

[0013] When the amitriptyline hapten has the structural formula shown in formula (I), the bromoester is tert-butyl 4-bromobutyrate;

[0014] When the amitriptyline hapten has the structural formula shown in formula (II), the bromoester is tert-butyl 6-bromohexanoate.

[0015] In one preferred embodiment, the weak base is triethylamine or anhydrous potassium carbonate.

[0016] In one preferred embodiment, the reaction is stirred at 50-70°C for 2-4 hours.

[0017] In one preferred embodiment, the separation and purification steps are as follows: the obtained product is extracted and subjected to silica gel column chromatography.

[0018] In one preferred embodiment, the extraction is performed by using an aqueous ethyl acetate solution to collect the ethyl acetate phase; in the aqueous ethyl acetate solution, the volume ratio of ethyl acetate to water is 1-2:1-2.

[0019] In one preferred embodiment, the developing solvent for silica gel column chromatography is a mixed solution of petroleum ether and ethyl acetate; the volume ratio of petroleum ether to ethyl acetate in the mixed solution is 2-4:1.

[0020] During silica gel column chromatography, the sample is added, and the product is collected by gradient elution with the developing solvent.

[0021] In one preferred embodiment, the molar ratio of 3-(10,11-dihydro-5H-dibenzo[a,d]cycloheptene-5-ylidene)-N-methyl-1-propane hydrochloride, the weak base, and the brominated ester is 1:2-3:1.5-2.

[0022] In one preferred embodiment, the solvent used for dissolution is dichloromethane; the volume is sufficient to dissolve the dried product, without excessive limitations. Generally, 3-10 ml is added.

[0023] In one preferred embodiment, the amount of trifluoroacetic acid added is 1-2 times the volume of dichloromethane.

[0024] In one preferred embodiment, the product obtained after reaction with trifluoroacetic acid is adjusted to pH 6-7, and then extracted and dried to obtain the amitriptyline hapten.

[0025] In one preferred embodiment, the extraction reagent is ultrapure water and ethyl acetate in a volume ratio of 1-2:1-2.

[0026] In the preparation of haptens in this invention, triethylamine is used as a catalyst, which improves the simplicity of the reaction process. The use of brominated esters for one-step reaction greatly reduces time costs. The moderate temperature avoids the formation of byproducts and greatly improves the yield.

[0027] Based on the same inventive concept, this invention also claims protection for an amitriptyline antigen, the structural formula of which is shown in formula (III) or (IV): ; where Z is the carrier protein.

[0028] In one preferred embodiment, the carrier protein is ovalbumin (OVA) or bovine serum albumin (BSA).

[0029] Based on the same inventive concept, the present invention also claims protection for a method for preparing the amitriptyline antigen, which is obtained by conjugating the amitriptyline hapten with a carrier protein.

[0030] In one preferred embodiment, the method for preparing the amitriptyline antigen includes: coupling a carrier protein to the carboxyl group of the amitriptyline hapten.

[0031] In one preferred embodiment, the method for preparing the amitriptyline antigen includes the following steps:

[0032] S1. After dissolving the amitriptyline hapten, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS), stir until homogeneous, and obtain solution A;

[0033] The carrier protein was dispersed evenly to obtain solution B;

[0034] S2. Add solution A dropwise to solution B and react at 0-4℃ for 6-12 hours. After the reaction is complete, dialyze to obtain the amitriptyline antigen.

[0035] In one preferred embodiment, the mass ratio of the amitriptyline hapten, N-hydroxysuccinimide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 1:1-2:1-2, and the molar ratio of the carrier protein to the amitriptyline hapten is 1:60-80.

[0036] In one preferred embodiment, the mass ratio of the amitriptyline hapten, N-hydroxysuccinimide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 1:1.5:1.5.

[0037] In one preferred embodiment, the carrier protein is uniformly dispersed in a phosphate buffer solution.

[0038] In one preferred embodiment, the dialysis temperature is 0-4°C, and dialysis is performed for 3-5 days using phosphate buffer solution, with the dialysis solution being changed 2-4 times per day.

[0039] Based on the same inventive concept, the present invention also claims protection for the use of the amitriptyline hapten or the amitriptyline antigen in the preparation of amitriptyline antibodies.

[0040] In one preferred embodiment, the amitriptyline hapten is shown as in formulas (I) and (II), and the amitriptyline antigen is shown as in formulas (III) and (IV).

[0041] Based on the same inventive concept, this invention also claims protection for an amitriptyline antibody, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, and the light chain variable region comprises light chain CDR1, light chain CDR2, and light chain CDR3, the sequence of heavy chain CDR1 is shown in SEQ ID NO.4, the sequence of heavy chain CDR2 is shown in SEQ ID NO.6, the sequence of heavy chain CDR3 is shown in SEQ ID NO.8, the sequence of light chain CDR1 is shown in SEQ ID NO.11, the sequence of light chain CDR2 is RAN, and the sequence of light chain CDR3 is shown in SEQ ID NO.13.

[0042] In one preferred embodiment, the heavy chain variable region is as shown in SEQ ID NO.1; and / or the light chain variable region is as shown in SEQ ID NO.2.

[0043] Based on the same inventive concept, the present invention also claims a kit for detecting amitriptyline, the kit comprising the amitriptyline antigen.

[0044] In one preferred embodiment, the kit comprises: an enzyme-labeled plate coated with the amitriptyline antigen, the amitriptyline antibody, enzyme-labeled secondary antibody, substrate chromogenic solution, stop solution, washing solution, diluent, and blocking solution.

[0045] In one preferred embodiment, the diluent is a phosphate buffer.

[0046] In one preferred embodiment, the washing solution contains 0.8-1.0 wt% Tween-20 and 0.02-0.05 wt% of a preservative in a phosphate buffer solution with a pH of 7.4.

[0047] In one preferred embodiment, the substrate colorimetric solution includes solution A and solution B, where solution A is urea peroxide and solution B is tetramethylbenzidine.

[0048] In one preferred embodiment, the terminating solution is 10% H2SO4.

[0049] In one preferred embodiment, the blocking solution is a 0.2 mol / L phosphate buffer containing 2% casein by mass, with a pH of 7.3.

[0050] Based on the same inventive concept, the present invention also claims a method for detecting amitriptyline, which uses the amitriptyline detection kit to detect the concentration of amitriptyline in a sample.

[0051] Compared with the prior art, the beneficial effects of the present invention are:

[0052] The amitriptyline hapten prepared by this invention has a better immunogenic effect compared to existing structures.

[0053] The artificial antigen prepared in this invention is used as an immunogen to immunize Balb / c mice, and a hybridoma cell line is prepared by chemical fusion. The resulting antibody has high titer, strong specificity, and high affinity. The ELISA method established using this antibody shows a LOD of 0.013 ng / mL and an IC50 of 0.865 ng / mL for amitriptyline, with a quantitative detection range of 0.07-470.3 ng / mL, high detection sensitivity, and a wide linear range. The antibody and detection method of this invention are simple, rapid, highly specific, have a wide linear range, and high sensitivity, showing good application prospects and broad development potential in the rapid and effective detection of amitriptyline. Attached Figure Description

[0054] Figure 1 This is a synthetic route diagram for the hapten A-4C of this invention.

[0055] Figure 2 This is a synthetic route diagram for the hapten A-6C of this invention.

[0056] Figure 3 This is a UV scan of the artificial antigen A-4C-BSA.

[0057] Figure 4 This is a UV scan of the artificial antigen A-6C-OVA.

[0058] Figure 5 This is a diagram showing the subtypes of amitriptyline monoclonal antibody.

[0059] Figure 6 This is a gel electrophoresis image of amitriptyline monoclonal antibody.

[0060] Figure 7 This is the standard curve for amitriptyline ELISA. Detailed Implementation

[0061] This invention is not limited to the specific embodiments listed below. Those skilled in the art can implement this invention using various other specific embodiments based on the content disclosed herein. Any modifications or alterations made to the design structure and concept of this invention fall within the protection scope of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0062] The experimental raw material, 3-(10,11-dihydro-5H-dibenzo[a,d]cycloheptene-5-ylidene)-N-methyl-1-propanamine hydrochloride, also known as nortriptyline hydrochloride, CAS number 894-71-3, was purchased from Aladdin. Its molecular weight is 299.84, and its structural formula is as follows: .

[0063] Example 1

[0064] Synthesis and Identification of Amitriptyline Hapten

[0065] 1. Synthesis and identification of amitriptyline hapten 4-((3-(10,11-dihydro-5H-dibenzo[a,d][7]benzocyclohepten-5-ylmethylene)propyl)(methyl)amino)butyric acid (A-4C)

[0066] (1) The synthetic route of 4-((3-(10,11-dihydro-5H-dibenzo[a,d][7]benzocyclohepten-5-ylmethylene)propyl)(methyl)amino)butyric acid (A-4C) is as follows Figure 1 As shown, the specific synthesis steps are as follows:

[0067] 1 g of 3-(10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5-ylidene)-N-methyl-1-propanamine hydrochloride was dissolved in acetonitrile. Triethylamine (1:3 molar ratio to the starting material) and tert-butyl 4-bromobutyrate (1:1.5 molar ratio to the starting material) were added, and the mixture was stirred at 60 °C. The reaction was monitored by TLC, and the reaction was stopped when the starting material spot disappeared. The developing solvent was petroleum ether-ethyl acetate (3:1, v / v). After reacting for 3 h, the mixture was extracted with ethyl acetate and primary water (1:1 v / v). The ethyl acetate phase was collected and purified by silica gel column chromatography. The developing solvent was a solution of petroleum ether-ethyl acetate (3:1, v / v). After adding the sample, the mixture was eluted with a gradient of developing solvents to obtain a relatively pure product. The solvent was removed by vacuum distillation. The product was then dissolved in 3 mL of dichloromethane, and trifluoroacetic acid (1:1 v / v ratio to dichloromethane) was added. The mixture was stirred at room temperature for 1 h. After the reaction, the pH was adjusted to 6-7 with a 1 mol / L sodium hydroxide aqueous solution. Extraction was then performed using ultrapure water and ethyl acetate at a volume ratio of 1:1. The resulting organic phase was collected, and the aqueous phase was repeatedly extracted with pure ethyl acetate. The organic phases were then combined, dried over anhydrous Na2SO4, filtered, and the solvent was removed by vacuum distillation. After vacuum concentration, a white powder was obtained, which was the hapten 4-((3-(10,11-dihydro-5H-dibenzo[a,d][7]benzocyclohepten-5-ylmethylene)propyl)(methyl)amino)butyric acid. Its structure is shown in formula (I).

[0068] Equation (Ⅰ).

[0069] (2) Identification of amitriptyline hapten 4-((3-(10,11-dihydro-5H-dibenzo[a,d][7]benzocyclohepten-5-ylmethylene)propyl)(methyl)amino)butyric acid (A-4C)

[0070] The hapten 4-((3-(10,11-dihydro-5H-dibenzo[a,d][7]benzocyclohepten-5-ylmethylene)propyl)(methyl)amino)butyric acid was identified by NMR and analyzed by mass spectrometry. The 1H NMR results are as follows: 1H NMR (600MHz, MeOD) δ 8.08 (d, J = 1.3 Hz, 1H), 7.31 – 7.19 (m, 4H), 7.18 – 7.10 (m, 3H), 7.04 (dd, J = 6.9, 1.9 Hz, 1H), 5.83 (t, J = 7.3 Hz, 1H), 3.36 (s, 4H), 3.32 (s, 2H), 3.18 (s, 1H), 3.08 (s, 2H), 2.92 (d, J = 1.9 Hz, 1H), 3.32 (s, 2H), 3.18 (s, 1H), 3.08 (s, 2H), 2.92 (d, J = 1.9 Hz, 1H), 3.32 (s, 2H), 3.32 (s, 2H), 3.18 (s, 1H), 3.08 (s, 2H), 3.32 ... 14.4 Hz, 1H), 2.77 (s, 3H), 2.57 (tt, J = 8.0, 4.9 Hz, 2H), 1.90 (d, J = 15.5 Hz, 2H).

[0071] Mass spectrometry results are as follows: MS: C 23 H 27 NO2: 349.47, ESI + [MH] + 350.24.

[0072] The mass spectrometry and NMR results show that 349.47 is the positive ion molecular peak of the hapten 4-((3-(10,11-dihydro-5H-dibenzo[a,d][7]benzocyclohepten-5-ylmethylene)propyl)(methyl)amino)butyric acid (A-4C). Its calculated relative molecular mass is 350.24, which matches the actual relative molecular mass. Furthermore, the proton NMR spectrum number corresponds to that of the amitriptyline hapten 4-((3) The number of proton atoms in the skeleton structure of 4-((3-(10,11-dihydro-5H-dibenzo[a,d][7]benzocyclohepten-5-ylmethylene)propyl)(methyl)amino)butyric acid (A-4C) indicates that the amitriptyline hapten 4-((3-(10,11-dihydro-5H-dibenzo[a,d][7]benzocyclohepten-5-ylmethylene)propyl)(methyl)amino)butyric acid (A-4C) was successfully prepared.

[0073] 2. Synthesis and identification of amitriptyline hapten 6-((3-(10,11-dihydro-5H-dibenzo[a,d][7]cyclononen-5-ylidene)propyl)(methyl)amino)hexanoic acid (A-6C)

[0074] (1) The synthetic route of 6-((3-(10,11-dihydro-5H-dibenzo[a,d][7]cyclononen-5-ylidene)propyl)(methyl)amino)hexanoic acid (A-6C) is as follows Figure 2 As shown, the specific synthesis steps are as follows:

[0075] 1 g of 3-(10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5-ylidene)-N-methyl-1-propane hydrochloride was fully dissolved in acetonitrile. Triethylamine (1:3 molar ratio to the starting material) and tert-butyl 6-bromohexanoate (1:1.5 molar ratio to the starting material) were added, and the mixture was stirred at 60 °C. The reaction was monitored by TLC, and the reaction was stopped when the starting material spot disappeared. The developing solvent was petroleum ether-ethyl acetate (3:1, v / v). After reacting for 3 h, the mixture was extracted with ethyl acetate and primary water (1:1 v / v). The ethyl acetate phase was collected and purified by silica gel column chromatography. The developing solvent was a petroleum ether-ethyl acetate (3:1, v / v) solution. After adding the sample, the mixture was eluted with a gradient of developing solvents to obtain a relatively pure product. The solvent was removed by vacuum distillation. The product was then dissolved in 3 mL of dichloromethane, and trifluoroacetic acid (1:1 v / v ratio to dichloromethane) was added. The mixture was stirred at room temperature for 1 h. After the reaction, the pH was adjusted to 6-7 with a 1 mol / L sodium hydroxide aqueous solution. Extraction was then performed using ultrapure water and ethyl acetate at a volume ratio of 1:1. The resulting organic phase was collected, and the aqueous phase was repeatedly extracted with pure ethyl acetate. The organic phases were then combined, dried over anhydrous Na₂SO₄, filtered, and the solvent was removed by vacuum distillation. The filtrate was concentrated under reduced pressure to obtain a white powder, which is the hapten 6-((3-(10,11-dihydro-5H-dibenzo[a,d][7]cyclononen-5-ylidene)propyl)(methyl)amino)hexanoic acid. Its structure is shown below:

[0076] Formula (II).

[0077] (2) Identification of amitriptyline hapten 6-((3-(10,11-dihydro-5H-dibenzo[a,d][7]cyclononen-5-ylidene)propyl)(methyl)amino)hexanoic acid (A-6C)

[0078] The 1H NMR results are as follows: 1H NMR (600 MHz, MeOD) δ 8.08 (s, 1H), 7.33 – 7.19 (m, 4H), 7.14 (dtd, J = 12.2, 7.2, 3.6 Hz, 3H), 7.07 – 7.02 (m, 1H), 5.83 (t, J = 7.3 Hz, 1H), 3.35 (s, 4H), 3.05 – 2.90 (m, 4H), 2.85 (s, 2H), 2.74 (s, 3H), 2.56 (q, J = 7.9 Hz, 2H), 1.61 (dp, J = 15.4, 7.6 Hz, 4H), 1.34 – 1.27 (m, 2H).

[0079] Mass spectrometry results are as follows: MS: C 25 H 31 NO2: 377.53, ESI + [MH] + 378.24.

[0080] The mass spectrometry and NMR results show that 377.53 is the positive ion molecular peak of the hapten 6-((3-(10,11-dihydro-5H-dibenzo[a,d][7]cyclononen-5-ylidene)propyl)(methyl)amino)hexanoic acid (A-6C). The calculated relative molecular mass is 378.24, which is consistent with the actual relative molecular mass. Furthermore, the proton NMR spectrum number corresponds to the proton NMR spectrum number on the skeleton structure of the amitriptyline hapten 6-((3-(10,11-dihydro-5H-dibenzo[a,d][7]cyclononen-5-ylidene)propyl)(methyl)amino)hexanoic acid (A-6C), indicating that the amitriptyline hapten 6-((3-(10,11-dihydro-5H-dibenzo[a,d][7]cyclononen-5-ylidene)propyl)(methyl)amino)hexanoic acid (A-6C) was successfully prepared.

[0081] Example 2

[0082] Preparation of amitriptyline artificial antigen

[0083] 1. Synthesis of artificial antigens

[0084] Weigh out 1 mol of the hapten 4-((3-(10,11-dihydro-5H-dibenzo[a,d][7]benzocyclohepten-5-ylmethylene)propyl)(methyl)amino)butyric acid prepared in Example 1, and dissolve it in 200 μL with N-hydroxysuccinimide (NHS) (0.8 mol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) (1.9 mol). In N,N-dimethylformamide (DMF), stir at room temperature in the dark for 2-4 hours to obtain hapten A-4C activation solution; add bovine serum albumin BSA (10 mg) to 1 mL of PBS buffer (0.01 mol / L, pH = 7.4); slowly add hapten A-4C activation solution dropwise to BSA PBS buffer solution, and react at 4℃ for 12 hours; after the reaction, dialyze with PBS buffer at 4℃ for 3 days, changing the dialysate 3 times a day. After dialyzing, amitriptyline artificial antigen A-4C-BSA is obtained, with the structure shown in formula (III). Amitriptyline artificial antigen A-4C-BSA is aliquoted into centrifuge tubes and stored at -20℃ for use.

[0085]

[0086] Formula (Ⅲ).

[0087] The PBS buffer formula is as follows: Na2HPO4·12H2O 2.90g, NaCl 8.50g, KCl 0.20g, KH2PO4 0.20g, and distilled water is added to bring the volume to 1000mL.

[0088] Based on the preparation method of artificial antigen A-4C, the hapten 4-((3-(10,11-dihydro-5H-dibenzo[a,d][7]cyclononen-5-ylidene)propyl)(methyl)amino)hexanoic acid (A-6C) was replaced with the same molar amount of amitriptyline hapten 6-((3-(10,11-dihydro-5H-dibenzo[a,d][7]benzocyclohepten-5-ylmethylene)propyl)(methyl)amino)butyric acid, and the same mass of chicken ovalbumin (OVA) was replaced with bovine serum albumin (BSA). The other steps were the same as those for artificial antigen A-4C, and the artificial antigen A-6C-OVA was prepared, with the structure shown in formula (Ⅳ).

[0089]

[0090] Formula (Ⅳ).

[0091] 2. Identification of artificial antigens

[0092] The haptens 4-((3-(10,11-dihydro-5H-dibenzo[a,d][7]benzocyclohepten-5-ylmethylene)propyl)(methyl)amino)butyric acid (A-4C), 6-((3-(10,11-dihydro-5H-dibenzo[a,d][7]cyclononen-5-ylmethylene)propyl)(methyl)amino)hexanoic acid (A-6C), chicken ovalbumin OVA, bovine serum albumin BSA, and the prepared artificial antigens A-4C-BSA and A-6C-OVA were identified by ultraviolet wavelength (150-400nm).

[0093] The results of the ultraviolet full-wavelength scanning identification are as follows: Figure 3 , Figure 4 As shown, comparing the highest absorbance values ​​of BSA, OVA, amitriptyline hapten A, A-4C-BSA, and A-6C-OVA before and after conjugation, it can be seen that the absorption curves of A-4C-BSA and A-6C-OVA are significantly different from those of BSA and OVA. The absorption curves of A-4C-BSA and A-6C-OVA show a shift at 280 nm that differs from those of BSA and OVA. Therefore, the absorption curves of A-4C-BSA and A-6C-OVA are the cumulative absorption peaks of BSA and OVA with amitriptyline hapten C, respectively. This demonstrates that amitriptyline hapten was successfully conjugated with BSA and OVA, and the present invention successfully prepared A-4C-BSA artificial antigen and A-6C-OVA artificial antigen.

[0094] Example 3

[0095] Preparation of amitriptyline monoclonal antibody

[0096] (1) Preparation of immunogen:

[0097] Immunogen for the experimental group: Take 50 μg of A-4C-BSA artificial antigen protein, dilute it with PBS to 50 μL, mix it with 50 μL of Freund's adjuvant in a 2.5 mL syringe, and emulsify it with an emulsifier until the emulsion does not spread on the surface of the water.

[0098] Control group immunogen: The succinic anhydride hapten A-DESG, prepared according to the method disclosed in the prior art CN109265534A, was conjugated with BSA according to the method in Example 1, resulting in artificial antigen A-DESG-BSA. 50 μg of the A-DESG-BSA artificial antigen protein was diluted to 50 μL with PBS and mixed with 50 μL of Freund's adjuvant in a 2.5 mL syringe. The mixture was emulsified using an emulsifier until the emulsion did not diffuse on the water surface during the water test.

[0099] Coating agent: A-6C-OVA (prepared in this invention).

[0100] (2) Immunization: Balb / C female mice (purchased from Zhuhai Baishitong) were selected as immunization animals, with 3 mice in each group. Immunogen was injected at multiple points on the abdomen and back of each mouse, 100 μL / mouse. The selection of immunization adjuvants and immunization cycle are shown in Table 1.

[0101] (3) Detection of antibody production in animals: One week after the third immunization and each subsequent immunization, blood was collected from the tail vein of mice, centrifuged, and antiserum was obtained. The antibody titer and inhibition rate of the antiserum were examined using ic-ELISA. The specific operating steps of the indirect competitive ELISA (ic-ELISA) method are as follows:

[0102] 1) Plate coating: Dilute the coating agent A-6C-OVA to a certain concentration using coating buffer (0.01mol / L carbonate buffer CB, pH 9.6) (adjust the coating concentration according to actual experimental needs), and then add it to a 96-well microplate, 100μL / well. Incubate in a 37℃ water bath for 12h, wash the plate twice, and pat dry.

[0103] 2) Blocking: Add blocking solution (5% skim milk powder), 120 μL / well, incubate in a 37℃ water bath for 3 h, discard the liquid in the plate, and invert in a 37℃ oven for 30 min.

[0104] 3) Competitive Reaction: Antiserum was serially diluted with 0.01 mol / L PBST solution at dilution factors of 1000, 2000, 4000, 8000, 16000, 32000, and 64000 (the dilution factors can be adjusted according to actual experimental conditions). The standard for detecting amitriptyline was diluted with 0.01 mol / L PBST solution to a specific concentration (the concentration of the detection drug was adjusted according to actual experimental needs). For each negative well, 50 μL of 0.01 mol / L PBST was added, followed by 50 μL of diluted antibody. For the negative control well, 100 μL of 0.01 mol / L PBST was added. For each positive well, 50 μL of diluted detection drug was added, followed by 50 μL of diluted antibody. For the positive control well, 50 μL of 0.01 mol / L PBST and 50 μL of diluted detection drug were added. After adding the liquid, incubate in a 37°C water bath for 40 minutes, wash the plate 5 times, and pat dry.

[0105] 4) Add secondary antibody: Dilute HRP-labeled goat anti-rabbit or goat anti-mouse (Sigma) 5000 times with 0.01 mol / L PBST, set 100 μL / well, incubate in a 37℃ water bath for 30 min, wash the plate 5 times, and pat dry.

[0106] 5) Color development: Add color development solution (TMB color development solution, Aladdin), 100 μL / well, and incubate in a 37℃ water bath for 10 min;

[0107] 6) Termination: Add stop solution (10% H2SO4), 50 μL / well, and read the absorbance value at 450 nm wavelength in a microplate reader.

[0108] 7) Result determination: Define the antiserum dilution factor corresponding to the negative well OD450 of 1.0~1.5 as the titer. Calculate the inhibition rate of the antiserum according to the following formula based on the OD450 value of the titer well and the corresponding OD450 value of the inhibition well.

[0109] Inhibition rate (%) = OD450 (valence pore OD) − OD450 (inhibition pore OD) / OD450 (valence pore OD).

[0110] The experimental results are shown in Table 2.

[0111] The concentration of amitriptyline was 1 µg / mL.

[0112] Experimental results show that, under the same coating antigen and immunization conditions, the antibodies induced by the A-4C-BSA and A-6C-OVA antigens prepared in this invention have significantly better titers and inhibition rates than antigens prepared by existing methods. This demonstrates that the structural design of the A-4C hapten in this invention leads to excellent immunization effects.

[0113] The hapten A-4C and its artificial antigen provided by this invention can increase the immunogenicity by more than 16 times and the antibody inhibition rate from about 20% to about 90% compared with the prior art, laying a solid foundation for the development of highly sensitive amitriptyline immunoassay products.

[0114] Example 4

[0115] Preparation of amitriptyline monoclonal antibody

[0116] (1) Preparation of immunogen: Take 50 μg of A-4C-BSA artificial antigen protein, dilute it with PBS to 50 μL, mix it with 50 μL of Freund's adjuvant in a 2.5 mL syringe, and emulsify it with an emulsifier until the emulsion does not spread on the water surface during the test;

[0117] (2) Immunization: Balb / C female mice were selected as immunized animals, and the immunogen was injected at multiple sites on the abdomen and back, 100 μL / mouse. The selection of immunization adjuvants and immunization cycle are shown in Table 3.

[0118] (3) Detection of antibody production in animals: One week after the third immunization and each subsequent immunization, blood was collected from the tail vein of mice, centrifuged, and antiserum was obtained. The antibody titer and inhibition rate of the antiserum were examined using ic-ELISA. The steps of ic-ELISA were the same as in Example 3.

[0119] (4) Cell fusion

[0120] ① Resuscitate myeloma cells: Take myeloma cells out of the -80℃ freezer and quickly place them in a 37℃ water bath to thaw. Use a pipette to remove the thawed cells and mix them with complete culture medium. Then place them in a 100mm culture dish and expand the culture to 5-6 dishes. When each dish is almost confluent, it can be used for cell fusion.

[0121] ②Boost immunization: Three days before formal cell fusion, mice with the best immunization effect were given a boost immunization once. The injection concentration was 1 mg / mL of diluted A-4C-BSA artificial antigen, and the injection volume was 100 µL. No adjuvant was required.

[0122] ③ Collecting myeloma cells: On the day of cell fusion, remove the supernatant from the myeloma cells in 6 culture dishes. Using a bent pipette, add approximately 25 mL of fresh basal culture medium, repeatedly blowing up the adherent myeloma cells in a specific direction until the bottom of the culture plate becomes transparent and no longer blurred. Collect this 25 mL of culture medium into a 50 mL centrifuge tube, seal it, and centrifuge at 800-1000 rpm for 8 minutes. Discard the supernatant after centrifugation and set aside for later use.

[0123] ④ Preparation of Immunosplenic Cells: Prepare approximately 25 mL of basal culture medium in a culture dish, placing a disposable cell grinding mesh inside. Use carbon dioxide to asphyxiate mice. After soaking in 75% ethanol for 4 minutes, transfer the mice to a clean bench and remove the spleen. Place the removed spleen in the disposable cell grinding mesh. First, use a syringe to draw culture medium and inject it into the spleen, repeating the injection to expel cells until the spleen changes color from red to transparent. Then, gently grind the spleen on the mesh using the syringe plunger, aspirating the culture medium to rinse the mesh. Collect this 25 mL of culture medium into a 50 mL centrifuge tube, seal the tube, and centrifuge at 1000 rpm for 7 minutes. Discard the supernatant after centrifugation and set aside for later use.

[0124] ⑤ Cell Mixing: Mix myeloma cells and immune spleen cells (after centrifugation and discarding the supernatant) in a separate centrifuge tube at a ratio of 1:5. Add 25 mL of basal culture medium, seal the tube, and centrifuge at 1000 rpm for 7 minutes. Discard the supernatant after centrifugation and set aside for later use.

[0125] ⑥ Cell Fusion: Discard the supernatant from the centrifuged and mixed myeloma cells and immune spleen cells. Gently loosen the precipitated cells with your fingers. Place the centrifuge tube in 37°C warm water. Use a pipette tip to draw 1 mL of preheated PEG2000 (37°C) and slowly add the PEG to the precipitated cells over 1 minute, gently stirring after each drop of PEG. Let stand for 0.5 minutes. Preheat the basal medium and add 1.5 mL over 1 minute, gently stirring along the wall. Then add the basal medium (Gibco) to a final volume of 20 mL over 5 minutes, gently stirring up and down to separate the PEG. Seal the centrifuge tube and centrifuge at 1000 rpm for 7 minutes. After centrifugation, discard the supernatant and add 200 mL of complete culture medium (the basal medium is prepared with 10% fetal bovine serum from Nanjing Novizan Biotechnology Co., Ltd.). Gently aspirate the liquid with a bent pipette and stir gently. Spread the complete culture medium containing fused cells into four 24-well plates, 2 mL / well.

[0126] ⑦ Cell screening: Perform monoclonal cell culture and screening according to the procedure in Table 4.

[0127] Cells were cultured in an incubator for approximately 10 days. Starting on day 7, cell growth and culture medium status were monitored daily. On day 10, or if cell growth was too rapid, a partial medium change was performed using HAT medium (Sigma). On day 13, a complete medium change was performed using HAT medium. On day 15, a partial medium change was performed using HT medium (Sigma). The cell supernatant was then subjected to the first icELISA test. On day 17, the HT medium was completely changed, and a second icELISA test was performed. On day 19, the HT medium was completely changed again, and a third icELISA test was performed. The results of the first three supernatant tests were compared, and wells with high titers were selected for limiting dilution. When performing icELISA on the limitedly diluted cell supernatant, titer and inhibition rate were used as indicators. Positive wells were selected, and the next round of limiting dilutions was performed. The icELISA test procedure was the same as in Example 3. The hybridoma cell line was successfully established when every well on each plate tested positive and the titer and inhibition were similar. The cells were then cryopreserved promptly.

[0128] ⑧ Monoclonal antibody preparation: Around 10 weeks of age, female Balb / c mice were selected, and 500 μL of liquid paraffin was injected intraperitoneally one week in advance. Once the hybridoma cell line had reached confluence in 100 mm cell culture dishes, the cells were aspirated with 1.0 mL of basal culture medium and collected in a disposable sterile syringe. The cells were then injected into the peritoneal cavity of the mice, with two mice injected per dish, and each mouse receiving 500 μL of cells. Approximately 7 days after cell injection, the mice's condition needed frequent observation. When the mice showed decreased activity and significant abdominal swelling, the ascites fluid was dissected, centrifuged at 12000 rpm for 15 min, and the supernatant fat and sublayer protein were removed. The ascites fluid was collected and stored at -20°C for later use.

[0129] 9. Purification of monoclonal antibodies

[0130] All solutions used for purification were filtered through a 0.22 μm pore size membrane and sonicated for 30 min (including ultrapure water, equilibration buffer PBS, and elution buffer Gly-HCl) to remove insoluble solids and air bubbles. The collected ascites fluid was diluted 1-fold with PBS, passed through a 0.45 μm pore size nylon microporous membrane to remove impurities, and then purified using an AKA protein purifier. The purification steps are as follows:

[0131] (1) System and column equilibration: Replace the protective solution in the column with equilibration buffer PBS and wash the column;

[0132] (2) Sample loading: Flow the ascites fluid through the proteinG affinity chromatography column at a rate of 1 mL / min until the UV detection line and Cond detection line return to the baseline and remain stable;

[0133] (3) Elution: Wash the protein G affinity chromatography column with 0.1 mol / L Gly-HCl elution buffer at pH 2.7 at a rate of 2 mL / min to elute the antibody adsorbed on the column. Adjust the pH of the collected liquid to about 7 with 1 mol / L Tris-HCl (pH 9.0).

[0134] (4) Dialysis: After pH adjustment, the solution was dialyzed in PBS solution at 4℃ for two days; the molecular weight cutoff for dialysis was 5000 kDa.

[0135] (5) Determination of monoclonal antibody purity: The purity of the purified antibody was determined by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE).

[0136] ⑩ Identification of monoclonal antibodies

[0137] (1) Antibody subtype identification

[0138] Mouse antibody subtypes are classified into IgG1, IgG2a, IgG2b, IgG3, IgM, and IgA. This experiment used a commercially available antibody subtype identification kit (Sigma) to identify antibody subtypes. The results are as follows: Figure 5 As shown, the results indicate that the antibody subtype is IgG1.

[0139] (2) Antibody purity identification

[0140] Take 8 μL of antibody and ascites fluid, and mix them separately with 2 μL of 5×SDS loading buffer. Heat at 100℃ for 10 min. Load 10 μL of the sample and 5 μL of standard protein marker onto the sample for SDS-PAGE gel electrophoresis. Stain with Coomassie Brilliant Blue solution, destain overnight on a shaker, and then image using a gel imaging system. The SDS-PAGE gel electrophoresis results are shown below. Figure 6 The theoretical molecular weight of IgG via non-reducing electrophoresis is 150 kDa. Reducing electrophoresis separates it into two 50 kDa heavy chains and a 25 kDa light chain. The electrophoresis results match the theoretical value, indicating successful purification and successful preparation of the monoclonal antibody. The monoclonal antibody was sent to Nanjing Zhongding Biotechnology Co., Ltd. for sequencing, and the results are shown in the table below.

[0141] The full length of the heavy chain variable region VH of the monoclonal antibody is: EVQLQESGAELMKPGASVKISCKAIGYTFSSYWIEWVKQRPGHGLEWIGEILRGRGSINYNEKFKGKATFTADTSSNTVHMQLSSLTSEDSAVYYCASYGNNRFDWGQGTLVTVSA (SEQ ID NO.1).

[0142] The full length of the light chain variable region (VL) of the monoclonal antibody is: DIVLTQTPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYAEFPYTFGGGAKLEIK (SEQ ID NO.2).

[0143] Example 5

[0144] Optimization of the combination of amitriptyline immunogen and coating agent

[0145] Artificial antigens A-4C-LF and A-6C-LF, using lactoferrin (LF) of equal mass as the carrier protein, and artificial antigen A-4C-OVA, using ovalbumin (OVA) of equal mass as the carrier protein, were prepared according to the preparation method of A-4C-BSA in Example 2, and all were successfully conjugated.

[0146] Using A-4C-BSA prepared in Example 2 as an immunogen, amitriptyline monoclonal antibody prepared by immunizing Balb / c mice according to the method in Example 3 was screened for coating antigens. A-4C-LF, A-6C-LF, A-4C-OVA and A-6C-OVA prepared in Example 2 were used as coating antigens, and the titer and inhibition rate of the monoclonal antibody were detected by icELISA.

[0147] The specific operating steps are as follows:

[0148] ① Dilute amitriptyline artificial antigens A-4C-LF, A-6C-LF, A-4C-OVA and A-6C-OVA to a concentration of 250 ng / mL with coating buffer (0.05 M carbonate buffer, pH 9.6), coat 96-well microplates, add 100 μL to each well, incubate overnight at 37°C, discard the coating buffer, and wash twice with PBST (0.01 M PBS, 0.06% Tween-20 (v / v));

[0149] ② Add 120 μL of blocking solution (5 wt% skim milk powder) to each well, seal at 37°C for 3 hours, discard the blocking solution, tap the plate, and dry in a drying oven at 37°C for later use;

[0150] ③ The amitriptyline monoclonal antibody prepared in Example 4 was diluted with PBST to 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000, and 1:512000, and blank control wells were set up (using PBST instead); 1 mg / mL amitriptyline was diluted 1000 times with PBST to 1 μg / mL;

[0151] Titer series: First, add 50 μL of PBST to each well, then add 50 μL of serially diluted amitriptyline monoclonal antibody to each well in sequence, with no antibody added to the last well, and replace it with 50 μL of PBST;

[0152] Inhibition column: First, add 50 μL of amitriptyline drug to each well, then add 50 μL of serially diluted amitriptyline monoclonal antibody to each well sequentially, except for the last well which is not filled with antibody and is replaced with 50 μL of PBST; incubate at 37°C for 40 min, wash 5 times, and plate.

[0153] ④ Add goat anti-mouse secondary antibody Ig-HRP (sigma) (5000-fold dilution), incubate at 37℃ for 30 min, wash 5 times, and plate.

[0154] ⑤ Add colorimetric solution (TMB colorimetric solution, Aladdin) and incubate at 37°C for 10 min;

[0155] ⑥ Add 10% H2SO4 to terminate the reaction and read the OD value at 450nm.

[0156] Valence is defined as OD 450 The antiserum dilution factor corresponding to approximately 1.0; Inhibition rate = (OD value of titer - OD value of inhibition) / OD value of inhibition * 100%.

[0157] The screening results of immunogens and coating agents are shown in Table 6.

[0158] Table 5 shows that different amitriptyline artificial antigens used as coating agents all exhibited certain titers and varying degrees of inhibitory effects on the analyte amitriptyline. Among them, the combination of immunogen and coating agent structure number 4 showed the best antiserum efficacy and inhibition rate against amitriptyline. With this combination, the amitriptyline monoclonal antibody not only specifically recognized the target analyte amitriptyline but also demonstrated good antibody sensitivity; the inhibition rates were higher than those of immunogen and coating agent combinations numbered 1, 2, and 3. Therefore, the combination of immunogen and coating agent structure number 4 is the optimal combination. Specifically, A-4C-BSA was used as the immunogen and A-6C-OVA as the coating agent.

[0159] Example 6

[0160] Establishment of an indirect competitive ELISA detection method for amitriptyline

[0161] 1. Experimental Methods

[0162] An indirect competitive ELISA method for detecting amitriptyline includes the following steps:

[0163] (1) Artificial antigen A-6C-OVA was used as the coating agent, diluted to 156.25 ng / mL with coating solution, and coated with 100 μL of the coating agent in each well of a 96-well microplate. The plate was incubated at 37°C overnight (12 h).

[0164] (2) Discard the coating solution, wash twice, and pat dry;

[0165] (3) Add 120 μL of blocking solution (5% skim milk powder) to each well and seal at 37°C for 3 hours;

[0166] (4) Discard the sealing liquid, pat the plate, dry at 37°C for 30 minutes, and then pack it in a self-sealing bag for later use;

[0167] (5) Dilute amitriptyline antibody with PBST at a dilution ratio of 1:256000, and dilute amitriptyline standard (Aladdin) to 1000 ng / mL, 333.3333 ng / mL, 111.1111 ng / mL, 37.03704 ng / mL, 12.34568 ng / mL, 4.115226 ng / mL, 1.371742 ng / mL, 0.457247 ng / mL, 0.152416 ng / mL, 0.050805 ng / mL, 0.016935 ng / mL, 0.005645 ng / mL, 0.001882 ng / mL, 0.000627 ng / mL, 0.000209075 ng / mL;

[0168] (6) Add 50 μL of amitriptyline diluent to each row (three sets in parallel), then add 50 μL of monoclonal antibody diluent per well, incubate at 37°C for 40 min, wash five times, and pat dry;

[0169] (7) Add 100 μL of goat anti-rabbit secondary antibody-HRP (sigma, 5000 times dilution) to each well, incubate at 37℃ for 30 min, wash five times, and pat dry;

[0170] (8) Add colorimetric solution (TMB colorimetric solution, Aladdin), 100 μL per well, and develop color for 10 min;

[0171] (9) Add 50 μL of 10% H2SO4 solution to terminate the reaction and read the OD value at 450 nm.

[0172] 2. Experimental Results

[0173] The standard curve for an indirect competitive ELISA used to detect amitriptyline is as follows: Figure 7 As shown, the half-maximal inhibitory concentration (WMC) of the antibody used to detect amitriptyline is 0.865 ng / mL, the quantitative detection range is 0.07-470.3 ng / mL, and the limit of detection is 0.013 ng / mL. This indicates that the antibody prepared by this invention for detecting amitriptyline can meet the detection requirements and has high recognition ability, strong specificity, and high detection sensitivity for amitriptyline.

[0174] Example 7

[0175] Specificity evaluation of amitriptyline antibodies

[0176] 1. Test Methods

[0177] The specificity of amitriptyline for detection was determined by cross-reactivity experiments with amitriptyline and its analogues. The specificity of the antibody was expressed as the cross-reactivity rate (CR), with a lower CR indicating stronger specificity. Amitriptyline and its analogues (clomipramine, citalopram, imipramine, and doxepin) were serially diluted and measured using an indirect competitive ELISA method, following the same procedure as in Example 6, to obtain the IC50 values ​​for each analogue. 50 The cross-reactivity rate (CR) of amitriptyline is calculated using the following formula:

[0178] CR(%) = IC 50 (Amitriptyline) / IC 50 (Similar substances) × 100%.

[0179] 2. Experimental Results

[0180] The cross-reactivity results of amitriptyline and its analogues are shown in Table 7.

[0181] The results showed that the antibody used to detect amitriptyline had a 100% cross-reactivity with amitriptyline, and the IC50 was [missing information]. 50 The concentration was 0.865 ng / mL, and the cross-reactivity rates to clomipramine, citalopram, imipramine, and doxepin were 1.9%, 0.5%, 6.65%, and 2.4%, respectively. This indicates that the amitriptyline antibody has good specificity.

[0182] Example 8

[0183] Development of an ELISA kit for detecting amitriptyline

[0184] 1. Construct a kit for detecting amitriptyline, the kit comprising the following components:

[0185] (1) Preparation of ELISA plates coated with coating antigen: The artificial antigen A-6C-OVA prepared in Example 2 was used as the coating antigen. The coating antigen was diluted to 156.25 ng / mL with coating buffer (carbonate buffer). The ELISA plates were coated with 100 μL / well and incubated overnight at 4°C in the dark. The liquid in the wells was poured off, and the plates were washed twice with washing buffer for 30 seconds each time and patted dry. Then, blocking buffer was added at 120 μL / well and incubated at 37°C in the dark for 1 h. The liquid in the wells was poured off and patted dry. After drying, the plates were vacuum sealed with aluminum foil for storage.

[0186] (2) Amitriptyline standard solution: 15 concentration gradients, namely 1000 ng / mL, 333.3333 ng / mL, 111.1111 ng / mL, 37.03704 ng / mL, 12.34568 ng / mL, 4.115226 ng / mL, 1.371742 ng / mL, 0.457247 ng / mL, 0.152416 ng / mL, 0.050805 ng / mL, 0.016935 ng / mL, 0.005645 ng / mL, 0.001882 ng / mL, 0.000627 ng / mL, and 0.000209075 ng / mL.

[0187] (3) Amitriptyline monoclonal antibody prepared in Example 5.

[0188] (4) The enzyme-labeled secondary antibody is horseradish peroxidase-labeled goat anti-mouse secondary antibody (Sigma).

[0189] (5) Substrate colorimetric solution: composed of solution A and solution B, where solution A is urea peroxide and solution B is tetramethylbenzidine.

[0190] (6) The stop solution is 10% H2SO4.

[0191] (7) The washing solution is a 0.2 mol / L phosphate buffer with a pH of 7.4, containing 0.8% Tween-20 by volume and 0.02% sodium azide preservative by mass. Before use, dilute the washing solution 20 times with water (i.e., add 1 part washing solution to 19 parts water, prepare fresh before use) to obtain the working solution of the washing solution.

[0192] (8) The diluent is 0.2 mol / L phosphate buffer; dilute the diluent 20 times with water before use (i.e., add 1 part of diluent to 19 parts of water, prepare fresh before use) to obtain the working solution of the diluent.

[0193] (9) The blocking solution is a 0.2 mol / L phosphate buffer with a pH of 7.3 and containing 2% casein by mass.

[0194] 2. Use the above ELISA kit to test the samples.

[0195] (1) Sample testing

[0196] Number the wells of the samples and standards from the ELISA kit sequentially, performing two parallel wells for each sample and standard, and record the positions of the standard and sample wells. Dilute the antibody with the required amount of diluent at a 1:40 volume ratio (i.e., add 1 part antibody to 40 parts diluent, prepare fresh before use) to obtain the antibody working solution. Dilute the enzyme-labeled secondary antibody with the required amount of diluent at a 1:10 volume ratio (i.e., add 1 part enzyme-labeled secondary antibody to 10 parts diluent, prepare fresh before use) to obtain the enzyme-labeled secondary antibody working solution.

[0197] Add 50 μL of standard or sample to the corresponding well, then add 50 μL of antibody working solution to the corresponding well, gently shake to mix, cover with a cover plate and incubate at 25°C in the dark for 40 min.

[0198] Shake off the liquid in the well and add 250 μL of washing working solution per well. Wash thoroughly 4-5 times, with 10-second intervals between each wash. Discard the washing working solution in the well and pat dry with absorbent paper (any air bubbles not removed after patting can be popped with an unused pipette tip).

[0199] Add 100 μL of enzyme-labeled secondary antibody working solution to the corresponding microwell, gently shake to mix, cover with a cover plate membrane, and incubate at 25°C in the dark for 30 min.

[0200] Shake off the liquid in the well and add 250 μL of washing working solution per well. Wash thoroughly 4-5 times, with 10-second intervals between each wash. Discard the washing working solution in the well and pat dry with absorbent paper (any air bubbles not removed after patting can be punctured with an unused pipette tip).

[0201] Add 50 μL of substrate development solution A per well, then add 50 μL of substrate development solution B per well, gently shake to mix, cover with a cover plate and incubate at 25°C in the dark for 10 min.

[0202] Add 50 μL of stop solution per well, gently shake to mix, set the microplate reader to 450 nm, and measure the OD value of each well.

[0203] (2) Drawing the standard curve

[0204] A standard curve was prepared by plotting B / B0 as the ordinate (B being the absorbance OD450 of standards at different concentrations, and B0 being the absorbance OD450 of the blank control wells) and the logarithm of the standard concentrations as the abscissa, and then fitting the curve using the Logistic function. Figure 7 As shown.

[0205] (3) Calculation of amitriptyline concentration in the sample

[0206] Serum samples from patients who had taken amitriptyline were used as samples, and each sample was tested three times. HPLC-MS / MS was used as the gold standard, and the HPLC-MS / MS method was based on existing technology (Li Tao, Zhao Xiaoliang, Gao Tianle, et al. Study on microdialysis sampling and HPLC-MS / MS quantification of free sinomenine, tetramethylpyrazine, gabapentin, acetaminophen, pregabalin and amitriptyline in rat blood and brain tissue fluid [J]. Acta Pharmaceutica Sinica, 2020, 55(9):9.). Recovery experiments were performed using the above-mentioned ELISA kit. In the detection, the average OD450 of the sample was substituted into the formula of the above standard curve to obtain the concentration of amitriptyline in the sample, and then multiplied by the corresponding dilution factor to obtain the actual concentration of amitriptyline in the test sample. The results are shown in Table 8.

[0207] As can be seen, the average recovery rate of the kit of the present invention is between 97.3% and 106.5%, and the coefficient of variation is less than 15%, indicating that the kit of the present invention has high accuracy.

[0208] In summary, this invention provides two amitriptyline haptens with appropriately long spacer arms, and uses them to prepare artificial antigens. The resulting amitriptyline monoclonal antibodies exhibit high titers, strong specificity, and high affinity, providing core raw materials for establishing a specific amitriptyline immunoassay method. The immunoassay method based on the amitriptyline artificial antigen and antibody provided by this invention has high specificity and high sensitivity, with a limit of detection (LOD) of 0.013 ng / mL and a half-inhibitory concentration (IC50). 50 The concentration is 0.865 ng / mL, the quantitative detection range is 0.07~470.3 ng / mL, and the cross-reactivity rate with amitriptyline analogues is low. It can be used for rapid qualitative and quantitative detection of amitriptyline in samples. The operation is simple and the detection results are accurate and reliable.

[0209] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of this invention are still within the scope of protection of this invention.

Claims

1. An amitriptyline antibody, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, and the light chain variable region comprises light chain CDR1, light chain CDR2, and light chain CDR3, characterized in that, The sequence of heavy chain CDR1 is shown in SEQ ID NO.4, the sequence of heavy chain CDR2 is shown in SEQ ID NO.6, the sequence of heavy chain CDR3 is shown in SEQ ID NO.8, the sequence of light chain CDR1 is shown in SEQ ID NO.11, the sequence of light chain CDR2 is RAN, and the sequence of light chain CDR3 is shown in SEQ ID NO.

13.

2. The amitriptyline antibody according to claim 1, characterized in that, The variable region of the heavy chain is shown in SEQ ID NO.1; and / or the variable region of the light chain is shown in SEQ ID NO.

2.

3. The amitriptyline antibody according to claim 1, characterized in that, It is prepared using amitriptyline hapten or amitriptyline antigen, and the structural formula of the amitriptyline hapten is shown in formula (I) or formula (II): ； The structural formula of the amitriptyline antigen is shown in formula (III) or (IV): ； Z represents the carrier protein.

4. The amitriptyline antibody according to claim 3, characterized in that, The carrier protein is chicken ovalbumin or bovine serum albumin.

5. A kit for detecting amitriptyline, characterized in that, The kit for detecting amitriptyline contains an amitriptyline antibody according to any one of claims 1-4.

6. The kit for detecting amitriptyline according to claim 5, characterized in that, The kit for detecting amitriptyline comprises: an enzyme-labeled secondary antibody, a substrate chromogenic solution, a stop solution, a washing solution, a diluent, a blocking solution, an enzyme-labeled plate coated with amitriptyline antigen, and an amitriptyline antibody according to any one of claims 1-4; The structural formula of the amitriptyline antigen is shown in formula (III) or (IV): ； Z represents the carrier protein.

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